Multi-protease approach for the improved identification and molecular characterization of small proteins and short open reading frame-encoded peptides

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

INSTRUMENT(S): Q Exactive

ORGANISM(S): Methanosarcina Mazei Go1

TISSUE(S): Cell Culture

SUBMITTER: Andreas Tholey  

LAB HEAD: Andreas Tholey

PROVIDER: PXD023921 | Pride | 2021-04-01

REPOSITORIES: Pride