Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Changes in glycoprotein expression are correlates of disease, but secreted glycoproteins cannot be accurately traced to their cell line of origin. Here, the authors develop a strategy to chemically tag and profile glycoproteins in a cell line-specific manner in co-culture systems and in vivo. Recent years have seen a stark increase in methods to probe the transcriptomes of tumour and host cell populations, providing some insight into their state within a multicellular conglomerate4. However, the relationship between transcriptome and proteome is still elusive5. In addition, posttranslational modifications (PTMs) heavily impact the plasticity of the proteome. Glycosylation is the most complex and most abundant PTM, but challenging to probe due to the non-templated nature of glycan biosynthesis6. Glycans are generated by the combinatorial interplay of >250 glycosyltransferases (GTs) and glycosidases, mostly in the secretory pathway7. A small number of glycoproteins aberrantly expressed in cancer, such as mucins, have been approved as diagnostic markers, but their discovery is a particular challenge9. This is especially true when in vivo or in vitro model systems are comprised of cell populations from the same organism that do not allow distinction of proteomes by amino acid sequence11. Methods to study the glycoproteome of a cell type in co-culture or in vivo are therefore an unmet need.Cancer is a multifactorial disease consisting of an interplay between host and tumour. Emulating the complexity of a tumour in cell monoculture is thus incomplete by design, requiring more elaborated co-culture systems or in vivo models12. MOE reagents are membrane-permeable monosaccharide precursors modified with chemical tags amenable to bioorthogonal chemistry13. Following incorporation into the glycoproteome, chemical tags are reacted with traceable enrichment handles or fluorophores, for instance by Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC)15. Many MOE reagents are based on analogues of sugars such as N-acetylgalactosamine  that are straightforward to chemically tag by replacing the acetamide with bioorthogonal N-acylamides  use UDP-GalNAc to form the linkage GalNAcα-Ser/Thr and thereby prime cancer-relevant O-GalNAc glycans20. Second, epimerisation at the GalNAc C4 position by the UDP-galactose-4-epimerase (GALE) yields UDP-N-acetylglucosamine (UDP-GlcNAc) that can be incorporated into different glycan subtypes, for instance Asn-linked N-glycans21. Certain chemical modifications at the N-acyl moiety can render GalNAc analogues recalcitrant to these metabolic processes. For instance, analogues of UDP-GalNAc with long alkyne-containing N-acyl substituents are not biosynthesised by the GalNAc salvage pathway and not used as substrates by Wild Type (WT)-GalNAc-Ts24. While being a substantial impediment to generating MOE reporters, we realised that overcoming these metabolic roadblocks might enable programmable bioorthogonal glycoprotein tagging. Such a strategy would allow for studying the glycoproteome in a cell-specific fashion, which is currently elusive despite the rapid advances in the development of new MOE reagents.Metabolic oligosaccharide engineering (MOE) produces chemical reporters of glycan subtypesdes Fig. . Unmodifdes Fig. 14,16–18.N-acyl substituents drastically reduces uptake by the native GalNAc salvage pathway and thereby reduces the background of bioorthogonal labelling in non-transfected cells. Only cells carrying the artificial pathway biosynthesise the corresponding UDP-sugars  that are then used by GTs to chemically tag the glycoproteome. We further expand the strategy with mutant GalNAc-Ts that are engineered to accept UDP-GalN6yne as a substrate. The combined use of an artificial biosynthetic pathway and engineered GalNAc-Ts enables GalN6yne-mediated fluorescent labelling of the cellular glycoproteome that is two orders of magnitude higher than in cells carrying neither component. We demonstrate that BOCTAG allows for programmable glycoprotein tagging in co-culture and mouse models. Moreover, the nature of the artificial biosynthetic pathway allowed for the use of readily available Ac4GalN6yne as a precursor with enhanced stability over previously used caged GalN6yne-1-phosphates as an essential pre-requisite for in vivo applications. We show that the chemical modification enters a range of glycan subtypes, supporting the use of BOCTAG to tag a large number of glycoproteins in complex biological systems.Here, we develop a technique called Bio-Orthogonal Cell-specific Tagging of Glycoproteins (BOCTAG). The strategy uses an artificial biosynthetic pathway to generate alkyne-tagged UDP-GalNAc and UDP-GlcNAc analogues from a readily available GalNAc precursor that is not accepted by the GalNAc salvage pathway. We find that a single methylene group between 5-carbon  and 6-carbon  N-acyl moiety of GalNAc  and a 5-carbon pent-4-ynoate chain  as GalNAc modifications due to their availability and previous use by us and others27. According to in vitro enzymatic assays detected by Liquid Chromatography-Mass Spectrometry (LC-MS), recombinant GALK2 accepted GalNAlk as a substrate, but only marginally accepted GalN6yne  from various source organisms converted GalN6yne to GalN6yne-1-phosphate almost quantitatively  to the corresponding UDP-sugars -mannosamine) that enters the pool of the sugar sialic acid was included as a positive control. Alkyne tags were visualised by in-gel fluorescence after cell lysis  and DMSO-fed NahK/mut-AGX1, respectively. The relative ratio of both metabolites was constant in all cell lines . To assess if the cellular glycome would be affected by variations in nucleotide-sugar concentrations, we performed lectin blotting on lysates of cells expressing NahK/mut-AGX1. Compared to mock-transfected cells, we found no differences in binding patterns with the four lectins ConA, MAL II, SNA and AAL, as well as the antibody RL2 which detects nucleocytoplasmic O-linked GlcNAc . In three independent experiments, in which both combinations of heavy and light SILAC labelling each were used proteome of cells transfected with NahK and mut-AGX1. K-562 cells transfected with NahK/mut-AGX1 or an empty plasmid (mock-transfected) were individually grown in heavy or light media in the presence of either Acsed Fig. , we foun4GalN6yne, Ac4ManNAlk or DMSO  that selectively use chemically tagged UDP-GalNAc analogues in glycosylation reactions33. We stably co-expressed WT- or BH-versions of GalNAc-T1 or T2 from plasmids also encoding NahK and mut-AGX1 in K-562 cells  LC/MS-MS analysis. We transfected cells with either a plasmid encoding NahK/mut-AGX1/BH-GalNAc-T2 (termed “BOCTAG-T2”) or empty plasmid , co-cultured murine and human cells overnight and subsequently fed the co-cultures with either Acads Fig. . On-beadies Fig. . We alsoies Fig. . The pre4GalN6yne, vehicle or Ac4MAnNAlk once daily for 5 consecutive days  mice, consisting of 4T1 cells expressing GFP and either BOCTAG-T2 (one fat pad) or no additional transgene . These mice were intraperitoneally injected with Acays Fig. . At the yne Fig. . In cont4GalN6yne-dependent chemical glycoproteome tagging . GalNAlk, GalN6yne, GalNAlk-1-phosphate, Ac3GalN6yne-1-P(SATE)2 and Ac4GalN6yne were prepared as reported24. Primers were from Merck KGaA . Restriction enzymes were from New England Biolabs. The plasmids pSBtet-GH and pSBbi-GH were a gift from Eric Kowarz 43. pTriEx6-His-GST-3C-MCS was an in-house construct modified from pTriEx-6 (Supplementary Data 19. The plasmid pCMV(CAT)T7-SB100 T7-SB100; RRI- D:Addgene_34879) was a kind gift from Zsuzsanna Izsvak42.The compounds AcAll sequences of oligonucleotides were reported in Supplementary Data Adobe Illustrator 2022 was used to assemble figures and to re-label plot axes.flashBAC™ system . Cells were harvested ) and resuspended in 50 mL cold Lysis Buffer  with cOmplete protease inhibitors  and BaseMuncher mix , and left at 4 °C for 1 h. Cells were then lysed by 6 min sonication  using a Sonifier 450  prior to ultra-centrifugation . The supernatant was collected and incubated overnight with 2 mL of GST-4B Sepharose slurry beads  pre-equilibrated in cold Lysis Buffer containing 10% (v/v) glycerol. The supernatant was then collected  and washed twice with 10 column volumes (CV) of same buffer. GST-4B Sepharose beads were then resuspended in 2 CV of Lysis Buffer containing 10% (v/v) glycerol and incubated with 100 µL of HRV 3 C protease 47 at 4 °C for 4 h. The supernatant was collected and the digestion was repeated three times. Supernatants were pooled and concentrated to 2 mL using a 30 kDa MWCO  Amicon® Ultra-15mL Centrifugal Filter Unit(Merck). The concentrated sample was injected onto an ÄKTA™ Pure system, running a Superdex™ S200 16/600 gel filtration column , collecting 1 mL fractions in Lysis Buffer containing 10% (v/v) glycerol. Fractions were pooled, concentrated using a 30 kDa MWCO Amicon® Ultra-15mL Centrifugal Filter Unit, concentration measured by Nanodrop® (1.94 mg/mL), sample diluted twofold in a freezing buffer  glycerol, 1 mM DTT) and stored at −80 °C until use.pDONR221 plasmids containing GALK1 or GALK2 were purchased from DNASU Plasmid Repository . The coding sequences in pDONR221 (HsCD00746245 for GALK1 and HsCD00039428 for GALK2) were cloned into BamHI/BglII-digested pTriEX with an N-terminal GST-3c cleavage site and a C-terminal FLAG-tag by using the In-Fusion HD Cloning Kit  and the following primers: inf_GALK1_F/R or inf_GALK2_F/R. Reaction was run for 30 cycles using the following protocol: 98 °C (30 s), 98 °C (10 s), 58 °C (15 s), 72 °C (3 min – return to step 2), 72 °C (5 min). Sf21 cells (ATCC CRL-1711) were transfected with pTriEx6-His-GST-3C-GALK1-FLAG and pTriEx6-His-GST-3C-GALK2-FLAG constructs using the Baculovirus system with a transfer plasmid method. Sf21 cells were infected for 3 days with recombinant baculovirus generated based on the 24. WT- and mut-AGX1 fragments were cloned into BamHI/BglII-digested pTriEX with an N-terminal GST-3c cleavage site and a C-terminal FLAG-tag by using In-Fusion HD Cloning Kit  and primers inf_AGX1_F/R. The reaction was run for 30 cycles using the following protocol: 98 °C (30 s), 98 °C (10 s), 58 °C (15 s), 72 °C (3 min – return to step 2), 72 °C (5 min). pTriEx6-His-GST-3C-WT-AGX1-FLAG and pTriEx6-His-GST-3C-mut-AGX1-FLAG were transfected and expressed in Sf21 cells by following the same procedure described above for pTriEx6-His-GST-3C-GALK1/2-FLAG.A pIRES-puro3 plasmid containing WT-AGX1 was used as template to generate mut-AGX1 by site-directed mutagenesis by using the primers AGX1_F383A_F/R and the Q5®-site direct Mutagenesis KitB. Longum, T. bernardi, A. phocae, C. sp FS41 and E. tayi NahKs were produced by Prozomix Ltd. 28. Reactions with NahK enzymes or human kinases GALK1 and GALK2 were run in 50 µL volume, containing GalN6yne or GalNAlk (5 mM), adenosine triphosphate , MgCl2 (10 mM), Tris-HCl pH 8 (100 mM) and kinase (20 µg). Reactions were run for 4 h at 37 °C. Reactions were diluted with 50 µL methanol, cooled to −20 °C for 2 h and centrifuged  to remove any precipitated enzyme. Supernatants were analysed by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) equipped with a ACQUITY UPLC BEH Glycan  1.7 µm 2.1 × 50 mm column  acetonitrile/water). Estimated conversion was obtained by analysing a 5 mM product standard  in the same analysis conditions as above, extracting product mass and comparing ion count with the ion count of the extracted mass of product in the samples.2 (5 mM), Tris-HCl pH 8 (75 mM), BSA (1 mM), uridine triphosphate , pyrophosphatase , recombinant WT- or mut-AGX1 (125 nM). Reactions were run for either 2 h or 16 h at 37 °C. 7 µL of each reaction were diluted with 7 µL of acetonitrile, cooled on ice for 30 min and centrifuged  to remove any precipitated enzyme. Supernatants were analysed by UPLC-MS equipped with an ACQUITY UPLC BEH Glycan 1.7 µm 2.1 × 50 mm column  acetonitrile/water). The percentage conversions of sugar-1-phosphate analogues to corresponding UDP-sugars was estimated by UV peak integration of products at 260 nm. The corresponding product peak areas were converted to concentration using standard curves for each of the products. The standard curves were constructed by running commercial  and synthetic  samples on UPLC, and plotting integrated area against concentration.In vitro enzymatic assays were run in 15 µL volume, containing GalNAlk- or GalN6yne-1-phosphate , MgClWT and pSBbi-AGX1F383A were cloned previously by GeneArt 18; NahK from Bifidobacterium longum (E8MF12) was cloned into pSBbi-AGX1WTand pSBbi-AGX1F383A plasmids by GeneArt, containing 2A self-cleaving peptides and a C-terminal HA tag, to give the plasmids pSBbi-AGX1WT-NahK and pSBbi-AGX1F383A-NahK.pSBbi-AGX124 into pSBbi-AGX1WT-NahK or pSBbi-AGX1F383A-NahK using an SfiI cloning strategy with primers inf_GalNAc-T1_F/R and inf_GalNAc-T2_F/R. This cloning strategy gave the following plasmids: pSBbi-AGX1WT-NahK-T1BH, pSBbi-AGX1F383A-NahK-T1WT, pSBbi-AGX1F383A-NahK-T1BH, pSBbi-AGX1WT-NahK-T2BH, pSBbi-AGX1F383A-NahK-T2WT, pSBbi-AGX1F383A-NahK-T2BH. AGX1, NahK and GalNAc-T1/T2 constructs were tagged with C-terminal FLAG, HA and VSV-G tags, respectively. The full plasmid sequences are reported in Supplementary Data WT- or ”Bump-and-Hole” (BH) engineered versions of GalNAc-T1 and T2 were cloned previously from pSBtet-plasmidsAll cells were screened for contamination by mycoplasma and other cell lines by the Crick Cell Services Science Technology Platform. K-562 cells (ATCC CCL-243) were propagated in RPMI (Thermo Fisher) with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL). 