Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ∼7.0 femtomole detection limits and linear spectral response for proteins fractionated over ∼4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5-6× increase in the number of unique monoisotopic masses observed <30 kDa and an ∼4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate pI compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different pI. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues.
ASJC Scopus subject areas
- Analytical Chemistry