title: Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring.
abstract: Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.
authors: Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, United States.
authors: Hsin-Yao
authors: HY
authors: Tang
authors: Lynn A
authors: LA
authors: Beer
authors: Kurt T
authors: KT
authors: Barnhart
authors: David W
authors: DW
authors: Speicher
mesh_terms: D000595:Amino Acid Sequence; D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D002402:Cathepsin D / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D010446:Peptide Fragments; C533922:CTSD protein, human; D002402:Cathepsin D