4T1 , murine MLg fibroblast (ATCC CCL-206) and MCF7 human breast cancer cells (ATCC HTB-22) were maintained in DMEM (Thermo Fisher) with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL).WT or pSBbi-AGX1F383A have been prepared previously18. K-562 cells were stably transfected with pSBbi-AGX1-NahK, pSBbi-AGX1-NahK-T1/T2, pSBtet-AGX1F383A-T2 constructs or empty pSBtet-GH24 using Lipofectamine LTX (Thermo Fisher) according to the manufacturer’s instructions, with a 20:1 (w/w) mixture of pSBbi and pCMV(CAT)T7-SB100 plasmid DNA. After 24 h, cells were harvested and selected in growth medium containing 150 µg/mL hygromycin B (Thermo Fisher) for 7–10 days to obtain stable cells. 4T1 (GFP), MLg and MCF7 cells were stably transfected with either pSBbi-AGX1F383A-NahK, pSBbi-AGX1F383A-NahK-T2BH or empty pSBbi-Hyg using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s specifications, with a 20:1 (w/w) mixture of pSBbi and pCMV(CAT)T7-pSB100 plasmid DNA. After 24 h, cells were harvested and selected in growth medium containing 100 µg/mL hygromycin B (Thermo Fisher) for 7–10 days to obtain stable cells.K-562 and 4T1 (GFP) stably transfected with pSBbi-GH, pSBbi-AGX124 with pSBbi-GH, pSBbi-AGX1WT, pSBbi-AGX1F383A, pSBbi-AGX1WT-NahK and pSBbi-AGX1F383A-NahK were fed with 100 µM (from a 100 mM stock solution in DMSO) membrane-permeable precursor Ac4GalN6yne, Ac4GalNAlk, Ac3GalN6yne-1-P(SATE)2 or DMSO. After 16 h, cells were harvested, centrifuged  and resuspended in PBS (1 mL). 0.9 mL cell suspension were transferred to O-ring tubes  and harvested. Zirconia/silica beads  were added at a volume comparable to the cell pellet volume, followed by 1:1 acetonitrile/water (1 mL). Cells were lysed using a bead beater  at 6 m/s for 30 s, and the cell lysate was cooled at 4 °C for 10 min. Samples were centrifuged , and the supernatant was transferred to a fresh tube. The solvent was evaporated by SpeedVac, and the residue was dissolved in Milli-Q water  containing 15 µM adenosyl diphosphate (ADP)-α-D-glucose . The solution was passed through a centrifuge filter  using a 3 kDa MWCO Amicon® Ultra-0.5 mL Centrifugal Filter Unit. The flow-through was evaporated by SpeedVac and the residue resuspended in 60 µL of MQ water. High Performance Anion Exchange Chromatography (HPAEC) was used to analyse lysates.K-562 cells  stably transfectedHPAEC was carried out using an ICS-6000 equipped with a quaternary pump and a conductivity detector  on an AS11 2 × 250 mm column and a 2 × 50 mm guard column (Thermo Fisher). Solvents were: A = water; B = 1 M NaOH. The gradient profile (0.25 mL/min flow rate) was as follows: 0 min 99.9% A, 0.1% B; 3 min 99.9% A, 0.1% B; 8 min 96.9% A, 3.1% B; 13 min 96.4% A, 3.6% B; 38 min 93% A, 7% B; 39 min 90% A, 10% B; 43 min 90% A, 10% B; 48 min 90% A, 10% B. Commercial or synthetic UDP-sugar standards (200–500 µM) were used as controls.WT, pSBbi-AGX1F383A, and pSBbi-AGX1F383A-NahK were fed with 10 µM (from a 50 mM stock solution in DMSO) membrane-permeable precursor Ac4GalN6yne or DMSO. After 16 h, cells were harvested, centrifuged  and resuspended in PBS (1 mL). 0.9 mL cell suspension were transferred to O-ring tubes  and harvested. Zirconia/silica beads  were added at a volume comparable to the cell pellet volume, followed by 1:1 acetonitrile/water (1 mL). Cells were lysed using a bead beater  at 6 m/s for 30 s, and the cell lysate was cooled at 4 °C for 10 min. Samples were centrifuged , and the supernatant was transferred to a fresh tube. The solvent was evaporated by SpeedVac, and the residue was dissolved in Milli-Q water  containing 15 µM ADP-α-D-glucose. The solution was passed through a centrifuge filter  using a 3 kDa MWCO Amicon® Ultra-0.5mL Centrifugal Filter Unit. The flow-through was evaporated by SpeedVac and the residue resuspended in 60 µL of MQ water. High Performance Ion-pair Reversed-phase Chromatography (IP RP HPLC) was used to analyse lysates.K-562 cells  stably transfected with pSBbi-GH, pSBbi-AGX12HPO4, 61.8 mM KH2PO4, 8mM (CH3)4N(HSO4); B = 80% buffer A, 20% Acetonitrile. The gradient profile (0.3 mL/min flow rate) was as follows: 0 min 100% A; 56 min 40% A; 64 min 100% A; 78 min 0% A; 103 min 0% A. Commercial or synthetic UDP-sugar standards (1.25–50 µM) were used as controls.IP RP HPLC was carried out using an Agilent 1260 Infinity HPLC equipped with a Diode Array Detector  on an Kinetex 5 µm EVO C18 100 Å 250 × 4.6 mm column . Solvents were: A = 38.1 mM K4GalN6yne or Ac4ManNAlk at the indicated concentrations or a corresponding volume of DMSO. Cells were grown for 20 h. Cells were harvested  in a V-shaped 96-well plate and washed twice with 2% (v/v) FBS in PBS . Cells were resuspended in Labelling Buffer (35 µL), treated with a solution of 200 µM CuSO4, 1.2 mM BTTAA , 5 mM sodium ascorbate, 5 mM aminoguanidinium chloride and 200 µM CF680-picolyl azide  in Labelling Buffer (35 µL), and incubated for 7 min at RT on an orbital shaker. The click reaction was quenched with 3 mM bathocuproinedisulfonic acid (BCS) in PBS (35 µL). Cells were centrifuged , washed twice with Labelling Buffer and then with PBS, and treated with 100 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) Sodium Dodecyl Sulfate (SDS), 1 mM MgCl2, and 100 mU/µL benzonase (Merck)) containing halt protease inhibitors (ThermoFisher). Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit (Thermo Fisher) was used to measure protein concentration. Loading buffer (a 1:1:1:0.5 (v/v/v/v) mixture of 1 M Tris-HCl pH 6.5, 80% (v/v) glycerol, 10% (w/v) SDS and 1 M dithiothreitol (DTT)) was added; samples were run on a 10% or 4–20% Criterion™ TGX™ Precast gel  for SDS-PAGE, and imaged on an Odyssey CLx imager . For protein quantification by densitometry, the signal in each lane was integrated in the 700 nm channel using Image Studio Pro software . Total protein was stained with Coomassie using Acquastain . Protein expression was assessed by Western blot, on a nitrocellulose membrane, with a different aliquot of the same set of samples, using antibodies against FLAG tag , HA tag , VSV-G tag  and GADPH .K-562 cells stably transfected with pSBbi-based plasmids were seeded at a density of 250,000 cells/mL into 6-well plates in growth medium without hygromycin. Cells were treated with Ac35 or 2 µL of a mixture of SialEXO and OpeRATOR . Samples were incubated for 2 h at 37 °C, briefly heated to 95 °C and cooled on ice. SDS-PAGE and in-gel fluorescence were performed as described above.For enzymatic treatment, lysates from cell surface-labelled cells (5 µg protein) were diluted to 20 µL with 50 mM Tris-HCl pH 7.5 and 150 mM NaCl. Samples were either left untreated, or treated with 2 µL of a 1:10 dilution in PBS of commercial PNGase F , 2 µL StcE (96.5 µg/mL solution in PBS)In-gel fluorescence and Western blot images were visualised and processed on Image Studio Lite software , cropped by Photoshop 2020 and assembled by Illustrator 2022 .4GalN6yne at the indicated concentrations or a corresponding volume of DMSO. Cells were grown for 20 h. Cells were harvested  in a V-shaped 96-well plate and washed twice with 2% (v/v) FBS in PBS . Cells were resuspended in Labelling Buffer (35 µL), treated with a solution of 200 µM CuSO4, 1.2 mM BTTAA, 5 mM sodium ascorbate, 5 mM aminoguanidinium chloride and 200 µM biotin-picolyl azide (Sigma Aldrich) in Labelling Buffer (35 µL), and incubated for 7 min at RT on an orbital shaker. The click reaction was quenched with 3 mM BCS in PBS (35 µL). Cells were centrifuged, washed three times with PBS, and treated with 100 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl2, and 100 mU/µL benzonase) containing halt protease inhibitors and 50 µM of PUGNAc (Sigma Aldrich). Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit was used to measure protein concentration. 40 µg of cell surface labelled proteins were divided in two aliquots of 20 µg each. 20 µg were directly loaded on the gel, while 20 µg were diluted to 17 µL with PBS and 3 µL of 10X CuAAC solution  were added. The click reaction was left 6 h at RT under shaking.K-562 cells stably transfected with pSBbi-based plasmids were seeded at a density of 250,000 cells/mL into 6-well plates in growth medium without hygromycin. Cells were treated with AcLoading buffer (a 1:1:1:0.5 (v/v/v/v) mixture of 1 M Tris-HCl pH 6.5, 80% (v/v) glycerol, 10% (w/v) SDS and 1 M DTT) was added; samples were incubated at 95 °C for 5 min and run on a 10% or 4-20% Criterion™ TGX™ Precast gel. After transferring proteins on a nitrocellulose membrane, the total protein amount was compared using the REVERT protein staining kit (LI-COR Biosciences) and biotinylation detected using IRDye 800CW Streptavidin (LI-COR) according to the manufacturer’s instructions.Western blot images were visualised and processed on Image Studio Lite software (LI-COR), cropped by Photoshop 2020 and assembled by Illustrator 2022.4GalN6yne at the indicated concentrations or a corresponding volume of DMSO. Cells were grown for 20 h. Cells were harvested  in a V-shaped 96-well plate and washed three times with PBS. Cells were treated with 100 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl2, and 100 mU/µL benzonase) containing halt protease inhibitors and 50 µM of PUGNAc. Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit was used to measure protein concentration.K-562 cells stably transfected with pSBbi-based plasmids were seeded at a density of 250,000 cells/mL into 6-well plates in growth medium without hygromycin. Cells were treated with Ac4, 6 mM BTTAA, 1 mM biotin-picolyl azide, 50 mM sodium ascorbate and 50 mM aminoguanidinium chloride) were added. The click reaction was left 6 h at RT under shaking.20 µg of proteins were diluted to 17 µL with PBS and 3 µL of 10X CuAAC solution (3 mM CuSOLoading buffer (a 1:1:1:0.5 (v/v/v/v) mixture of 1 M Tris-HCl pH 6.5, 80% (v/v) glycerol, 10% (w/v) SDS and 1 M DTT) was added; samples were incubated at 95 °C for 5 min and run on a 10% or 4–20% Criterion™ TGX™ Precast gel. After transferring proteins on nitrocellulose membrane, the total protein amount was assessed using the REVERT protein staining kit and biotinylation detected using IRDye 800CW Streptavidin (LI-COR) according to the manufacturer’s instructions.Western blot images were visualised and processed on Image Studio Lite software (LI-COR) cropped by Photoshop 2020 and assembled by Illustrator 2022.4GalN6yne at 10 µM or a corresponding volume of DMSO. Cells were grown for 20 h. Cells were harvested  in a V-shaped 96-well plate and washed three times with PBS. Cells were treated with 100 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl2, and 100 mU/µL benzonase) containing halt protease inhibitors and 50 µM of PUGNAc. Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit was used to measure protein concentration.K-562 cells stably transfected with pSBbi-based plasmids were seeded at a density of 250,000 cells/mL into 6-well plates in growth medium without hygromycin. Cells were treated with Ac20 µg of proteins were diluted to 17 µL with PBS and loading buffer (a 1:1:1:0.5 (v/v/v/v) mixture of 1 M Tris-HCl pH 6.5, 80% (v/v) glycerol, 10% (w/v) SDS and 1 M DTT) was added; samples were incubated at 95 °C for 5 min and run on a 10% or 4-20% Criterion™ TGX™ Precast gel. After transferring proteins on a nitrocellulose membrane, the total protein amount was assessed using the REVERT protein staining kit. The membrane was blocked 30 min at RT with Intercept (TBS) Blocking buffer (LI-COR).O-GlcNAc-containing glycoproteins were detected by overnight staining at 4 °C with mouse anti-RL2  in Intercept (TBS) Blocking buffer (LI-COR) followed by anti-mouse secondary antibody according to the manufacturer’s instructions.2 in 0.05% (v/v) Tween-20 (PBS) incubation buffer.Prior to blot staining with biotinylated lectin, endogenous biotinylated proteins were blocked by using the Endogenous Avidin/Biotin blocking kit  according to the manufacturer’s instructions. To assess the glycan distribution, 1 h incubation in 0.05% (v/v) Tween-20 (PBS) at RT with 20 µg/mL of the following biotinylated lectins was performed: Concanavalin A , Aleuria Aurantia Mushrooms , Maackis Amurensis II , Sambucus Nigra . ConA staining required 0.1 mM of CaClThe binding of biotinylated lectins was detected by using IRDye 800CW Streptavidin (LI-COR) according to the manufacturer’s instructions.Blot images were visualised and processed on Image Studio Lite software (LI-COR) cropped by Photoshop 2020 and assembled by Illustrator 2022.F383A-T2 constructs were seeded at a density of 250,000 cells/mL into 6-well plates in growth medium without hygromycin. Cells were treated with different concentration of sterile-filtered doxycycline or a corresponding volume of PBS. After 24 h, media was replaced with fresh doxycycline-containing media and incubated for further 24 h. Cells were harvested  in a 96-well plate and washed twice with PBS. Cells were treated with 100 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl2, and 100 mU/µL benzonase) containing halt protease inhibitors and 50 µM of PUGNAc. Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit was used to measure protein concentration.K-562 cells stably transfected with pSBtet-GH and pSBtet-AGX120 µg of proteins were diluted to 17 µL with PBS and loading buffer (a 1:1:1:0.5 (v/v/v/v) mixture of 1 M Tris-HCl pH 6.5, 80% (v/v) glycerol, 10% (w/v) SDS and 1 M DTT was added; samples were incubated at 95 °C for 5 min and run on a 4–20% Criterion™ TGX™ Precast gel. After transferring proteins on nitrocellulose membrane, the total protein amount was assessed using the REVERT protein staining kit and GalNAc-T2 expression levels were determined by 3 h incubation at RT with rabbit anti-VSV-G  followed by anti-rabbit secondary antibody according to the manufacturer’s instructions. MAL II binding was determined as described in the section above. Blot images were visualised and processed on Image Studio Lite software (LI-COR) cropped by Photoshop 2020 and assembled by Illustrator 2022.F383A-NahK, were either collected for RNA extraction (unfed) or fed with DMSO, 10 µM Ac4GalN6yne or 10 µM Ac4GalNAc. After overnight feeding (16 h), cells were collected for RNA extraction. Unfed and fed pellets were collected on different days. Biological replicates of each sample were collected on the same day.K-562 cells , transfected with pSBbi-GH or pSBbi-AGX1RNA was extracted from fed and unfed cells by using RNeasy Mini kit , DNase I (Thermo Fisher) and QIAshredder (Qiagen) according to the manufacturer’s instructions. mRNA capture and library preparation were performed by the Advanced Sequencing Facility at the Francis Crick Institute using the KAPA mRNA HyperPrep Kit . Technical triplicate libraries were sequenced on an Illumina HiSeq 4000 platform at the facility to generating on average 12 million 101 bp single-end reads per sample.48 before alignment. Reads were mapped and subsequent gene-level counted using RSEM 1.3.049 and STAR 2.5.250 against the human genome GRCh38 using annotation release 86, both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package (version 1.24.0)51 within the R programming environment (version 3.6.1)52. The following pairwise comparison were performed: pSBbi-AGX1F383A-NahK samples vs pSBbi-GH samples; pSBbi-AGX1F383A-NahK-DMSO-fed samples vs GH-pSBbi-DMSO-fed samples; pSBbi-AGX1F383A-NahK-GalNAc-fed samples vs pSBbi-GH- GalNAc-fed samples; pSBbi-AGX1F383A-NahK-GalN6yne-fed samples vs pSBbi-GH-GalN6yne-fed samples; and a likelihood ratio test analysis for each cell type across all feeding regimes with the contrast function, from which genes differentially expressed  between different conditions were determined. Gene lists were used to look for pathways, biological processes, cellular components and molecular functions enrichment using the Broad’s GSEA software (version 3.0) with genesets from MSigDB (version 7.1)53.Raw reads were quality and adapter trimmed using cutadapt (version 1.5)F383A-NahK  were individually grown in heavy and light media for 6 doublings, sufficient to achieve a labelling efficiency of >95%, before being fed with either DMSO or 10 μM Ac4GalN6yne. For each feeding sample, 5,000,000 cells in 30 mL of feeding media were used.K-562 cells stably transfected with pSBbi-GH and pSBbi-AGX112C/14N light lysine/arginine K0/R0 . Heavy media contained RPMI (Thermo Fisher) with 10% (v/v) dialysed FBS, proline (0.1 mg/mL) and 13C6, 15N2 heavy lysine/arginine K8/R10  that replaced natural lysine and arginine.Light media contained RPMI (Thermo Fisher) with 10% (v/v) dialysed FBS, proline (0.1 mg/mL. Thermo Fisher) and g, 5 min) and washed twice with PBS (200 µL). After being transferred into a V-shaped 96-well plate, cells were lysed with 200 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl2, and 100 mU/µL benzonase) containing 50 µM PUGNAc. Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit was used to measure protein concentration.After 20 h, cells were centrifuged  were added. The click reaction was left 6 h at RT under shaking. Samples were treated with 3 mL cold methanol  and left 24 h at −80 °C for protein precipitation.Heavy and light lysates were mixed 1:1 (0.25 mg each), normalised up to 250 µL with PBS and incubated for 2 h at RT with 300 µL of Neutravidin beads slurry , previously washed with PBS (2 × 200 µL), to remove endogenous biotinylated proteins. The supernatant was collected, diluted to 270 µL with PBS and 30 µL of 10X CuAAC solution  and supernatant discarded; pellets were washed twice with cold methanol (3 mL) and centrifuged between washes . Supernatant was completely removed  and samples were resuspended in 250 µL 0.1% (w/v) Rapigest (Waters) in PBS and sonicated in a water bath for 25 min. Samples were centrifuged , supernatants were transferred to new tubes and pellets were treated with 250 µL of 6 M urea in PBS. Samples were sonicated for 25 min and centrifuged again . The pellets were resuspended with 250 µL of PBS, sonicated for 25 min and centrifuged again. Rapigest, urea and PBS supernatants were then combined and incubated with 350 µL of dimethylated Neutravidin Beads slurry54 (previously washed twice with 200 µL of PBS) for 2 h at RT. Supernatant was discarded and beads were washed with 1% (w/v) Rapigest (3 × 350 µL), 6 M urea in PBS (6 × 350 µL), AmBic  and 40% (v/v) LCMS-grade acetonitrile (4 × 100 µL). Beads were resuspended in 100 µL of AmBic containing 10 mM DTT and then incubated at 50 °C for 15 min. Beads were washed with AmBic (2 × 350 µL) and 100 µL of 20 mM iodoacetamide in AmBic was then added. Samples were kept for 30 min in the dark. Iodoacetamide was then quenched by adding DTT 10 mM . The beads were washed with AmBic (3 × 350 µL), then resuspended in 100 µL of AmBic and 300 ng of Lys-C Mass Spec Grade (Promega) were added to beads followed by overnight incubation at 37 °C. The supernatant was transferred to a new tube and 200 ng of Trypsin gold Mass Spec Grade (Promega) were added. The digestion was left for 8 h at 37 °C. Peptides were desalted by UltraMicroSpin™  according to the manufacturer’s protocol and vacuum-dried by SpeedVac to remove any traces of organic solvents.Samples were then centrifuged  coupled to an UltiMate 3000 RSLCnano (Thermo Fisher). The samples (15 μL) were loaded via autosampler isocratically onto a 50 cm, 75 µm PepMap RSLC C18 column after pre-concentration onto a 2 cm, 75 µm Acclaim PepMap100 m nanoViper.Dried peptides were resuspended in 16 µL of 0.1% (v/v) formic acid in LCMS-grade water, sonicated for 15 min in a water bath, vortexed briefly and harvested 5 min at 18,000The column was held at 40 °C using a column heater in the EASY-Spray ionization source (Thermo Fisher). The samples were eluted at a constant flow rate of 0.275 μL/min using a 240 min gradient. Solvents were: A = 5% (v/v) DMSO, 95% (v/v) 0.1% formic acid in water; B = 5% (v/v) DMSO, 20% (v/v) 0.1% formic acid in water, 75% (v/v) 0.1% formic acid in acetonitrile.The gradient profile was as follow: 0 min 98% A, 2% B; 5 min 98% A, 2% B; 55 min 98% A, 2% B; 190 min 70% A, 30% B; 213 min 60% A, 40% B; 213 min 5% A, 85% B; 213 min 5% A, 85% B; 225 min 98% A, 2% B: 240 min 98% A, 2% B.m/z, 120 K resolution, 4 × 105 ion inject target, and 50 ms maximum inject time. Dynamic exclusion was set to exclude for 45 s with a repeat count of 1. Charge states 2–6 with an intensity >1 × 104 were selected for fragmentation at top speed for 3 s. Selected precursors were fragmented using HCD at 30% nCE with 1.2 Da isolation window, 1 × 104 inject target, and 100 ms maximum inject time. MS2 scans were taken in the ion trap at a rapid scan rate.MS1 scans were collected with a mass range from 350 to 1500 55 for quantification and identification by using Homo sapiens FASTA protein sequences database from UniProt  for the database search56. The protein groups table were uploaded into Perseus (version 1.6.14.0)57 to allow for data transformation and visualization, and into RStudio for statistical analysis.Raw MS files were loaded into MaxQuant software (version 1.6.5.0)52 was used for statistical analysis. Differentially enriched proteins were determined using empirical Bayes moderated t-test (two-sided) by limma package. Multiple-testing corrections were performed using the Benjamini-Hochberg procedure to calculate adjusted p values . Protein hits were filtered using a p value of 0.1.Search parameters included standard group-specific parameter type with multiplicity of 2 and maximum labelled of 3. Specific cleavage specificity of R and K, with two missed cleavages were allowed. Methionine oxidation and N-terminal acetylation were set as variable modifications with a total common max of 5. Cysteine carbamidomethylation was set as a fixed modification. Minimum peptide length was set at 7 amino acid units. Mass tolerance was set at 20 ppm for MS1s, 10 ppm for HCD MS2s. Peptide- and protein-level FDR were set at 0.01. R programming environment (version 4.1.3)F383A-NahK stably transfected 4T1 (GFP-expressing) and fibroblast MLg cells were seeded into a µ-Plate 24 Well Black  at a density of 30,000 cells in 350 μL growth medium (DMEM with 10% (v/v) FBS, penicillin (100 U/mL), streptomycin (100 µg/mL)) without hygromycin. Cells were