Accurate Quantitative Proteomic Analyses Using Metabolic Labeling and High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)

Stable
isotope labeling by amino acids in cell culture (SILAC)
is routinely used to profile changes in protein and peptide abundance
across different experimental paradigms. As with other quantitative
proteomic approaches, the detection of peptide isotopomers can be
limited by the presence of interference ions that ultimately affect
the quality of quantitative measurements. Here, we evaluate high field
asymmetric waveform ion mobility spectrometry (FAIMS) to improve the
accuracy and dynamic range of quantitative proteomic analyses using
SILAC. We compared quantitative measurements for tryptic digests of
isotopically labeled protein extracts mixed in different ratios using
LC–MS/MS with and without FAIMS. To further reduce sample complexity,
we also examined the improvement in quantitative measurements when
combining strong cation exchange (SCX) fractionation prior to LC–MS/MS
analyses. Using the same amount of sample consumed, analyses performed
using FAIMS provided more than 30% and 200% increase in the number
of quantifiable peptides compared to LC–MS/MS performed with
and without SCX fractionation, respectively. Furthermore, FAIMS reduced
the occurrence of interfering isobaric ions and improved the accuracy
of quantitative measurements. We leveraged the application of FAIMS
in phosphoproteomic analyses to profile dynamic changes in protein
phosphorylation in HEK293 cells subjected to heat shock for periods
up to 20 min. In addition to the enhanced phosphoproteomic coverage,
FAIMS also provided the ability to separate phosphopeptide isomers
that often coelute and can be misassigned in conventional LC–MS/MS
experiments.