Proteoform analysis is a critical step in understanding the pathological mechanisms in disease formation and progression. Most analytical techniques fail to accurately detect features such as Post Translational Modifications (PTM) and products of proteolytic cleavage. Current methods, such as PTM enrichment kits are for single targeted modifications, exemplifying a clear need for an analytical technique that is able to detect PTMs and products of proteolytic cleavage in an untargeted manner.
In this study we use 2D-PAGE as a form of physical and chemical fractionation for total proteome screening of PTM’s. The two dimensions of separation (mass and isoelectric point) allow a significant reduction in sample complexity while also enriching proteoforms with similar chemical properties together. This rapid, multiplexed method, fractionates a proteome into 80 samples for mass spectrometry analysis, thereby reducing sample preparation time from months to less than a day and only requiring ~24 hours of instrument time per sample. Using this method, we are able to identify PTM abundance changes, glycans, and proteolytic cleavage products without the use of enrichment kits.
Protein extracts from mouse lung from a chronic obstructive pulmonary disorder (COPD) smoke model were purified, reduced and alkylated and separated by 2D-PAGE. Coomassie-stained gels were rapidly destained, cut, in-gel digested, and purified using SDB-RPS stage tips all in a 96 well format followed by short gradient LC-MS/MS. We detected 6925 proteins, 3965 protein groups, 52,274 peptides including 22,617 modified peptides with 122 known post-translational modifications, including biologically relevant modifications such as acetylation, ubiquitinylation, formylation, methylation, cyclization of glutamine and sulphonation. Different peptides from the same protein in different mass locations on the 2D gel indicate clear products of proteolytic cleavage, with non-tryptic cleavage being present. The clear discernment of different proteoforms and their modified sequences are confirmed by their different spatial locations on the gel.