Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach.
Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.
['Percy|Andrew J|AJ|', 'Yang|Juncong|J|', 'Chambers|Andrew G|AG|', 'Mohammed|Yassene|Y|', 'Miliotis|Tasso|T|', 'Borchers|Christoph H|CH|']
D000465:Algorithms D015415:Biomarkers D002138:Calibration D019295:Computational Biology D057225:Data Mining D030562:Databases, Protein D057166:High-Throughput Screening Assays D006801:Humans D013058:Mass Spectrometry D011237:Predictive Value of Tests D011506:Proteins D020543:Proteome D040901:Proteomics D012015:Reference Standards D015203:Reproducibility of Results D012984:Software
Standardization Biomarker Quantitation Proteomics Internal standards Plasma MRM