treated with either DMSO, 50 μM Ac4GalN6yne or 50 μM Ac4ManNAlk.For monoculture samples, non-transfected and pSBbi-AGX1F383A-NahK stably transfected MLg cells were seeded into a µ-Plate 24 Well Black (Thistle Scientific Ltd) at a density of 30,000 cells in 350 μL growth medium without hygromycin. After 4 h, 3000 4T1 (GFP) cells were plated on the top of 30,000 MLg cells (1:10 ratio). The co-culture samples were grown 72 h before feeding with either DMSO, 50 μM Ac4GalN6yne or 50 μM Ac4ManNAlk.For co-culture samples, non-transfected and pSBbi-AGX14, 5 mM sodium ascorbate, 5 mM aminoguanidinium chloride and 200 μM biotin-picolyl azide. The reaction was carried out for 3 min at RT, the supernatant was aspirated and cells were washed with ice-cold PBS (4 × 200 μL). Cells were incubated for 20 min at RT with 20 μg/mL Streptavidin-AlexaFluor647  in 1% (w/v) BSA in PBS solution in the dark. After washing with ice-cold PBS (4 × 200 μL), cells were fixed 20 min with cold 4% (v/v) formaldehyde (Thermo Fisher) in 100 mM sodium phosphate buffer pH 7.4, at RT in the dark. The reaction was quenched by 5 min incubation with 50 mM ammonium chloride and the cells were washed with PBS (3 × 200 μL). Cells were permeabilised with 0.1% (v/v) Triton X-100 in PBS for 10 min at 4 °C and washed with PBS (3 × 200 μL). Cells were blocked in a solution of 10% (v/v) normal donkey serum , 1% (w/v) BSA and 0.1% (v/v) Tween-20 in PBS for 1 h at RT. GFP expression was detected by incubation with goat anti-GFP  in a solution containing 5% (v/v) donkey serum in 1% (w/v) BSA in PBS. Cells were washed with PBS (3 × 200 μL) and incubated for 30 min at RT with AlexaFluor488 anti-goat secondary antibody  in a solution containing 1% (v/v) normal donkey serum in 1% (w/v) BSA in PBS. Cells were then incubated with AlexaFluor568 Phalloidin  in 1% (w/v) BSA and 0.1% (v/v) Tween-20 followed by PBS washing (3 × 200 μL) and DAPI incubation  in 1% (w/v) BSA and 0.1% (v/v) Tween-20 in PBS for 30 min at RT. After washing with PBS (3 × 200 μL), circle CoverSlips with 15 mm diameter (Thermo Fisher) were mounted onto each well by using 20 μL of ProLong Gold Antifade Mountant (Invitrogen).Cells were incubated for 16 h. Medium was aspirated, and cells were washed with ice-cold 2% (v/v) FBS in PBS (2 × 200 μL). Cells were then treated with 200 μL of a freshly prepared CuAAC solution containing 300 μM BTTAA, 50 μM CuSOThe confocal acquisition was made on a Zeiss LSM710 Invert microscope. The images were acquired using a Plan Apochromat 40×/1.3 Oil objective. Monoculture samples were imaged with an acquisition zoom of 1.3, so the corresponding resulting pixel size was 0.159 μm. For the co-culture samples, three-dimensional images were acquired with an acquisition zoom of 0.6, so the corresponding resulting pixel size was 0.219 μm  and 0.7 μm (z).58.A sequential scan to spectrally separate the fluorescence of DAPI, AlexaFluor647, AlexaFluor488 and AlexaFluor568 was used. In addition, the transmitted light channel was activated to visualise the cell morphology. Images were visualised and processed with Fiji and Zen  softwareF383A-NahK-T2BH and pSBbi-Hyg plasmids were individually plated or co-cultured (1:1 ratio) overnight in DMEM with 10% (v/v) FBS, penicillin (100 U/mL), streptomycin (100 µg/mL) and hygromycin B (100 µg/mL). For each mono- and co-culture sample, two 15 cm petri dishes containing a total of 6,000,000 cells were prepared.Murine 4T1 (GFP-expressing) and human MCF7 cells stably transfected with pSBbi-AGX14GalN6yne. FBS-free media was used for feeding samples addressed to secretome glycoproteins labelling.After overnight growing, the media was discarded and replaced with fresh media containing either DMSO or 10 μM Acg, 5 min) to remove any cell debris. The secretome was then concentrated up to 300 µL and media replaced with PBS by using a 10 kDa MWCO Amicon® Ultra-15 Centrifugal Filter Unit. Pierce™ BCA Protein Assay kit was used to measure protein concentration. 300 µg of each sample was used for the next step.FBS-free media was collected and centrifuged  containing 50 µM PUGNAc. Cells were lysed for 20 min at 4 °C on an orbital shaker and centrifuged . Supernatant was transferred to a new plate and Pierce™ BCA Protein Assay kit was used to measure protein concentration.Media was discarded and cells were washed with PBS (5 mL). Cells were detached from the petri dish by 10 min incubation at RT with 8 mM EDTA in PBS (10 mL) and then washed twice with PBS. Cells were lysed with 200 µL of ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl4, 6 mM BTTAA, 1 mM DADPS-biotin-picolyl azide , 50 mM sodium ascorbate and 50 mM aminoguanidinium chloride) were added. The click reaction was left 6 h at RT under shaking. Samples were treated with 3 mL cold methanol  and left 24 h at −80 °C for protein precipitation.For each lysate, 300 µg of protein was brought to 250 µL with PBS and incubated for 2 h at RT with 300 µL of Neutravidin beads slurry, previously washed with PBS (2 × 200 µL), to remove endogenous biotinylated proteins. Both secretome and lysate samples were diluted to 270 µL with PBS and 30 µL of 10X CuAAC solution  formic acid in LCMS-grade water 30 min at RT on a rotator. The supernatant was collected and the acid-cleavage treatment was repeated a second time. Beads were washed with LCMS-grade acetonitrile. The wash and the acidic supernatants were combined together and 200 ng of Trypsin gold Mass Spec Grade were added. The digestion was left 8 h at 37 °C. Glycopeptides were dried by SpeedVac and then resuspended in AmBic containing 2% (v/v) LCMS-grade acetonitrile and 0.1% (v/v) formic acid.Peptides and glycopeptides were desalted by UltraMicroSpin™ according to the manufacturer’s protocol and vacuum-dried by SpeedVac to remove any traces of organic solvents.g.Dried peptides and glycopeptides were resuspended in 16 µL of 0.1% (v/v) formic acid in LCMS-grade water, sonicated for 15 min, vortexed briefly and centrifuged for 5 min at 18,000 Sample mixtures were analysed by nanoflow LC-MS/MS using an Orbitrap Eclipse with ETD (Thermo Fisher) coupled to an UltiMate 3000 RSLCnano (Thermo Fisher).The sample (15 μL for glycopeptide fractions and 3 injections of 5 μL each for peptide fractions) was loaded via autosampler isocratically onto a 50 cm, 75 µm PepMap RSLC C18 column (ES903) after pre-concentration onto a 2 cm, 75 µm Acclaim PepMap100 m nanoViper.The column was held at 40 °C using a column heater in the EASY-Spray ionization source (Thermo Fisher). The samples were eluted at a constant flow rate of 0.275 μL/min using a 120 and 140 min gradient for peptides and glycopeptides, respectively. Solvents were: A = 5% (v/v) DMSO, 95% (v/v) 0.1% formic acid in water; B = 5% (v/v) DMSO, 20% (v/v) 0.1% formic acid in water, 75% (v/v) 0.1% formic acid in acetonitrile.The gradient profile was as follows: 0 min 98% A, 2% B; 5 min 98% A, 2% B; 5.5 min 92% A, 8% B; 93 min 60% A, 40% B; 94 min 5% A, 95% B; 104 min 5% A, 95% B; 105 min 98% A, 2% B; 120 min 98% A, 2% B.The gradient profile was as follows: 0 min 98% A, 2% B; 6 min 98% A, 2% B; 114 min 60% A, 40% B; 115 min 95% A, 5% B; 119 min 5% A, 95% B; 120 min 98% A, 2% B: 140 min 98% A, 2% B.m/z, 120 K resolution, 4 × 105 ion inject target, and 50 ms maximum inject time. Dynamic exclusion was set to exclude for 20 s with a repeat count of 1. Charge states 2–6 with an intensity greater than 1 e4 were selected for fragmentation at top speed for 3 s. Selected precursors were fragmented using HCD at 30% nCE 1.2 Da isolation window, 1 × 104 inject target, and 100 ms maximum inject time. MS2 scans were taken in the ion trap at a rapid scan rate.MS1 scans were collected with a mass range from 350 to 1500 m/z, 120 K resolution, 4 × 105 ion inject target, and 50 ms maximum inject time. Dynamic exclusion was set to exclude for 10 s with a repeat count of 3. Charge states 2–6 with an intensity >1 × 104 were selected for fragmentation at top speed for 3 s. Selected precursors were fragmented using HCD at 28% nCE with 2 Da isolation window, 5 × 104 inject target, and 54 ms maximum inject time before collection at 30 K resolution in the Orbitrap. For precursors from 300 to 1000 m/z, presence of 3 oxonium ions over 5% relative abundance triggered a charge calibrated ETD scan to be collected in the ion trap with a 3 Da isolation window, 1 × 104 inject target, and 100 ms maximum injection time.MS1 scans were collected with a mass range from 300 to 1500 55 for quantification and identification by using Homo sapiens  and Mus musculus  FASTA protein sequences database from UniProt as a reference database56. For peptides, search parameters included specific cleavage specificity of R and K, with two missed cleavages allowed. Methionine oxidation and N-terminal acetylation were set as variable modifications with a total common max of 5. Carbamidomethyl cysteine was set as a fixed modification. Minimum peptide length was set at 7 amino acid units. Mass tolerance was set at 20 ppm for MS1s, 10 ppm for HCD MS2s. Peptide- and protein-level FDR were set at 0.01. The protein groups and peptides tables were uploaded into Perseus (version 1.6.14.0)57 to allow for data transformation, visualization and statistical analysis. Differentially enriched peptides were determined using Welch’s T-test (two-sided). Peptide hits were filtered using an eightfold enrichment and p value of 0.1.Raw MS files were loaded into MaxQuant software (version 1.6.5.0)m/z to account for the chemical modification. A maximum of two variable modifications were allowed per peptide. For each sample, variable modifications were searched against a focused FASTA file that exclusively contains protein sequences find in that sample. All identifications with |logP| >3 that contained chemically modified glycans were manually validated and localised using a combination of HCD and ETD information.Data evaluation was performed with Byonic™ . For glycopeptide analysis, search parameters included semi-specific cleavage specificity at the C-terminal site of R and K, with two missed cleavages allowed. Mass tolerance was set at 10 ppm for MS1s, 20 ppm for HCD MS2s, and 0.2 Da for ETD MS2s. Carbamidomethyl cysteine was set as a fixed modification. Variable modifications included methionine oxidation (common 1), asparagine deamidation (common 1), and a custom database of O-glycans that included HexNAc, HexNAc-NeuAc, HexNAc-Hex, HexNAc-Hex-NeuAc, HexNAc2-Hex-NeuAc and HexNAc-Hex-NeuAc2 with an additional 287.1371 59. Briefly, cell pellets  were subjected to sonication in the presence of CHAPS detergent (Thermo Fisher), reduced in 4 M guanidine-HCl (Pierce), carboxymethylated, and digested with Trypsin gold Mass Spec Grade. The digested glycoproteins were then purified by an Oasis HLB Sep-Pak (Waters). N-glycans were released by peptide N-glycosidase F  digestion, whereas O-glycans were released by reductive elimination. Released N- and O-glycans were permethylated using the sodium hydroxide procedure and purified by C18-Sep-Pak (Waters).For N -and O-glycan structural analysis, all cells were treated as described previouslyMS and MS/MS data were acquired using a 4800 MALDI-TOF/TOF (Applied Biosystems) mass spectrometer. Permethylated samples were dissolved in 10 μL of methanol, and 1 μL of dissolved sample was premixed with 1 μL of matrix  aqueous Acetonitrile), spotted onto a target plate, and dried under vacuum.60. The proposed assignments for the selected peaks were based on 12C isotopic composition together with knowledge of the biosynthetic pathways. The proposed structures were then confirmed by data obtained from MS/MS analysis experiments.The MS and MS/MS data were processed using Data Explorer 4.9 Software (Applied Biosystems). The processed spectra were subjected to manual assignment and annotation with the aid of a glycobioinformatics tool, GlycoWorkBenchNOD-SCID IL2Rgnull (NSG) strain mice (strain nomenclature NOD.Cg-PrkdcSCID Il2rgtm1Wjl/Sz) were obtained from the Jackson Laboratory