Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS–QTOF Instrument

• Published in: Journal of proteome research Vol. 21; no. 8; pp. 2036 - 2044
• Main Authors: Guergues, Jennifer, Wohlfahrt, Jessica
• Format: Journal Article
• Language: English
• Published: American Chemical Society 05.08.2022
• Subjects: fractionation; humans; peptides; proteome; spectroscopy; ultra-performance liquid chromatography; timsTOF Pro; gas-phase fractionation; proteomics; DDA-PASEF; spectral library; ion mobility; DIA-PASEF
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.2c00336

Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm2), followed by parallel accumulation–serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min gradient. This result corresponded to ∼19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single-range TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by the average and median coefficient of variation values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.