Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues

Abstract

Small open reading frame encoded peptides (SEPs), also called microproteins, play a vital role in biological processes. Plenty of their open reading frames are located within the non-coding RNA (ncRNA) range. Recent research has demonstrated that ncRNA-encoded polypeptides have essential functions and exist ubiquitously in various tissues. To better understand the role of microproteins, especially ncRNA-encoded proteins, expressed in different tissues, we profiled the proteomic characterization of five mouse tissues by mass spectrometry, including bottom-up, top-down, and de novo sequencing strategies. Bottom-up and top-down with database-dependent searches identified 811 microproteins in the OpenProt database. De novo sequencing identified 290 microproteins, including 12 ncRNA-encoded microproteins that were not found in current databases. In this study, we discovered 1,074 microproteins in total, including 270 ncRNA-encoded microproteins. From the annotation of these microproteins, we found that the brain contains the largest number of neuropeptides, while the spleen contains the most immunoassociated microproteins. This suggests that microproteins in different tissues have tissue-specific functions. These unannotated ncRNA-coded microproteins have predicted domains, such as the macrophage migration inhibitory factor domain and the Prefoldin domain. These results expand the mouse proteome and provide insight into the molecular biology of mouse tissues.

Proteomics‐driven identification of short open reading frame‐encoded peptides

“…Due to their low abundance and PSM, the spectra counting is not suitable for quantification. The peak intensity will be a more reliable label-free quantification strategy [38,50]. The absence of the MS2 spectrum may be caused by miss fragmentation, so the precursor should be checked to make sure the SEPs do not exist.…”

Microproteins: Overlooked regulators of physiology and disease

“…Some of the most commonly used methods for microprotein enrichment include solid-phase extraction, urea-tricine polyacrylamide gel electrophoresis (PAGE), acid precipitation, and molecular weight cut-off (MWCO) filtration. 34 , 36 These methods have resulted in the identification of several microproteins, 37 , 38 , 39 , 40 and when combined with sequencing-based or bioinformatic approaches, they may be the most robust approach for high confidence microprotein discovery and identification. 34 This was recently highlighted with the implementation of comprehensive proteogenomic approaches combining peptidomics and RNA sequencing to filter out known peptides and exclusively identify new microproteins.…”

The Emerging Role of uORF-Encoded uPeptides and HLA uLigands in Cellular and Tumor Biology

“…Recent developments in LC-MS workflow, such as improvements in peptide enrichment techniques, have allowed for the identification of 762 non-canonical ORFs from lncRNAs in human and mouse tissues [ 9 ]. The combination of different MS-based strategies, including de novo sequencing strategies, led to the discovery of 1074 micropeptides from murine liver, brain, spleen, kidney, and heart [ 48 ].…”