Proteomics

Dataset Information

1

Accurate quantitative proteomic analyses using metabolic labeling and High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)


ABSTRACT: 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 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 co-elute and can be misassigned in conventional LC-MS/MS experiments.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo sapiens  

TISSUE(S): Tissue Not Applicable To Dataset

DISEASE(S): Not Available

SUBMITTER: Francis McManus  

LAB HEAD: Pierre Thibault

PROVIDER: PXD012924 | Pride | 2019-05-28

REPOSITORIES: Pride

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Publications

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

Pfammatter Sibylle S   Bonneil Eric E   McManus Francis P FP   Thibault Pierre P  

Journal of proteome research 20190417 5


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 rang  ...[more]

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