Multiplexing complexome profiling to foster routine protein complex profiling in medical research
Activity: Talk or presentation › Guest lectures › Research
Authors
- Sergio Guerrero Castillo - Speaker
Related Research units
Description
Background: Complexome profiling (CP) is an omics approach to comprehensively study the composition, abundance, stability
and dynamics of protein complexes. It identifies and quantifies protein complexes, biochemically separated into numerous
fractions of increasing molecular mass, by label-free (LF) bottom-up proteomics. Despite its potential, its use is restricted by the
large number of fractions to measure and the extensive time for mass spectrometry runs.
Isobaric peptide labeling has been used in bottom-up proteomics to compare quantitatively protein abundances between
biological samples or conditions. Quantifications in fragment spectra are based on low m/z reporter ions specific for each sample,
in contrast to LF that uses precursor intensities to build ion chromatograms for quantification. Peptide labeling offers also the
advantage of measuring multiple samples in a single LC-MS/MS run, in comparison to LF, which requires separate runs for each
sample. However, the compatibility of labeling reagents with complexome analysis, using reporter ion quantifications for
reconstructing protein migration profiles has not been assessed.
Case Study / Methods: Here, we implemented isobaric labeling reagents to the standard CP workflow. Multiplexed complexome
profiles of mitochondrial fractions from cells recovering from chloramphenicol treatment were compared to LF profiles of the same
set of samples.
Results: Multiplexed CP showed patterns of OXPHOS complexes and assembly intermediates that were consistent in composition
and abundance with profiles obtained by LF-CP. Quantifications of complexes unaffected by chloramphenicol exhibited less
variation than in the LF method.
Conclusion / Discussion: The implemented workflow decreased the number of samples and measuring times without
compromising protein identification or quantification reliability. Incorporation of tandem mass tags enabled an efficient and robust
CP analysis to foster a broader use of protein complex profiling in biomedical research and diagnostics.
and dynamics of protein complexes. It identifies and quantifies protein complexes, biochemically separated into numerous
fractions of increasing molecular mass, by label-free (LF) bottom-up proteomics. Despite its potential, its use is restricted by the
large number of fractions to measure and the extensive time for mass spectrometry runs.
Isobaric peptide labeling has been used in bottom-up proteomics to compare quantitatively protein abundances between
biological samples or conditions. Quantifications in fragment spectra are based on low m/z reporter ions specific for each sample,
in contrast to LF that uses precursor intensities to build ion chromatograms for quantification. Peptide labeling offers also the
advantage of measuring multiple samples in a single LC-MS/MS run, in comparison to LF, which requires separate runs for each
sample. However, the compatibility of labeling reagents with complexome analysis, using reporter ion quantifications for
reconstructing protein migration profiles has not been assessed.
Case Study / Methods: Here, we implemented isobaric labeling reagents to the standard CP workflow. Multiplexed complexome
profiles of mitochondrial fractions from cells recovering from chloramphenicol treatment were compared to LF profiles of the same
set of samples.
Results: Multiplexed CP showed patterns of OXPHOS complexes and assembly intermediates that were consistent in composition
and abundance with profiles obtained by LF-CP. Quantifications of complexes unaffected by chloramphenicol exhibited less
variation than in the LF method.
Conclusion / Discussion: The implemented workflow decreased the number of samples and measuring times without
compromising protein identification or quantification reliability. Incorporation of tandem mass tags enabled an efficient and robust
CP analysis to foster a broader use of protein complex profiling in biomedical research and diagnostics.
31.08.2022
