Shot-gun proteomics: why thousands of unidentified signals matter

Abstract

Mass spectrometry-based proteomics has become a constitutional part of the multi-omics toolbox in yeast research, advancing fundamental knowledge of molecular processes and guiding decisions in strain and product developmental pipelines. Nevertheless, post-translational protein modifications (PTMs) continue to challenge the field of proteomics. PTMs are not directly encoded in the genome; therefore, they require a sensitive analysis of the proteome itself. In yeast, the relevance of post-translational regulators has already been established, such as for phosphorylation, which can directly affect the reaction rates of metabolic enzymes. Whereas, the selective analysis of single modifications has become a broadly employed technique, the sensitive analysis of a comprehensive set of modifications still remains a challenge. At the same time, a large number of fragmentation spectra in a typical shot-gun proteomics experiment remain unidentified. It has been estimated that a good proportion of those unidentified spectra originates from unexpected modifications or natural peptide variants. In this review, recent advancements in microbial proteomics for unrestricted protein modification discovery are reviewed, and recent research integrating this additional layer of information to elucidate protein interaction and regulation in yeast is briefly discussed.

Proteome dynamics during transition from exponential to stationary phase under aerobic and anaerobic conditions in yeast

“…However, as no or minimal differences were observed in the other components of V-ATPase between the MG and control strains, the molecular mechanism leading to the slightly higher abundance of Fba1 in the MG strain remains unclear. Many factors can alter the functionality of proteins, including post-translational modifications, protein localization or interactions with other proteins or biomolecules [80, 81]. A recent study has suggested that phosphorylation regulates the activity of many glycolytic enzymes [7].…”

Metaproteomics to understand how microbiota function: The crystal ball predicts a promising future

“…Nevertheless, the number remains very large compared to those resulting from proteomics analysis performed on human cell lines, where at best less than 1% of that number is managed in the initial extract. Post‐translational modifications of proteins can complexify the picture further through the introduction of multiple proteoforms (den Ridder et al, 2020 ). The resulting peptide space is illustrated in Figure 2 .…”

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“…Moreover, tools for data mining and interpretation in proteomics may be more complex due to the great variety of signals, such as the possible number of targets for protein modifications and changes in interactions or interactors [217], the variety of analytical approaches [218,219] and of bioinformatics pipelines [220,221], and the associated statistical analyses [222,223], particularly in relation to peptide assembly and subsequent proteome reconstruction. These aspects are crucial to achieve informative and reliable findings, and also taking into account that a great number of fragmentation spectra in a typical shot-gun proteomics experiment remain unidentified and that this large portion of unidentified spectra could originate from unexpected modifications or natural peptide variants [224]. Consequently, proteomics data sharing, starting from raw data to value added information and associated mining tools, would require large investment, infrastructure and coordination [216].…”