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

Abstract

Accumulating evidence has shown that a large number of short open reading frames (sORFs) also have the ability to encode proteins. The discovery of sORFs opens up a new research area, leading to the identification and functional study of sORF encoded peptides (SEPs) at the omics level. Besides bioinformatics prediction and ribosomal profiling, mass spectrometry (MS) has become a significant tool as it directly detects the sequence of SEPs. Though MS-based proteomics methods have proved to be effective for qualitative and quantitative analysis of SEPs, the detection of SEPs is still a great challenge due to their low abundance and short sequence. To illustrate the progress in method development, we described and discussed the main steps of largescale proteomics identification of SEPs, including SEP extraction and enrichment, MS detection, data processing and quality control, quantification, and function prediction and validation methods.

Identification of Microproteins in Saccharomyces cerevisiae under Different Stress Conditions

“…The function of SEPs can be validated by a genetic approach (knockdown and knockout) and determining the protein interaction by affinity purification mass spectrometry or proximity-labeling approach. 53 In summary, our results contribute to a deeper understanding of the S. cerevisiae proteome and provide supporting data for the functional characterization of SEPs responding to environmental stress. These SEPs might help understand the stress response and improve the fermentation usage of yeast.…”

“…We found that many SEPs carried mitochondrial transfer peptides, suggesting that these SEPs might be functionally located in the mitochondria. The function of SEPs can be validated by a genetic approach (knockdown and knockout) and determining the protein interaction by affinity purification mass spectrometry or proximity-labeling approach . In summary, our results contribute to a deeper understanding of the S.…”

Proteoforms expand the world of microproteins and short open reading frame-encoded peptides

“…It should be noted that TDP for the analysis of SEPs employs many of the strategies developed for peptidomic analyses. 44 , 55 Essentially both TDP and peptidomics target (small) intact proteins/peptides without the use of enzymatic digestion and the differentiation between small protein and large peptide is largely semantic.…”

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis

“…Therefore, sample preparation is a prerequisite for thorough analysis of PTMs proteins [12][13][14][15][16].…”

“…In addition, owing to the weaker ionization efficiency of PTMs proteins than non‐PTMs proteins, the analysis and identification of samples is more difficult. Therefore, sample preparation is a prerequisite for thorough analysis of PTMs proteins [12–16].…”