Proteoform analysis is a critical step in understanding the pathological mechanisms in disease onset and progression. Most analytical techniques fail to accurately detect proteoform-specific features, including post-translational modifications (PTMs) and the products of proteolytic cleavage, especially those in low abundance. Current methods rely on expensive enrichment kits targeting a single type of modification, thus limiting the discovery of novel PTMs or proteolytic cleavage products. There is a clear need for an analytical technique that is able to identify individual proteoforms with their respective PTMs and proteolytic cleavage events without the need for enrichment at the peptide level.
In this study, we used Integrative Top-Down Proteomics (iTDP), utilising 2D-PAGE as a form of physical and chemical fractionation for proteoform resolution. The two dimensions of separation (isoelectric point followed by mass) significantly reduces sample complexity while also enriching for individual proteoforms. This fractionates a proteome into 63 samples for mass spectrometry analysis, requiring ~24 hours of instrument time per sample. Using this method, we are able to identify individual proteoforms and their respective PTMs that make them distinct proteoforms, such as phosphorlylation, acetylation, glycosylation, and proteolytic cleavage products, without the use of enrichment kits.
Protein extracts from mouse liver were analysed by iTDP. Entire gels were cut into 63 grid pieces, in-gel digested, and peptides purified using SDB-RPS stage tips in a 96-well format, followed by short gradient LC-MS/MS using a Thermo Q Exactive Plus. Across the gel 5326 ORF products were detected, consisting of 36314 peptides of which 15015 were modified by more than 62 post-translational modifications, including phosphorylation, acetylation, ubiquitinylation, methylation, and proteolytic cleavage. In comparison, a single-injection shotgun-based workflow using the same sample detected 2826 ORF products, 9182 peptides including 3247 modified peptides, and 23 different types of PTMs were detected.
Different peptides from the same ORF product at different mass locations on the 2D gel indicate proteoforms generated by proteolytic cleavage, with non-tryptic cleavage being present. The clear discernment of different proteoforms and their modified sequences is confirmed by their different spatial locations on the gel.
Compared to traditional techniques, this approach provides a more comprehensive proteoform-level analysis of the proteome, offering broad utility for studying proteoforms in liver diseases or related pathologies.