and bred at the Francis Crick Institute Biological Resources Facility in individually vented cages under specific-pathogen-free conditions at light/dark cycle 7-7, 21 °C and at 50% humidity. When performing the mammary fat pad injection of cancer cells into the mice, the mice were anaesthetised with inhaled isoflurane.All animals in the experiments discussed here were performed under project license (P83B37B3C), approved by the UK Home Office, and in accordance with The Francis Crick Institute animal ethics committee guidelines. Tumour burden limit for the given experiments is 12 mm × 12 mm. Such maximal tumour size was not exceeded.F383A-NahK-T2BH were resuspended in 50 μL of growth-factor-reduced Matrigel (BD Biosciences) and injected into the left hind fat pad of 9 female NSG mice (6–8 weeks old), with GFP-expressing 4T1 cells transfected with empty pSBbi-Hyg injected into the right hind fat pad of the mouse as an internal control . Body weight and tumour volume were monitored every other day before compound treatment and did not vary markedly between groups. Mice were randomly assigned to treatment groups based on tumour size.GFP-expressing 4T1 cells transfected with pSBbi-AGX13 were randomly assigned to receive intratumoral injection with 50 μL of vehicle (5% (v/v) DMSO/PEG-400; n = 1) or Ac4GalN6yne (6 mg/mL in 5% (v/v) DMSO/PEG-400; n = 2) once daily for 3 consecutive days. On the fourth day the tumours were harvested, flash-frozen and homogenised for Western blot analysis. In the case of the BOCTAG-T2 tumours, an additional piece was prepared for tumour digestion, protein expression analysis and metabolic cell surface labelling (see below for procedures).11 days post injection, 3 mice harbouring tumours of roughly 200–300 mmn = 1), Ac4GalN6yne (40 mg/mL in 5% (v/v) DMSO/PEG-400; n = 3) or Ac4ManNAlk (40 mg/mL in 5% (v/v) DMSO/PEG-400; n = 2) once daily for 5 consecutive days. On the sixth day, the tumours were harvested, flash-frozen and homogenised for Western blot analysis. In the case of the BOCTAG-T2 tumours, an additional piece was prepared for tumour digestion, protein expression analysis and metabolic cell surface labelling (see below for procedures).13 days post tumour cell injection, 6 mice were randomly divided into three groups and intraperitoneally injected with 100 μL of vehicle (5% (v/v) DMSO/PEG-400; 2, and 100 mU/µL benzonase) containing 50 µM of PUGNAc using a Precellys© homogeniser . The samples were allowed to stand for 10 min and subsequently centrifuged  to remove cell debris. The supernatant was collected and the protein concentration determined by BCA assay.Tumour pieces were homogenised in ice-cold Lysis Buffer  Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgClTM BCA Protein assay kit.To remove endogenous biotinylated proteins, the samples were diluted up to 250 µL with PBS and incubated 2h at RT with 300 µL of Neutravidin-Coated Magnetic Beads slurry previously washed with PBS (3 × 200 μL). The supernatant was subsequently collected and the protein concentration determined by Pierce4, 5 mM sodium ascorbate, 5 mM aminoguanidinium chloride and 100 μM biotin-picolyl azide and incubated overnight at RT on an orbital shaker.The collected supernatant (30 μg protein) was then treated with a freshly prepared CuAAC solution containing 600 μM BTTAA, 300 μM CuSOLoading buffer was subsequently added to the samples and these were run on a 4–20% Criterion™ TGX™ Precast gel for SDS-PAGE. After transferring proteins on a nitrocellulose membrane, the total protein amount was assessed using the REVERT protein staining kit and biotinylation detected using IRDye 800CW Streptavidin according to the manufacturer’s instructions.2 or MgCl2) for 2 h at 37 °C. The cell suspension was then filtered using a 100 μm cell strainer and the reaction quenched by adding an equal volume of growth medium (DMEM with 10% (v/v) FBS, penicillin (100 U/mL), streptomycin (100 µg/mL)) to the filtered cell suspension. The samples were subsequently centrifuged  and the pellet resuspended in 10 mL growth medium. The cell suspension was then plated on a 10-cm dish and incubated overnight at 37 °C. After 24 h, the cells were washed once with PBS and treated with fresh growth medium. The cells were subsequently propagated in fresh growth medium with 50 μg/mL hygromycin B at 37 °C with 5% CO2 for 10 days.The BOCTAG-T2 tumours were manually minced with a scalpel and scissors until they became a smooth paste with no visible clumps and then incubated with 1.5 mL of a freshly prepared digestion solution  and incubated with 8 mM EDTA in PBS (1.5 mL) for 5 min. The cells were transferred to a 1.5 mL centrifuge tube, harvested  and resuspended in ice-cold Labelling Buffer (0.2 mL). The metabolic cell surface labelling and in-gel fluorescence analysis was subsequently performed as explained above.For protein expression analysis and metabolic cell surface labelling, the cells were seeded at a density of 500,000 cells/mL in 1.6 mL growth medium without hygromycin into 6-well plates and allowed to attach overnight. The cells were then treated with 50 μM AcProtein expression was assessed by Western blot using antibodies against FLAG tag , HA tag , VSV-G tag  and GADPH .Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary data 1Supplementary data 2Supplementary data 3Supplementary data 4Reporting Summary


Micropterus salmoides) (MsFcγRIα). Without a transmembrane segment or a cytoplasmic tail, MsFcγRIα was identified as a soluble form protein and widely distributed in the spleen, head kidney, and intestine. The native MsFcγRIα was detected in the serum of Nocardia seriolae-infected largemouth bass and the supernatants of transfected HEK293 cells. Additionally, it was verified that the transfected cells’ surface secreted MsFcRIα could bind to largemouth bass IgM. Moreover, the expression changes of MsFcγRIα, Syk, and Lyn indicated that MsFcγRIα was engaged in the acute phase response to bacteria, and the FcγR-mediated phagocytosis pathway was activated by Nocardia seriolae stimulation. Furthermore, recombinant MsFcγRIα could enhance both reactive oxygen species (ROS) and phagocytosis to Nocardia seriolae of leukocytes, presumably through the interaction of MsFcγRIα with a complement receptor. In conclusion, these findings provided a better understanding of the function of soluble FcγRs in the immune response and further shed light on the mechanism of phagocytosis in teleosts.Fc receptors (FcRs) are key players in antibody-dependent cellular phagocytosis (ADCP) with their specific recognition of the Fc portion of an immunoglobulin. Despite reports of FcγR-mediated phagocytosis in mammals, little is known about the effects of soluble FcγRs on the immune response. In this study, FcγRIα was cloned from the largemouth bass ( Fc receptors (FcRs), specific for the constant fragment crystallizable (Fc) region of immunoglobulin (Ig), are widely expressed in leukocytes, such as monocytes, macrophages, neutrophils, NK cells, and B lymphocytes . In geneCtenopharyngodon idellus) [Paralichthys olivaceus) [Oreochromis niloticus) [Labrus bergylta) [Micropterus salmoides) [Plecoglossus altivelis) [Ictalurus punctatus) [FcRs for IgG (FcγR), IgE (FcεR), IgA (FcαR) and IgM  have been found in mammals ,11. Diffidellus) , floundeivaceus) , Nile tiloticus) , ballan ergylta)  and larglmoides)  have beetivelis)  and FcRInctatus) . The in-Soluble forms of FcRs (sFcRs) are generated either by proteolytic cleavage of the membrane FcRs or alternative splicing of transmembrane (TM)-encoding exon . DistingMicropterus salmoides) is an economically freshwater fish with commercial value [Nocardia seriolae (N. seriolae) [The largemouth bass  ,25,26. Ieriolae) , and theFcγRIα (MsFcγRIα) gene was cloned, and then the sequence and structural characteristics were analyzed. The recombinant MsFcγRIα ((r)MsFcγRIα) protein was expressed in Escherichia coli and HEK293 cells. Moreover, a polyclonal antibody against MsFcγRIα was prepared to identify the localization in HEK293 cells, the native expression in N. seriolae-infected largemouth bass serum, and the distribution in largemouth bass tissues. The mRNA relative expression of MsFcγRIα and its downstream genes (Syk and Lyn) were investigated in vitro and in vivo upon N. seriolae and lipopolysaccharide (LPS) infection. In addition, the binding of transfected-MsFcγRIα to purified IgM was examined. Concurrently, the regulation of (r)MsFcγRIα on respiratory burst, phagocytosis, and transcriptional level of phagocytosis-related and complement system genes were detected. This research will broaden our understanding of the sFcRs functions in teleost.In this study, the largemouth bass MsFcγRIα consisting of 1272 bp (GenBank accession No. OK258092) was successfully cloned from the head kidney cDNA of largemouth bass (57SS) was observed in MsFcγRIα. The secondary structure of MsFcγRIα was composed of a signal peptide (resides: aa 1–19) at N-terminate and two Ig domains: Ig domain 1 (D1) consisting of 83 aa (aa 35–117) and Ig domain 2 (D2) consisting 84 aa (aa 125–208) (50-Cys93) (β-strand were found in MsFcγRIα D1 and D2 (The ORF of uth bass . It encouth bass . The the125–208) A. D1 con0-Cys93) B and was1 and D2 B, and th1 and D2 C.Lates calcarifer) FcRIL (54.58% identity) and highly identical to large yellow croaker (Pseudosciaena croce) FcRLα (49.60% identity). Phylogenetic tree analysis showed that MsFcγRIα clustered independently into one branch in fish  was localized on the cell membrane and cytoplasm B. All ofIHC was performed to detect the expression of secretory MsFcγRIα protein in the immune tissues of largemouth bass. The PBS solution was employed in the negative control to replace anti-MsFcγRIα PcAb. IHC results  showed tThe binding of expressed MsFcγRIα on transfected HEK293 cells to purified MsIgM was confirmed by flow cytometric, indirect immunofluorescence, and co-immunoprecipitation (CO-IP) assay. Results of flow cytometric assay indicated that after 0 h, 6 h, 12 h, and 24 h incubation with MsIgM, the MsFcγRIα-expressed HEK293 cells combined with 3.54 ± 0.22%, 19.70 ± 0.90%, 26.33 ± 0.61% and 40.27 ± 0.24% MsIgM, respectively B demonstTo exclude the effect of EGFP protein on the binding of MsIgM to MsFcγRIα, recombinant MsFcγRIα-pcDNA3.1(+) was constructed and transfected into HEK293 cells. Following incubation with MsIgM, the interactional protein in cell lysates was precipitated by anti-MsIgM mAb (MM06H). Western blotting was detected by MM06H and anti-MsFcγRIα PcAb, respectively. Results C showed The results indicated that the purified MsIgM could interact with the surface-expressed MsFcγRIα on transfected HEK293 cells.MsFcγRIα in healthy largemouth bass tissues was explored by qRT-PCR. The results  and N. seriolae (1 × 108 CFU/mL) were used to challenge the largemouth bass. And the identification of N. seriola was shown in MsFcγRIα increased gradually and reached the peak value at 72 h p.i. (p < 0.01); Syk and Lyn expression levels reached the maximum at 48 h p.i. (p < 0.01), which was earlier than MsFcγRIα. In the head kidney, the highest mRNA expression of Syk was noticed at 6 h p.i. (p < 0.01) with N. seriolae stimulation; the mRNA expression of MsFcγRIα and Lyn reached the highest expression level at 24 h p.i. (p < 0.01) with N. seriolae or LPS stimulation. The peak value of gene expression with LPS stimulation was higher than with N. seriolae stimulation in the spleen, while the opposite is true in the head kidney MsFcγRIα-treated leukocytes was 33.87 ± 1.577%, which was increased significantly (p < 0.01) compared to the control group with 15.43 ± 0.12% (p < 0.01) than the pET-32a (37.27 ± 0.480%) or PBS-treated group (37.13 ± 0.318%) (To explore the effect I(r)MsFcγRIα on reactive oxygen species (ROS) and phagocytosis to  ± 0.12% A. Meanwh 0.318%) B. It sugSyk (p < 0.01), MsFcγRIα (p < 0.01), ATG3 (p < 0.01), MARCKS (p < 0.01), Lyn (p < 0.05) as well as genes engaged in the complement system, including