multiFLEX-LF: A Computational Approach to Quantify the Modification Stoichiometries in Label-Free Proteomics Data Sets

In
liquid-chromatography–tandem-mass-spectrometry-based
proteomics, information about the presence and stoichiometry of protein
modifications is not readily available. To overcome this problem,
we developed multiFLEX-LF, a computational tool that builds upon FLEXIQuant,
which detects modified peptide precursors and quantifies their modification
extent by monitoring the differences between observed and expected
intensities of the unmodified precursors. multiFLEX-LF relies on robust
linear regression to calculate the modification extent of a given
precursor relative to a within-study reference. multiFLEX-LF can analyze
entire label-free discovery proteomics data sets in a precursor-centric
manner without preselecting a protein of interest. To analyze modification
dynamics and coregulated modifications, we hierarchically clustered
the precursors of all proteins based on their computed relative modification
scores. We applied multiFLEX-LF to a data-independent-acquisition-based
data set acquired using the anaphase-promoting complex/cyclosome (APC/C)
isolated at various time points during mitosis. The clustering of
the precursors allows for identifying varying modification dynamics
and ordering the modification events. Overall, multiFLEX-LF enables
the fast identification of potentially differentially modified peptide
precursors and the quantification of their differential modification
extent in large data sets using a personal computer. Additionally,
multiFLEX-LF can drive the large-scale investigation of the modification
dynamics of peptide precursors in time-series and case-control studies.
multiFLEX-LF is available at https://gitlab.com/SteenOmicsLab/multiflex-lf.