Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics

Top-down proteomics (TDP) aims to
delineate proteomes in a proteoform-specific
manner, which is vital for accurately understanding protein function
in cellular processes. It requires high-capacity separation of proteoforms
before mass spectrometry (MS) and tandem MS (MS/MS). Capillary isoelectric
focusing (cIEF)-MS has been recognized as a useful tool for TDP in
the 1990s because cIEF is capable of high-resolution separation of
proteoforms. Previous cIEF-MS studies concentrated on measuring the
protein’s mass without MS/MS, impeding the confident proteoform
identification in complex samples and the accurate localization of
post-translational modifications on proteoforms. Herein, for the first
time, we present automated cIEF-MS/MS-based TDP for large-scale delineation
of proteoforms in complex proteomes. Single-shot cIEF-MS/MS identified
711 proteoforms from an Escherichia coli (E. coli) proteome consuming only
nanograms of proteins. Coupling two-dimensional size-exclusion chromatography
(SEC)-cIEF to ESI-MS/MS enabled the identification of nearly 2000
proteoforms from the E. coli proteome.
Label-free quantitative TDP of zebrafish male and female brains using
SEC-cIEF-MS/MS quantified thousands of proteoforms and revealed sex-dependent
proteoform profiles in brains. Particularly, we discovered several
proteolytic proteoforms of pro-opiomelanocortin and prodynorphin with
significantly higher abundance in male zebrafish brains as potential
endogenous hormone proteoforms. Multilevel quantitative proteomics
(TDP and bottom-up proteomics) of the brains revealed that the majority
of proteoforms having statistically significant difference in abundance
between genders showed no abundance difference at the protein group
level. This work represents the first multilevel quantitative proteomics
study of sexual dimorphism of the brain.