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Reviews

Current strategies and findings in clinically relevant post-translational modification-specific proteomics

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Abstract

Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research.

Financial & competing interests disclosure

The authors were supported by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen and the Deutsche Forschungsgemeinschaft (DFG ZA 639/4-1). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues
  • LC-mass spectrometry (MS)–based post-translational modification (PTM) research enables the quantification of hundreds to thousands of PTMs in a single experiment. However, the link between aberrant PTM patterns and disease or during drug treatment is still poorly understood and is one of the main goals of LC-MS–based PTM research.

  • Typical workflows for analyzing PTMs are conducted on the peptide level, which allows specific enrichment of modified peptides from the bulk of non-modified peptides, prior to LC-MS analysis. Thus, the low abundance of PTMs can be overcome in the light of the dynamic range of mammalian cells.

  • State-of-the-art workflows enable quantitative analysis from less than 100 µg of cell lysate. Whereas in cell culture-based experiments sufficient sample is readily available, clinical applications with patient samples require highest sensitivity and robustness.

  • In clinical proteomics, label-free quantification, super-SILAC and chemical labels can be employed for large-scale quantitative discovery. Modified peptides that may serve as biomarkers can be validated with larger cohorts using targeted MS methods such as multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM). These can also be designed for diagnostic purposes.

  • Aberrant protein phosphorylation has been connected to a wide variety of diseases. Nowadays, various sensitive phosphopeptide enrichment methods such as Ti4+-IMAC, TiO2-MOAC and electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC) are established. Particularly, ERLIC is a simple and sensitive method for enrichment and simultaneous fractionation of both singly and multiphosphorylated peptides.

  • Glycosylation is an extremely heterogeneous group of PTMs that is characterized by the attachment of complex carbohydrate structures to proteins. Various cancer biomarkers are glycoproteins and several studies indicate that both glycosylation levels and glycan structures are potential biomarkers. Although dedicated enrichment methods are available for glycopeptides, site-specific analysis of glycosylation structure is challenging. Therefore, most studies focus on monitoring glycosylation sites rather than resolving glycan structures.

  • Proteolytic processing is a ubiquitous, non-reversible PTM. Generated ‘neo’-N-termini can be enriched using methods such as combined fractional diagonal chromatography (COFRADIC), terminal amine isotopic labeling of substrates (TAILS) and charge-based fractional diagonal chromatography (ChaFRADIC). These enable the identification of protease substrates as well as their distinct cleavage sites. Charge-based fractional diagonal chromatography has been demonstrated to be a sensitive and straightforward method that might be applicable for clinical proteomics. The analysis of ‘neo’-C-termini, however, is still more challenging.

  • The role of PTM crosstalk is still not well understood. The current limitations in performing real large-scale PTM crosstalk analyses render the development of adequate clinical biomarker assays that target peptides with different modifications extremely challenging and rather unlikely for the near future.

Notes