Common errors in mass spectrometry‐based analysis of post‐translational modifications

Abstract

Mass spectrometry (MS) is a powerful tool to analyze complex mixtures of proteins in a high-throughput fashion. Proteome analysis has already become a routine task in biomedical research with the emergence of proteomics core facilities in most research institutions. Pst-translational modifications (PTMs) represent a mechanism by which complex biological processes are orchestrated dynamically at the systems level. MS is rapidly becoming popular to discover new modifications and novel sites of known PTMs, revolutionizing the current understanding of diverse signaling pathways and biological processes. However, MS-based analysis of PTMs has its own caveats and pitfalls that can lead to erroneous conclusions. Here, we review the most common errors in MS-based PTM analyses with the goal of adopting strategies that maximize correct interpretation in the context of biological questions that are being addressed. Finally, we provide suggestions that should help mass spectrometrists, bioinformaticians and biologists to perform and interpret MS-based PTM analyses more accurately.

Identification and Mapping of Phosphorylated Isoforms of the Major Storage Protein of Potato Based on Two- Dimensional Electrophoresis

“…The finding of differentially phosphorylated isoforms is particularly relevant because increase (or decrease) in phosphorylation status without a parallel change in the amount of protein has been considered to be a useful indicator for a specific functional change [39,40,45]. In this regard, systematic follow-up studies on VSPs will be needed to assess whether their degradation takes place through a phosphorylation-dependent regulatory mechanism, as it occurs in common bean during dry-to-germinating seed transition [10].…”

“…Elucidating whether changes in abundance of protein phosphorylation reflect either changes in phosphorylation status or changes in the abundance of the protein itself is a major challenge in the interpretation of quantitative phosphoproteomics studies [39,40]. Thus, phosphopeptide enrichment methods prior to high-resolution MS permit the identification of low-abundance phosphoproteins but prevent joint quantitation of phosphorylation status and abundance of proteins [39].…”

“…In addition, patatin is a family of immunologically indistinguishable isoforms with similar structural properties and thermal conformational stability [21,22]. Overall, extensive heterogeneity in molecular mass (40)(41)(42)(43)(44)(45) and isoelectric point (4.5-5.2) seems to be the most salient differential molecular features among isoforms [21][22][23][24][25].…”

An Attempt to find Changes of Post-Translational Modifications on Serum Proteins between Embryonic 15.5 Fetuses and Newborn Rats

“…However, PTM analysis using MS still has a number of challenges [25] to surmount the most common errors that can occur during MS-based analysis of PTMs and lead to incorrect data interpretation and erroneous conclusions [26,27]. More reliable PTMs may be identified based on the enrichment of modified proteins or peptides in the future.…”

Literature Survey on Framework for Generating MS-MS Spectra for MS Spectrometry Data

“…Other problems can be solved through improvements in current bioinformatics pipelines such as integration of different PTM databases for better and more complete annotation. [2] Christopher J. Mitchell, Min-Sik Kim, Chan Hyun Na, and Akhilesh Pandey proposed paper where Quantitative mass spectrometry data necessitates an analytical pipeline that captures the accuracy and comprehensiveness of the experiments. Currently, data analysis is often coupled to specific software packages, which restricts the analysis to a given workflow and precludes a more thorough characterization of the data by other complementary tools.…”