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

Abstract

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.

Simplified and Efficient Quantification of Low-abundance Proteins at Very High Multiplex via Targeted Mass Spectrometry

“…Similarly high levels of multiplexing might be possible with data independent acquisition methods with internal standards. Data independent acquisition has in recent years reemerged as an analytic technique as a result of faster, more sensitive MS systems, improved on-board computerized instrument control, and more sophisticated software for data acquisition and analysis (22,23).…”

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

“…This includes an additional value to the analyses we performed, as they are still valuable for future studies of yet undiscovered PTMs. Even though SWATH™-MS is somewhat less sensitive than SRM (28), an SRM experiment of a histone mixture analysis has two major drawbacks: (1) the method is programmed to monitor a given list of peptides, and thus cannot be used for further analysis of analytes not on the inclusion list; (2) specifically for histone peptides such method would require a complex and long list of transitions. For instance, considering only the 41 peptide proteoforms we analyzed for histone H3 (supplemental Table S1), we would need 123 transitions to monitor three fragment ions for each peptide proteoform, and 492 transitions considering all fragment ions we used to quantify each peptide proteoform with SWATH™.…”

Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)*

“…This technique presents specific advantages in an unsupervised set up, such as an extensive coverage of the proteome, with no a priori hypothesis. However, the method in its current implementation presents limited selectivity and sensitivity for complex samples compared with TDA methods (20). Alternatively, the modification of the chromatographic conditions of peptide separations was also explored as a mean to increase the scale of TDA experiments, In the context of SRM analyses, the use of long chromatographic columns (typically Ͼ 30 cm) in conjunction with optimized shallow gradients (typically Ͼ 3 h) was shown to allow the measurement of 400 pairs of SIL and endogenous peptides with acceptable dwell times (between 8 and 100 ms) (21).…”