Introduction: The term proteoform is used to describe the base sequence of a protein plus or minus any chemical modifications that the protein might have. This term is given after post translational modifications (PTM’s) occur on the base sequence. Detection of PTM’s is typically done with enrichment kits or by using open or predictive search algorithms to see which post-translational modifications are present within the peptides of a proteoform. The problem that is faced with these approaches is that when multiple proteins are obtained from a sample, digested, and cleaved into peptides we can’t know which protein from a sample had which modifications that we detect.
The focus of this work was to investigate a method to detect modified peptides in the context of their parent proteins without requiring an enrichment kit, by separating proteins based on their isoelectric point. Our hypothesis that this method should allow for the determination of the context of modified peptides
Method: Mouse lungs were obtained from surplus tissue from a previous larger study. Protein extraction and solubilisation was carried out using a novel in house method followed by isoelectric focusing. Focused IPG strips were transferred to a gel and run using 1D SDS page. Then stained with Coomassie Brilliant Blue. For this pilot study the top row of each gel was used and cut into 12 equal size pieces. Proteins were digested using an In Gel Digestion Protocol. Samples were stagetipped and then prepared for analysis using a mass spectrometer.
Results: The densitometry report results showed that the extraction method was successful. Overall 2500 proteoforms were identified with >20 different modifications found including acetylation of lysine and phosphorylation, from just a single strip from the top of a 2D gel This new method greatly reduces the number of proteins that are analysed per spot to ~20. Which provides greater chances that the modified peptides are within the 12-15 dominant ions that are picked up by mass spectrometry analysis using a Top N method. In addition, this method allows the modifications to be detected alongside their respective parent protein. Hence the method allows for post-translational modifications to be detected simultaneously. Further study is currently being analysed to look at the PTM’s present in two separate whole gels (192 total samples).