Protein post-translational modifications (PTMs) play important roles in biological systems and help shed light on important physiological and pathological processes. Still, most PTMs remain underinvestigated. Protein O-sulfation (sometimes also referred to as O-sulfonation) is such a PTM (tyrosine-sulfation is the most studied in the literature, but serine- and threonine O-sulfation have also been reported to exist), due to lack of analytical methods. Two are the main challenges associated with high-throughput shotgun sulfoproteomics: sample preparation and sulfopeptide identification by mass spectrometry. Detection and identification of sulfation by mass spectrometry, taking into consideration both sulfation site localization and selectivity to distinguish it from the nearly isobaric phosphorylation, is the second main challenge in sulfoproteomics.
The main goal of this project proposal is to provide a large scale workflow for identification of sulfoproteins by shotgun proteomics. It is organized into two sub-goals: the first one is to extend the analytical method developed on sulfopeptide standards to a complex real-world sample and optimize it for recovery of unknown sulfopeptides first obtained by tryptic digestion of a standard sulfoprotein (fibrinogen), then to serum and cell protein digests. The whole analytical workflow, comprising sulfopeptide enrichment and nanoHPLC MS/MS with high resolution, would fill a gap which currently exists for the characterization of this neglected PTM, for which no large scale analytical method exists for complex proteomes, despite the potential interests for the biological activity.
Despite tyrosine sulfation being described some fifty years ago, still the modification is not extensively investigated. However, the interest is blooming in the field, due to the important biological significance of this PTM and the recent description of new strategies for detection and isolation of sulfopeptides. Still, the research in this field is not as advanced as for other modifications, especially phosphorylation, and there is need for enrichment strategies of sulfopeptides, as they are tools which, together with MS analysis, would provide information on protein sulfation by methods which are comprehensive, faster and more suitable for large scale analysis than radioactive labeling.
In this sense, in the previous work[1] we demonstrated that sulfopeptides are compatible with a typical proteomics sample preparation, with tryptic digestion and are also stable during protein dephosphorylation. Sulfopeptide recovery and selectivity were comparable to the ones obtained for phosphopeptide analysis, which is promising in this field. In fact, the number of works describing enrichment strategies for sulfopeptides is very limited in the literature, and the extension of our method to complex tryptic digests including native sulfoproteins would improve the current analytical methods available for the study of sulfation. Methods currently described in the literature are complex, with peptide derivatization protocols[2-4], which are also not compatible with automated bioinformatics peptide identification, or they address intact peptides but are limited at the recovery of standard sulfopeptides or sulfoproteins[5,6]. There are only two exceptions, i.e. Ga-immobilized metal ion affinity chromatography (IMAC)[7], which was applied to frog skin secretions and which had a very low selectivity, and antibodies[8], which were applied to crude tissue samples but are very expensive. Our method would overcome both drawbacks. Fe-IMAC showed a good selectivity and is far cheaper than antibodies[1]. As the method was developed on standard non-tryptic peptides, the extension to tryptic peptides (standards first, then obtained from native sulfoproteins) would provide a powerful enrichment system compatible with state of the art proteomics analysis.
At the same time, detection by MS still needs more investigation addressing sulfopeptide ionization in the positive ionization mode, specifically addressing tryptic sulfopeptides, as this enzyme is commonly employed to prepare protein digests used in proteomics analysis. While the study of sulfopeptides MS analysis has been addressed to find a way to differentiate them from phosphopeptides, this is usually achieved on non-tryptic standards[9]. Moreover, as far as MS analysis of sulfopeptides is concerned, there are some common preconceptions as such analytes would be very unstable and lose the sulfate directly in-source, which resulted in impossibility in even presume a possible sulfation on a peptide. This concept is, however, not absolute. As far as the literature is concerned, to the best of our knowledge, the in-source instability is rarely reported and we did not observe it for non-tryptic standards. Therefore, the optimization of the MS condition for sensitive detection of tryptic sulfopeptides would provide additional knowledge in this field. There is need to understand if the conventional positive ionization mode is compatible with tryptic sulfopeptides or if the negative ionization mode is necessary for detection of these analytes.
Together, enrichment and MS detection would provide a new analytical method for the identification of sulfopeptides by shotgun proteomics. Such method would fill a gap in analytical methods currently available for shotgun proteomics analysis of PTMs and would provide a tool to shed light on the biological function of sulfation in living beings.
1-Capriotti A. L. et al., Anal. Chem. 2020, 92, 7964-7971
2-Yu, Y. et al., Nat. Methods 2007, 4, 583-588
3-Kim, J. S. et al., J. Am. Soc. Mass Spectrom. 2011, 22, 1916-1925
4- Robinson, M. R. et al., Anal. Chem. 2016, 88, 11037-11045
5-Shinde, S. et al., Angew. Chemie - Int. Ed. 2012, 124, 8451-8454
6-Amano, Y.et al., Anal. Biochem. 2005, 346, 124-131
7-Balderrama, G. D. et al., Rapid Commun. Mass Spectrom. 2011, 25, 1017-1027
8- Hoffhines, A. J. et al., J. Biol. Chem. 2006, 281 (49), 37877-37887
9-Guangming, G. et al., J. Am. Soc. Mass Spectrom. 2018, 29, 455-426