C1R (p < 0.01), C3 (p < 0.01), C5 (p < 0.01) and C7 (p < 0.01) were significantly up-regulated.To further investigate the effect of MsFcγRIα on mRNA transcription of leukocytes, the relative expression levels of immune-related genes in leukocyteItimulated with (r)MsFcγRIα (5 μg/mL) were determined by qRT-PCR. The results  indicateFcRs carried out functions of interaction with immune complexes and hematopoietic cells have been extensively studied in mammalian leukocytes . HoweverN. seriolae. Moreover, the results of IHC showed that native MsFcγRIα was remarkably distributed among the extracellular matrix in the largemouth bass spleen, head kidney, and intestine. A line of results collectively pointed to MsFcγRIα as a soluble protein that could be secreted through its signal peptide. Both membrane and soluble FcγRIα (CD64) have been found in humans [Early studies have unveiled that most mammalian FcRs are membrane-bound proteins with TM domain or glycophosphatidyl inositol (GPI) ,32,33. Wn humans . HoweverIn previous studies, soluble FcRs have participated in mammalian B cell proliferation and antibody production . Their rMsFcγRIα is expressed predominantly in largemouth bass lymphoid tissues, including the spleen, head, kidney, and intestine. It is also detected in nonhemopoietic tissues such as the brain, liver, gill, and skin with relatively low expression levels, which was coincident with the expression of IpFcRI in channel catfish [MsFcγRIα, Syk, and Lyn) after N. seriolae infection were higher than in LPS infection within 72 h. Still, the tendency in the spleen showed otherwise, suggesting that the head kidney may be more sensitive to N. seriolae infections than the spleen in the acute phase response. Therefore, in the current study, as in many previous studies [N. seriolae infections, the expression level of Syk and Lyn began to increase at 24 h p.i. However, the beginning time of MsFcγRIα was 48 h p.i, later than Syk and Lyn. The gradual increase of MsFcγRIα may attribute to the activation of the FcγR-mediated phagocytosis pathway after crosslinking of ITAM in MsFcγRIα [Syk in this study declined at 48–72 h p.i. Furthermore, the expression level of Syk was distinctly higher than Lyn and MsFcγRIα, indicating that Syk and Lyn have different functions in the transduction of phagocytic signal generated by MsFcγRIα [Syk is necessary for phagocytosis of leukocytes [ catfish . The spl catfish ,44 and p catfish . Interes studies ,47,48, lMsFcγRIα ,50. Onceukocytes .Syk, Lyn, MsFcγRIα, ATG3, and MARCKS was up-regulated remarkably, indicating that soluble MsFcγRIα could facilitate the endocytosis of leukocytes. This up-regulation is the opposite of the recognized inhibitory effect of soluble FcRs through competing with membrane FcRs for Ig binding [C1R, C3, C5, and C7) with (r)MsFcγRIα stimulation in this study. However, the scarcity of information on the complement system in largemouth bass has seriously hindered the study of the phagocytosis mechanism in teleosts.It has been elaborately investigated that the endocytosis of FcRs is dependent on clathrin and dynamin. Conversely, the cytoplasmic domain of FcRs in phagocytosis is unessential . Therefo binding ,34. Howe binding . On the  binding . Thus, wMsFcγRIα successfully cloned in this study was structurally conserved with FcRs homologs in fish and mammals and highly expressed in lymphoid tissues of largemouth bass. Soluble form MsFcγRIα was detected in N. seriolae-infected largemouth bass serum and the supernatants of transfected HEK293 cells, as well as the extracellular matrix of the intestine, spleen, and head kidney. MsFcγRIα could bind to IgM and play a crucial role in the early stage of immune response to bacteria. In addition, MsFcγRIα promoted the ROS activation and phagocytosis of leukocytes which may be related to the interaction of MsFcγRI with the complement receptor. This study lays a foundation for investigating soluble FcRs in ADCP and will be conducive to exploring the phagocytosis mechanism of leukocytes in teleost.In summary, N. seriolae isolated from largemouth bass was identified by Gram’s stain, PCR, and DNA sequencing, cultured in Brain Heart Infusion (BHI) broth for four days at 25 °C, and enumerated before experiments. The healthy largemouth bass (15 ± 5 g) were purchased from a fish farm at Foshan, Guangdong province, China, and fed twice daily with commercial feed. The fish were randomly divided into a density of 45 fish per tank and acclimated in an automatic filtering aquaculture system at 26 ± 1 °C for 21 days. Prior to any animal experiment, the fish were anesthetized with 20 mg / L of eugenol. HEK 293 cells were collected from the laboratory of Dr. Li [f Dr. Li  and cultMsFcγRIα assembled from a largemouth bass transcriptome data (NCBI Accession No.: PRJNA806622) was cloned. Primers, MsFcγRIα-OF and MsFcγRIα-OR (Escherichia coli (E. coli) DH5α. Finally, three positive clones were sequenced by TSINGKE Biotechnology company .The open reading frame (ORF) of cγRIα-OR  were deshttp://www.snapgene.com/ (accessed on 13 March 2019)). Multiple alignments of amino acid sequences were performed with DNAMAN 8.0 software . The similarity of the amino acid sequences was carried out by NCBI protein BLAST programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 14 March 2019)). Phylogenetic trees were constructed by MEGA 6.0 software using the neighbor-joining (NJ) method with 1000 bootstrap replicates. The domain architecture was predicted by SMART program (http://smart.embl-heidelberg.de/ (accessed on 20 April 2019)). N-glycosylation sites were predicted by the NetNGlyc 1.0 server ), and the signal peptide was analyzed by SignaIP ). Conserved cysteine residues were predicted by ExPASy-PROSITE (https://prosite.expasy.org/ (accessed on 22 January 2021)). Protein tertiary structure was predicted online via the ExPASy website (https://swissmodel.expasy.org/ (accessed on 25 January 2021)). Transmembrane domain prediction was conducted using TMHMM-2.0 ). GPI-anchorage site was predicted by the big-PI Predictor (https://mendel.imp.ac.at/gpi/gpi_server.html (accessed on 28 January 2021)).Bioinformatics analysis of MsFcγRIα was performed as the following methods. DNA sequences were analyzed by SnapGene Viewer  expression levels of largemouth bass intraperitoneally injected with 100 μL LPS (50 ng/μL) , 100 μL N. seriolae (1 × 108 CFU/mL), and 100 μL PBS (as the control group), respectively, were determined by quantitative real-time PCR (qRT-PCR). The spleen and head kidney tissues were isolated from three individual fish at 0, 3, 6, 12, 24, 48, and 72 h post-injection (h p i), then snap-frozen in liquid nitrogen and stored at −80 °C before RNA extraction.To study the mRNA expression of ® Total RNA Kit II , and cDNA was synthesized from 1 μg of total RNA with the HiScript®IIQ RT Supermix Kit . The 1:10 of diluted cDNA as templates for the gene expression analysis by qRT-PCR according to the manufacturer’s instructions . Primers designed using Primer Primier 5.0 are listed in β-actin using the 2−ΔΔCt method [Total RNA was extracted using E.Z.N.A.t method .MsFcγRIα, Lyn, and Syk under antigen stimulation, the leukocytes were isolated from the head kidney of largemouth bass by leukocytes isolation kit  as described in the manufacturer’s introduction. Briefly, the cell suspension from tissues was overlaid gently on the Ficoll for leukocytes, then centrifuged at 450× g for 25 min at 20 °C. Leukocytes were harvested from the intermediate layer and washed three times in RPMI-1640 . Prior to resuspending the leukocytes in RPMI-1640 with 10% fetal bovine serum  and 1% penicillin/streptomycin , the activity of cells was defined using 0.4% trypan blue . Then 1 mL of the cell suspension (1 × 107 cells/mL) incubated with LPS (50 μg/mL), formalin-inactivated N. seriolae (1 × 108 CFU/mL) and PBS (100 μL/mL) at 25 °C, respectively. Leukocytes were collected at 0, 3, 6, 12, 24, 36, 48, and 72 h post-injection (h p i) and centrifuged at 500× g for 10 min at 4 °C, then snap-frozen in liquid nitrogen and stored at −80 °C before RNA extraction.To detect the expression rules of E. coli TSsetta (DE3) , induced by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) with O.D. 600 of 0.6 and cultured at 26 °C for 12 h. The protein purification was performed according to the protocol of His-tag Protein Purification Kit  and dialyzed at 4 °C in urea with gradient concentrations, as well as PBS. Eventually, the purified recombinant MsFcγRIα ((r)MsFcγRIα) was concentrated by PEG2000  and identified by SDS-PAGE gel electrophoresis.The CDS sequence encoding mature protein but excluding the signal peptide was amplified by expression primers (MsFcγRIα-32a-F and MsFcγRIα-32a-R) . The ampg, 4 °C for 10 min, then purified by Melon Gel Monoclonal IgG Purification Kit . Prior to being equally stored at −80 °C, the specificity was characterized by Western blotting.To prepare polyclonal antibody (PcAb) against protein MsFcγRIα (anti-MsFcγRIα), healthy six-week-old female BALB/C mice were immunized with the purified recombination protein. The mice were intraperitoneally injected with 50 μg/100 μL MsFcγRIα per mouse each time at two-week intervals for four times. Except for the first-time immunization, the MsFcγRIα was emulsified with complete Freund’s adjuvant , and each subsequent immunization was carried out with incomplete Freund’s adjuvant  instead. Four days after the final immunization, blood was obtained from the retro-orbital of the mice following the isoflurane anesthesia, then collected the serum by centrifugation at 5000× 2O2 and 85% methanol for 15 min. Following the blocking with 1% BSA for 1 h, the sections were overnight incubated with anti-MsFcγRIα PcAb at a dilution of 1:500 at 4 °C, and PBS was used as a negative control. After washing with PBS, slides were incubated with the horseradish peroxidase-conjugated (HRP) goat anti-mouse immunoglobulin G (IgG) secondary antibody  at a dilution of 1:2000 for 1 h at 37 °C. DAB detected the signal expression of MsFcγRIα under the microscope , then slides were counterstained in 50% Hematoxylin and washed with 0.02% ammonia water. After dehydration by 70% ethanol and 95% ethanol, slides were incubated in xylene for 5 min and sealed with neutral gum. PBS solution instead of anti-MsFcγRIα PcAb was used as a negative control. Images were obtained with a Leica DM2500 microscope .Immunohistochemistry (IHC) was performed as previously described with some modifications . Briefly5 cells/well) by Lipofectamine 3000 . After 24 h, transfected cells were stained with DiI by Cell Plasma Membrane Staining Kit (Red Fluorescence)  at 37 °C for 20 min and 10 μg/mL DAPI  for 10 min. Cells were observed under a laser scanning confocal microscope .To locate the expression of MsFcγRIα, MsFcγRIα containing signal peptide was amplified using the primers, MsFcγRIα-EGFP-F and MsFcγRIα-EGFP-R . PCR prov/v) and Alexa Fluor® 647 goat anti-mouse IgG  (1:2000 v/v) with flow cytometry , and laser scanning confocal microscope . The supernatants and cell lysates were collected independently to determine secretory MsFcγRIα by Western blotting.Furthermore, to test whether MsFcγRIα could generate a secretory protein, the recombinant eukaryotic plasmid (MsFcγRIα-pcDNA3.1(+)) was constructed. Primers (MsFcγRIα-3.1-F and MsFcγRIα-3.1-R) used to amplify the full length of MsFcγRIα CDS were listed in ® Protein A column  according to the previous procedure [6 cells/mL) were harvested and washed with PBS prior to incubation with anti-MsIgM mAb of largemouth bass (MM06H) (1:1000 v/v)  for 1 h at 37 °C. After washing with PBS, the cells were labeled with Alexa Fluor® 647 goat anti-mouse IgG  for 1 h at 37 °C. Finally, cells were resuspended in PBS following the three times washing and analyzed by flow cytometry . Meanwhile, 500 μL of transfected cells settled onto a 15 mm glass confocal dish  were treated with 20 μg/mL MsIgM and labeled with Alexa Fluor® 647 goat anti-mouse IgG  as mentioned above. After staining with 10 μg/mL DAPI , fluorescence images were acquired using a laser scanning confocal microscope .To establish whether the surface-expressed MsFcγRIα could bind to largemouth bass IgM (MsIgM), the IgM was purified from largemouth bass serum by HiTraprocedure . The introcedure ,42. Plas6 cells/well) were transfected with 2.5 μg MsFcγRIα-pcDNA3.1(+) per well and treated with 20 μg/mL MsIgM for 24 h as described above. Following careful washing with pre-cooling PBS, the cells were lysed with 200 μL immunoprecipitation lysis buffer  with 1 mm PMSF  on ice. Cellular debris was removed by centrifugation at 12,000× g for 5 min at 4 °C. The cell supernatants were incubated overnight with MM06H mAb (1:1000) at 4 °C. Non-immune mouse serum instead of MM06H mAb was performed as a negative control. 