SWATH-MS advanced from using 32x25 m/z windows to cover the 400-1200 m/z MS1 range, to utilize variable windows (vW), with widths dependent upon peptide precursor density in given MS1 m/z ranges, leading to deeper proteome coverage, especially in the tryptic peptide rich region of 600-800 m/z. For plasma biomarker studies, despite deploying vW-SWATH, information extraction commonly only penetrates a small proportion of large plasma protein assay libraries available in community repositories. Therefore, we explored gas phase fractionation (GPF) SWATH to narrow MS1 windows and determine the impact on quantitative proteome profiling.
Tryptic digests of human plasma and SW480 cell lysates were analysed by 60 min GPF-SWATH and vW-SWATH on 6600 triple TOF (SCIEX). 400–600 m/z and 600–800 m/z with 4Da fixed windows, and 800-1200 m/z with 8Da windows were chosen for GPF-SWATH. The vW-SWATH method used 100 windows from 400–1200 m/z. A high pH fractionation assay library was used for SW480 cells and an online repository assay library for plasma.
GPF-SWATH was optimized with SW480 lysates. Information extraction from vW-SWATH using the high pH assay library revealed quantitative information for 2,600 proteins (9,800 peptides). Analysis using GPF-SWATH resulted in ~3,500 proteins (30% increase) and ~15,500 peptides (60% increase). Striking improvements were seen for 400–600 m/z with >2-fold increased peptide extraction and 1.5-fold increases for 600–800 m/z and 800–1200 m/z regions. Using an online repository assay library vW-SWATH extracted quantitative information for 300 proteins (1,500 peptides) from plasma. With GPF-SWATH, proteins with quantitative information increased by 25%, peptides increased by 20%. Proteins unique to GPF-SWATH are less abundant cellular proteins, shed membrane proteins and extracellular matrix proteins.
GPF-SWATH of SW480 cells and human plasma demonstrates quantitation improvements over vW-SWATH and is a useful approach to take advantage of large assay libraries for deeper proteome quantitation.