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/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.