40 μL protein A+G agarose  was added into the mixture for incubation with slow rotation at 4 °C for 3 h. After five times washing with PBS by centrifugation at 1000× g for 5 min at 4 °C, the samples were boiled with 40 μL 1× SDS loading buffer for 5 min and used for immunoblot analysis with MM06H mAb and anti-MsFcγRIα PcAb.Co-immunoprecipitation experiment was performed to confirm the interaction between the native MsFcγRIα with MsIgM according to the previous study with some modifications . Brieflyw/v) skim milk in TBST  for 1 h at 37 °C, then incubated with MM06H mAb (1:1000) or anti-MsFcγRIα PcAb (1:500 v/v) for overnight at 4 °C in TBST containing 0.5% (w/v) skim milk. Then, the membranes were washed with TBST three times and incubated with HRP-conjugated goat anti-mouse IgG (1:5000)  in TBST containing 0.5% (w/v) skim milk for 1 h at 37 °C. After washing three times with TBST, the membranes were stained with ECL detection reagents . Staining was stopped with distilled water, and blots were analyzed with a gel imager .For Western blotting analysis, protein samples were separated using 12% SDS-PAGE gels and transferred onto a 0.22 μm pore size polyvinylidene difluoride (PVDF) membranes  by 160 mA for 90 min. The PVDF membranes were blocked in 5%  and then stimulated with 5 μg/mL recombinant MsFcγRIα-PET32a ((r)MsFcγRIα) or equal PET32a protein or PBS in 24-well plates containing 1 mL culture medium per well at 25 °C for 3 h. Following three times washing with PBS, the cells were harvested and stained with 10 μM DCFH-DA at 37 °C for 20 min in the dark. The fluorescence intensity was determined by flow cytometry . Each experiment was conducted in triplicates for each independent sample.According to the manufacturer’s instructions, reactive oxygen species (ROS) were detected by a ROS assay kit . Briefly, the leukocytes isolated from largemouth bass were resuspended and adjusted to 1 × 106 cells/well with RPMI1640 culture medium  and then incubated with 5 μg/mL (r)MsFcγRIα or equal PET32a or PBS at 25 °C for 3 h. The N. seriolae was inactivated by 0.2% formaldehyde at 4 °C overnight, then labeled with 0.2 mg/mL FITC  at 4 °C for 12 h. The FITC-labeled N. seriolae was added to cells at a cell-to-bacteria ratio of 1:20 for incubation at 20 °C or without bacteria treatment as a negative control. After 6 h, the non-ingested bacteria were removed by ficoll kit, and cells were harvested by centrifugation at 450× g for 20 min at 20 °C. Following three washing steps with PBS, cells were resuspended in PBS. Flow cytometry  analyzed the phagocytic ability of leukocytes. The relative gene expression of leukocytes after (r)MsFcγRIα stimulation was determined by qRT-PCR as described in Phagocytosis assay was performed as described in the previous study with some modifications ,60. Briep > 0.05, * significant at p < 0.05, and ** significant at p < 0.01. The results of gene relative expression were presented as mean ± standard deviation (SD), and the percentage of cells was presented as mean ± SEM.Statistical analysis was conducted by Statistical Product and Service Solution (SPSS) software , GraphPad Prism software , and Flowjo V10 software . The statistical significance was analyzed using one-way ANOVA and considered no significant at


Scientific Reports 10.1038/s41598-022-13618-y, published online 09 June 2022Correction to: The original version of this Article contained typographical errors.Parts of Figure 1, 2, 3 and Figure 5 did not display correctly.The original Figure Additionally, equations 1, 18 and 19 contained an error, where “-β2” should read “β2”.The original Article has been corrected.


Page 1: This article was published on 6 August 2020 with Timileyin Adediran’s surname misspelled as “Adedrian” in the byline.Volume 9, no. 32, e00691-20, 2020,


Furthermore, in patients with T2DM, hypomagnesemia is associated with worst glycaemic control. Bariatric surgery (BS) remains the most effective treatment in severe obesity and also provides resolution/improvement of T2DM. Our aim is to evaluate the association between Mg supplementation post-BS and Mg serum levels with diabetes status after BS.Low Magnesium (Mg) dietary intake has been associated with increased risk of type 2 diabetes We performed an observational study on patients with obesity and T2DM who underwent BS. Data was assessed pre-BS and one-year post-BS.vs 6.4 ± 1.0% (p < 0.001), fasting plasma glucose 146.2 ± 58.8 mg/dL vs 117.5 ± 36.6 mg/dL (p < 0.001) and were under a greater number of anti-diabetic drugs 1.0 (IQR 0–2.0) vs 1.0 (IQR 0–1.0) (p = 0.002). These findings persisted at one-year post-BS. At the first-year post-BS, 58.4% of the patients had total remission of T2DM and 4.1% had partial remission. Patients without Mg deficiency at one-year post-BS had higher rates of total and partial remission. Higher serum Mg levels at baseline is an independent predictor of total T2DM remission (p < 0.0001). The optimal cut-off of baseline Mg to predict total T2DM remission was 1.50 mg/dL with a sensitivity of 73% and a specificity of 58% (area under ROC = 0.65). Patients that were under Mg supplementation post-BS had serum Mg values, glycaemic control and total remission of T2DM similar to patients non-supplemented.We included a total of 403 patients with T2DM. At baseline, 43.4% of the patients had Mg deficiency. Pre-BS, patients with Mg deficiency had poorer glycaemic control – HbA1c 7.2 ± 1.6% In patients with T2DM submitted to BS, higher Mg serum levels at baseline and 1-year after BS were associated with better glycaemic control and higher rates of total T2DM remission at the first year post-BS.The online version contains supplementary material available at 10.1186/s12902-022-01210-4. Magnesium (Mg) acts as a cofactor of enzymes involved in glucose metabolism, protein production and nucleic acid synthesis , 2. Mg bMg deficiency is relatively common in general population , 6 and tDietary Mg is mostly absorbed by the duodenum and jejunum via passive paracellular transport. The intestinal absorption is not directly proportional to Mg intake, but depends on body’s Mg status . Serum omellitus (T2DM)  vs 1.0 [IQR 0–1.0] (p = 0.002). Although patients with hypomagnesemia had higher insulin resistance (HOMA-IR 9.0 ± 9.2 vs 7.6 ± 6.3) this had not statistical significance (p = 0.92).In Table n = 392) vs 1.53 ± 0.17 mEq/L (n = 403). 11 patients were withdrawn due to missing Mg serum values, HbA1c or FPG. Regarding glycaemic profile, we found results overlapping the baseline findings. Patients with Mg deficiency had higher HbA1c 5.8 ± 0.7% vs 5.5 ± 0.6% (p = 0.01), higher FPG 96.8 ± 25.4 mg/dL vs 89.8 ± 15.8 mg/dL (p = 0.02) and took a higher number of anti-diabetic drugs 1.0 [IQR 0.5–2.0] vs 1.0 [IQR 0–1.0] (p = 0.04). Insulin resistance was not significantly higher in Mg-deficient patients – HOMA-IR 1.8 ± 1.2 vs 1.6 ± 1.1 (p = 0.48).At 1-year post-BS, the prevalence of Mg deficiency was lower when compared with pre-surgical moment (19.1% vs 43.4% of patients) and the mean serum Mg was higher compared to baseline – 1.62 ± 0.14 mEq/L . After surgery, 78.0% of patients were under Mg supplementation.In Table n = 229) of the patients had total remission of T2DM and 4.1% (n = 16) had partial remission of T2DM. Concerning serum Mg levels, patients with total remission had higher levels when compared with patients that didn’t remit—1.65 ± 0.13 mEq/L vs 1.58 ± 0.16 mEq/L (p < 0.001).At 1-year post-BS, 58.4% (n = 273), around 62% had total remission. These patients had higher Mg serum values compared to patients that had no T2DM remission—1.64 ± 0.14 mEq/L vs 1.59 ± 0.16 mEq/L (p = 0.01). In patients submitted to SG, we had 51.3% of total T2DM remission. Higher serum Mg values were observed in the group of patients achieving T2DM total remission.In Table vs 48.3% (p = 0.18).Patients that were under Mg supplementation post-BS did not have higher rates of total T2DM remission – 62.0% p < 0.001]. In our analysis, type of surgery (SG vs RYGB) was not an independent predictor of T2DM remission .In Table The optimal cut-off of baseline Mg to predict total T2DM remission was 1.50 mg/dL with a sensitivity of 73% and a specificity of 58% (area under ROC = 0.65).In this study, we report four main findings. First, patients with Mg deficiency (pre and post-BS) had worst glycaemic control in pre and post-BS evaluations. Second, patients with normal serum Mg levels post-BS had higher chances of T2DM total remission on first year post-BS. Third, serum Mg values at baseline were an independent predictor of T2DM remission (cut-off was 1.50 mg/dL). Fourth, patients under Mg supplementation post-BS did not differ from patients without Mg supplementation on glycaemic control, insulin sensitivity and T2DM total remission on first year post-BS.When we focus on Mg dietary intake (either by food or supplements), many epidemiological studies and, more recently, randomized controlled trials, demonstrated an inverse relation between higher dietary Mg content and T2DM –19. In pWhile some previous studies have reported higher prevalence of Mg deficiency after BS , an imprAs we stated previously, in patients with diabetes poorly controlled, hypomagnesemia is relatively common –30. In ovs RYGB) was not an independent predictor of remission.Regarding T2DM remission rates, patients with normal serum Mg values post-BS had higher T2DM total and partial remission rates. When remission rates were evaluated according to the type of surgery, patients with total remission had higher Mg serum levels independently of being submitted to RYGB or SG. In agreement with our results, a recent long-term cohort study  showed tThere are limitations that should be addressed in our study. As an observational study, the possibility of confounding factors  cannot be excluded. As we stated previously, a possible explanation to Mg supplementation analysis did not reach statistical significance was that patients in the supplemented group were doing Mg supplementation either by multivitamin complex or Mg salt, with different doses , formulations and, possibly, compliance. Also, Mg food-derived intake could not be assessed. Higher consumption of Mg-rich foods has demonstrated to reduce T2DM risk and improve glycaemic control and blood lipids in patients with T2DM [To the best of our knowledge, this is the first study aiming to assess the relationship between Mg supplementation/Mg serum levels with glycaemic control and T2DM remission rates after BS. Furthermore, establishing a cut-off point for baseline serum Mg level to predict T2DM remission and the elevated number of patients are additional strengths of our study.In the future, there is a need to carry out prospective studies to analyze the effect of Mg supplementation in post-bariatric patients with T2DM.In patients with T2DM submitted to BS higher Mg serum levels at baseline and 1-year after surgery were associated with better glycaemic control and higher rates of total T2DM remission at the first year post-BS. The optimal cut-off of baseline Mg to predict total T2DM remission was 1.50 mg/dL.Additional file 1. Supplementary information.


