Multi-protease Approach for the Improved Identification and Molecular Characterization of Small Proteins and Short Open Reading Frame-Encoded Peptides

• DOI: 10.1021/acs.jproteome.1c00115
• Publication Date: 2021-03-24T18:27:33Z
• Authors: Philipp T. Kaulich; Liam Cassidy; Jürgen Bartel; Ruth A. Schmitz; Andreas Tholey
• Keywords: Proteome; Bottom-up proteomics

The identification of proteins below approximately 70-100 amino acids in bottom-up proteomics is still a challenging task due to the limited number peptides generated by proteolytic digestion. This includes short open reading frame-encoded (SEPs), which are subset small that were not previously annotated or alternatively encoded. Here, we systematically investigated use multiple proteases (trypsin, chymotrypsin, LysC, LysargiNase, and GluC) GeLC-MS/MS analysis improve sequence coverage identified for proteins, with focus on SEPs, archaeon Methanosarcina mazei. Combining data all proteases, 63 additional 28 SEPs at least two unique peptides, while only 55 22 SEP could be using trypsin only. For 27 12 complete was achieved. Moreover, five incorrectly predicted translation start points potential vivo processing identified, confirming previous top-down study this organism. results show clearly multi-protease approach allows molecular characterization SEPs. LC-MS data: ProteomeXchange PXD023921.