title: Improvement of Quantitative Measurements in Multiplex Proteomics Using High-Field Asymmetric Waveform Spectrometry.
abstract: Quantitative proteomics using isobaric reagent tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provides a convenient approach to compare changes in protein abundance across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC-FAIMS-MS/MS analyses of human and yeast protein digests led to significant reductions in interfering ions, which increased the number of quantifiable peptides by up to 68% while significantly improving the accuracy of abundance measurements compared to that with conventional LC-MS/MS. The improvement in quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment.
authors.affiliation: Institute for Research in Immunology and Cancer, ‡Department of Chemistry, Université de Montréal , C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Pfammatter
authors.affiliation: Institute for Research in Immunology and Cancer, ‡Department of Chemistry, Université de Montréal , C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.affiliation: Institute for Research in Immunology and Cancer, ‡Department of Chemistry, Université de Montréal , C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D000068598:Data Accuracy; D005656:Fungal Proteins / Q000032:analysis; D057809:HEK293 Cells; D018869:Heat-Shock Response; D006801:Humans; D008722:Methods; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D011506:Proteins
keywords: heat-shock; high-field asymmetric waveform spectrometry (FAIMS); isobaric labeling; quantitative proteomics; tandem mass tag