Recent work shows that superficially porous resins for reversed phase liquid chromatography (SPLC) combined with high resolving power mass spectrometry (MS) can efficiently characterize hundreds of label-free intact proteins (top-down analysis) from complex starting materials such as cell lysate. SPLC provides typical elution periods of 6 seconds for intact proteins above 200 kDa, with a wide linear dynamic range (>10,000), low attomole detection limits, and 5 ng loading capacity. Herein SPLC resins were adapted for high peak capacity separations of closely related intact protein proteoforms generated from alternative splicing events, single nucleotide polymorphisms, and post translational modifications (PTMs) in an online, high throughput setting. Results to date characterized a model protein, murine myelin basic protein (MBP), an intrinsically disordered protein that harbors a large number of PTM sites on each individual splice variant.   SPLC solvent conditions, ion pairing reagents, and elution gradients were optimized for MBP proteoform separations with results showing that the differential hydrophobicity of the PTMs harbored on each MBP splice variant facilitated chromatographic separation (e.g., oxidized proteoforms eluted before methylated or dimethylated proteoforms). The estimated peak capacity of MBP proteoforms is 300-500 for the 14 kDa splice variant. The dominant forms of the 14 kDa species were interrogated by MS/MS to acquire fragmentation data that was used to search against a custom shotgun annotated database including likely MBP proteoforms. Differences in PTM profiles were noted across splice variants, suggesting unique processing events for each variant. Traces of individual fragment intensities observed during elution correlated with the intensities of the intact proteoforms that harbored them. Our work will utilize this SPLC MS platform to rapidly characterize MBP isolated from brain and spinal cord in murine models of neurodegenerative disease, bringing intact protein chromatography to the timeframe associated with peptide chromatography.

Highly efficient characterization of highly modified protein variants using superficially porous reversed phase liquid chromatography

247th National Meeting (2014)

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