Bottom‐up and top‐down proteomic approaches for the identification, characterization, and quantification of the low molecular weight proteome with focus on short open reading frame‐encoded peptides

Abstract

The recent discovery of alternative open reading frames creates a need for suitable analytical approaches to verify their translation and to characterize the corresponding gene products at the molecular level. As the analysis of small proteins within a background proteome by means of classical bottom-up proteomics is challenging, method development for the analysis of small open reading frame encoded peptides (SEPs) have become a focal point for research. Here, we highlight bottom-up and top-down proteomics approaches established for the analysis of SEPs in both pro-and eukaryotes. Major steps of analysis, including sample preparation and (small) proteome isolation, separation and mass spectrometry, data interpretation and quality control, quantification, the analysis of post-translational modifications, and exploration of functional aspects of the SEPs by means of proteomics technologies are described. These methods do not exclusively cover the analytics of SEPs but simultaneously include the low molecular weight proteome, and moreover, can also be used for the proteome-wide analysis of proteolytic processing events.

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

“…In past decades, the most widely employed MS data acquisition mode is undoubtedly data dependence acquisition (DDA), in which MS selects top N abundant precursor ions for fragmentation and produces MS/MS spectra. Most MS identifications of SEPs depend on DDA [28, 96, 119‐47]. Ma et al.…”

“…In this way, additional evidence should be provided for further verification of SEPs. Comparing the retention time and MS/MS spectra of the detected endogenous peptides with that of synthesized ones is often needed [96,142]. Other than verification on the proteome level, additional evidence can also be obtained by ribosome profiling [64] or RT-PCR [143].…”

In Depth Exploration of the Alternative Proteome of Drosophila melanogaster

“…Although ribosome profiling approaches clearly established the binding of ribosome to alternative ORFs, it is in fact difficult to deduce the productive translation of the ORFs, resulting in the expression of stable proteins (Patraquim et al, 2020). Mass spectrometry is generally the method of choice for large scale identification of proteins and peptides (Cassidy et al, 2021). MS data demonstrating the genome-wide expression of AltProts are still limited to few species (Fabre et al, 2021).…”

In depth exploration of the alternative proteome of Drosophila melanogaster

“…Although ribosome profiling approaches clearly established the binding of ribosome to alternative ORFs, it is in fact difficult to deduce the productive translation of the ORFs, resulting in the expression of stable proteins (Patraquim et al, 2020). Mass spectrometry is generally the method of choice for large scale identification of proteins and peptides (Cassidy et al, 2021). MS data demonstrating the genome wide expression of AltProts is still limited to few species (Fabre et al, 2021).…”