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

Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm(2)), 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 similar to 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 similar to 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 PXDO33129.