The high performance is ascribed to the synergistic effect of the metal/metal oxide and hollow architecture, which is favorable for breaking the H–OH bond, forming hydrogen atoms, and enabling charge transport. These results indicate that the employed approach is promising for fabricating cost-effective catalysts for hydrogen production in alkaline media.Metal-organic frameworks (MOFs) constitute a class of crystalline porous materials employed in storage and energy conversion applications. MOFs possess characteristics that render them ideal in the preparation of electrocatalysts, and exhibit excellent performance for the hydrogen evolution reaction (HER). Herein, H–Ni/NiO/C catalysts were synthesized from a Ni-based MOF hollow structure via a two-step process involving carbonization and oxidation. Interestingly, the performance of the H–Ni/NiO/C catalyst was superior to those of H–Ni/C, H–NiO/C, and NH–Ni/NiO/C catalysts for the HER. Notably, H–Ni/NiO/C exhibited the best electrocatalytic activity for the HER, with a low overpotential of 87 mV for 10 mA cmThe online version contains supplementary material available at 10.1186/s40580-023-00354-w. Hydrogen  are ideal for the synthesis of hollow-structured catalysts because of the level of morphological control they provide via changing the linkers and reaction conditions. In addition, MOFs possess a high porosity, which increases the active site density. Xue et al. employed ZIF-67 as a template to prepare CoSe2–FeSe2 catalysts with a hollow architecture for the HER under alkaline conditions [2 hollow microspheres as highly stable catalysts for the HER [Hollow-structured materials have also been considered as potential electrocatalysts for the HER. Hollow structures possess thin shells with inner and outer surfaces to afford a larger surface area and more exposed active sites that promote reactant adsorption and redox reactions, providing these surfaces with locally incompatible cofactors for oxidative and reductive reactions , 36. In tructure . Ganesan the HER . The eff the HER , who hignditions . Tian et the HER . As ment−2 and good durability. The approach employed herein could serve as a novel route for the synthesis of hollow-structured catalysts for the HER and other applications.In this study, we used a Ni-MOF hollow structure to prepare a Ni/NiO/C hollow-structured composite through the two-step route of carbonization and oxidation. This material showed enhanced HER activity compared with Ni/C, NiO/C, and Ni/NiO/C with a non-hollow structure. Moreover, it exhibited a very low overpotential of 87 mV at a current density of 10 mA cm3)2·6H2O), benzene-1,3,5-tricarboxylic acid , N, N-dimethylformamide (DMF), and polyvinylpyrrolidone  and ethanol (C2H5OH) were purchased from Sigma Aldrich. Deionized water (DI) was prepared using a Millipore Milli-Q apparatus.Nickel nitrate hexahydrate (Ni(NO3)2·6H2O (216 mg) and H3BTC (75 mg) were dissolved in a mixed solvent containing 5 mL of DMF, 5 mL of C2H5OH, and 5 mL of DI for 30 min under magnetic stirring. Subsequently, 750 mg of PVP, which acted as a micelle stabilizer, was added to this mixture under magnetic stirring for 2 h to form a homogeneous solution. This solution was transferred into a 25 mL Teflon-lined stainless-steel reactor and heated to 150 °C for 10 h. The obtained light-green Ni-MOF crystals were washed by C2H5OH and dried at 60 °C for 10 h.Typically, a Ni-MOF hollow structure was synthesized according to a reported study with a slight modification . Ni(NO3)2 gas to form the hollow architecture of H–Ni/C. Subsequently, this powder was oxidized in air at 250 °C for 20 min to obtain the H–Ni/NiO/C composite. H–NiO/C was obtained by heating the Ni-MOF hollow structure at 450 °C for 2 h. In addition, a non-hollow structure of NH–Ni/NiO/C was fabricated from a Ni-MOF non-hollow structure in the same manner as for the synthesis of the H–Ni/NiO/C composite.The Ni-MOF powders were placed into an alumina crucible and heated at 450 °C for 2 h under a NThe morphological structure of the as-synthesized products was confirmed using scanning electron microscopy  and transmission electron microscopy . X-ray diffraction (XRD) tests were performed with a Bruker D8-Advance diffractometer. Raman curves of the composites were tested on a LabRAM-HR Evolution spectrometer with a 532 nm laser. The elemental composition was determined using X-ray photoelectron spectroscopy .2H5OH for 30 min. Subsequently, a certain volume of the homogeneous suspension was coated on the surface of a glassy carbon electrode (diameter of 3 mm), with a catalyst loading of 0.30 mg cm−2. Polarization curves were collected with IR correction at a scan rate of 2 mV s−1. IR corrections for resistance were conducted using electrochemical impedance spectroscopy (EIS) in the potentiostatic mode from 100,000 to 0.1 Hz. Cyclic voltammetry (CV) was performed to determine the electrochemical double-layer capacitance (Cdl) in the non-faradaic voltage region at 20, 40, 60, 80, 120, and 140 mV s−1. The reported potentials were converted to those of the reversible hydrogen electrode (RHE) using the following relation: ERHE = EHg/HgO + 0.098 + 0.059pH.The HER performance was evaluated using various electrochemical techniques with the help of an Ivium 55,630 instrument. The electrochemical system contained a graphite rod as the counter electrode, Hg/HgO electrode as the reference electrode, and 1 M KOH as the electrolyte. The working electrode was fabricated as follows: the as-synthesized materials (30 mg) were sonicated in a mixture containing 0.1 mL Nafion (5 wt%) and 3 mL CHerein, we used a Ni-MOF hollow structure to fabricate H–Ni/NiO/C via a two-step process involving pyrolysis and oxidation, as schematically shown in Fig. position , 47–49. mentclass2pt{minimmentclass2pt{minimmentclass2pt{minim−1, whereas that for H–Ni/NiO/C exhibits two peaks at 500 and 1060 cm−1. The Raman peaks at 360 and 500–510 cm−1 can be indexed to the one-phonon (1P) transverse optical (TO) and longitudinal optical (LO) vibrations of the Ni–O bonds. The other peaks are assigned to the second-order vibrational modes of 2PTO, 2PTO+LO, and 2PLO, respectively [LO increased, whereas the intensity of 2PLO decreased in the H–Ni/NiO/C. Because the intensity of 1PLO and 2PLO was related to the defect states associated with Ni vacancies [LO [LO could be ascribed to a large number of Ni vacancies in H–Ni/NiO/C, which was formed at 250 °C. H–NiO/C created at 450 °C shows a low intensity of 1PLO mode, which was attributed to the decelerated Ni vacancies. Also, the intensity of 2PLO was accelerated due to the improved crystalline quality of the H–NiO/C sample [Figure ectively , 54. Intacancies , 56. Accacancies , which dcies [LO . TherefoC sample .Fig. 3a 2+ 2p1/2 and Ni2+ 2p3/2, as depicted in Fig. 2+ 2p3/2 at 853.7 and 855.5 eV, and a Ni2+ 2p3/2 satellite at 860.8 eV. Also, the peaks at 870.9 eV and 872.4 eV correspond to the multiplet-split Ni2+ 2p1/2 and Ni2+ 2p3/2 of NiO phases, respectively [3/2 and 869.9 eV for metallic Ni 2p1/2 , the H–OH bond breaks to form H atoms that are absorbed on the surface of the catalyst. The Heyrovsky reaction (Tafel slope ≈ 40 mV dec−1) describes electrochemical desorption, whereas the Tafel reaction (Tafel slope ≈ 30 mV dec−1) expresses chemical desorption to form hydrogen. Based on the reported Tafel slopes, the Volmer and Heyrovsky reactions occur when using the investigated catalysts.The HER performances of H–Ni/C, H–Ni/NiO/C, H–NiO, and NH–Ni/NiO/C were investigated in a 1.0 M potassium hydroxide medium. As displayed in Fig. olecules , 62, 63.olecules , 64. Morolecules , 65, 66.ct). Note that the Rct of H–Ni/NiO/C is 75 Ω, which is much lower than that of H–Ni/C (189 Ω), H–NiO (229.4 Ω), and NH–Ni/NiO/C (133.4 Ω). The lowest Rct of H–Ni/NiO/C is indicative of the fastest interfacial charge transport kinetics of this catalyst during the electrocatalytic process. Typically, the electrochemical surface area (ECSA) can be predicted from Cdl, which is derived from the CV profiles in the non-faradaic potential region. The CV profiles of H–Ni/C , H–NiO (1.08 mF cm−2), and NH–Ni/NiO/C (3.69 mF cm−2), implying its larger ECSA as well as superior HER performance  and hydroxyl ions (OH‒), whereas Ni moieties recombine Hads to produce H2. This synergistic effect helps H–Ni/NiO/C achieve a higher HER activity than bare electrocatalysts.EIS was performed on the catalysts to confirm the reaction kinetics of the electrodes. Figure f H–Ni/C 89 Ω, H–Nf H–Ni/C 89 Ω, H–Nf H–Ni/C 89 Ω, H–Nf H–Ni/C 89 Ω, H–N/NiO/C 3.9 mF cm−2f H–Ni/C 89 Ω, H–N/NiO/C 3.9 mF cm−2−2. In addition, this catalyst exhibited remarkable stability after 12 h test. The good HER activity was assigned to the emerging effect of the metal/metal oxide and hollow morphological structure, which facilitated H–OH bond breakage, the recombination of intermediate hydrogen, and electron transfer. The empirical findings of this study provide a novel understanding of the use of MOFs for the fabrication of economical catalysts for H2 production.In conclusion, Ni/NiO/C hollow-structured composites were successfully fabricated from Ni-MOF precursors and utilized as efficient electrocatalysts for the HER in alkaline media. The experimental results confirmed that H–Ni/NiO/C exhibited better HER activity than H–Ni/C, H–NiO/C, and non-hollow NH–Ni/NiO/C catalysts. Notably, H–Ni/NiO/C exhibited a very low overvoltage of 87 mV at a current density of 10 mA cmAdditional file 1: Figure S1. (a) XRD pattern and (b) FE-SEM image of the Ni-MOF hollow structure. Figure S2. SEM images of (a) H–Ni/C and (b) H–NiO/C. Figure S3. (a) TEM, and (b) Field emission TEM images of H–Ni/C. Figure S4. (a) XPS survey spectrum. High-resolution XPS profiles of H–NiO/C: (b) Ni 2p, (c) O 1s, and (d) C 1s. Figure S5. High-resolution XPS profiles of H–Ni/C: (a) Ni 2p, and (b) C 1s. Figure S6. (a) Polarization curves of the H–Ni/NiO/C-10min, H–Ni/NiO/C-20min, and H–Ni/NiO/C-30min catalysts at a scan rate of 2 mV s-1 in a 1 M KOH solution. (b) Tafel plots of the same catalysts derived from (a). Figure S7. Cyclic voltammograms (0.1–0.2 V) of (a) H–Ni/C, (b) H–Ni/NiO/C, (c) H–NiO/C, and (d) NH–Ni/NiO/C at various scan rates (20–120 mV s-1) in a 1 M KOH solution. Figure S8. (a) XRD pattern and (b) SEM images of H–Ni/NiO/C after 2000 CV cycles. Table S1. Comparison of the HER performance of different samples in an alkaline solution. Table S2. Comparison of the catalytic activity of the Ni/NiO/C hollow structure with those of nickel-based catalysts for the HER in an alkaline solution.