Quantitative measurements of N ‐linked glycoproteins in human plasma by SWATH ‐ MS

SWATH ‐ MS is a data‐independent acquisition method that generates, in a single measurement, a complete recording of the fragment ion spectra of all the analytes in a biological sample for which the precursor ions are within a predetermined m/z versus retention time window. To assess the performance and suitability of SWATH ‐ MS ‐based protein quantification for clinical use, we compared SWATH ‐ MS and SRM ‐ MS ‐based quantification of N ‐linked glycoproteins in human plasma, a commonly used sample for biomarker discovery. Using dilution series of isotopically labeled heavy peptides representing biomarker candidates, the LOQ of SWATH ‐ MS was determined to reach 0.0456 fmol at peptide level by targeted data analysis, which corresponds to a concentration of 5–10 ng protein/mL in plasma, while SRM reached a peptide LOQ of 0.0152 fmol. Moreover, the quantification of endogenous glycoproteins using SWATH ‐ MS showed a high degree of reproducibility, with the mean CV of 14.90%, correlating well with SRM results ( R 2 = 0.9784). Overall, SWATH ‐ MS measurements showed a slightly lower sensitivity and a comparable reproducibility to state‐of‐the‐art SRM measurements for targeted quantification of the N ‐glycosites in human blood. However, a significantly larger number of peptides can be quantified per analysis. We suggest that SWATH ‐ MS analysis combined with N ‐glycoproteome enrichment in plasma samples is a promising integrative proteomic approach for biomarker discovery and verification.