Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Biological Significance and Analysis of Tyrosine Sulfation
Eva Klement1, Eva Hunyadi-Gulyas1 and Katalin F. Medzihradszky1,2
Overview of Protein Sulfation
Classical analysis of proteins and peptides using a wide array of enzymatic digestions, simple sample fractionation methods, and organic chemistry reactions yielded quite sophisticated results: not only the amino acid sequences were deciphered but also a wide variety of post-translational modifications (PTMs) were identified. O-sulfation of tyrosine residues is one of these PTMs. (Note: Sometimes, it is mistakenly referred to as sulfonation although that indicates a carbon-sulfur linkage.) The presence of sulfated Tyr in different proteins and polypeptides has been reported from the 1950s [1-3]. It has been demonstrated that the modification can be removed by acid hydrolysis  and was later shown that arylsulfatases recognize and remove the sulfate linked to Tyr residues . With the introduction of [35S]-labeling, gradually it became obvious that the modification is linked to the secretory pathway and the deri- vatization must occur in the trans-Golgi network [5-8]. The first proteomics- style Tyr-sulfation experiment was also performed using radioactive labeling and classical analytical techniques resulting in the detection of fibrinogen, a-fetoprotein, and fibronectin with sulfated Tyr residues .
Approximately 30 years after the first reports on Tyr sulfation, the two protein tyrosine sulfotransferase (PTST) enzymes performing the modification, using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfo-donor, were identified [10, 11]. Tyr sulfotransferases have also been described in plants [12, 13] and, recently, in a Gram-negative bacterium .
Numerous articles attest to the biological importance of Tyr sulfation: it has been shown that the sulfation of Factor V and Factor VIII is important for
Analysis of Protein Post-Translational Modifications by Mass Spectrometry, First Edition. Edited by John R. Griffiths and Richard D. Unwin.
© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.
optimal activity and thrombin cleavage [15-17]; sulfation has been implicated in protein-protein interaction [18-21]; and modified Tyr residues play important role(s) in ligand-receptor interactions [22-25]. Tyrosine sulfation modulates the activity of cytokine receptors , facilitates HIV entry in the cell [27, 28], and participates in b oth autoimmune processes and inflammatory responses .
Besides the modification of Tyr residues, the sulfation of Ser, Thr, and Cys side chains has also been reported [30-32]. The origin and biological role of these modifications will not be discussed in this chapter; however, it should be noted that they display the same chromatographic and mass spectrometry behavior as their Tyr-modified counterparts.
In addition, we would like to draw attention to the fact that the sulfation of the aforementioned residues, including Tyr, can be achieved by exposing the proteins/digests to certain chemicals, for example, thiosulfates, during sample preparation. Artifactual peptide sulfation during silver staining has been reported .
While working on this manuscript we extracted all human UniProt entries that were assigned as sulfated. The search yielded only 50 entries. All assignments based on “sequence analysis" “similarity” or “curated" were removed. Supporting references were scrutinized, and as a result reliable human sulfation data included in the UniProt database as of May 2015 are listed in Table 9.1.
More than half of these sites were identified by mutation and radioactive labeling. Mass spectrometry analyses were performed on purified proteins, and frequently a combination of different tools had to be applied. Perhaps the best example for a dedicated study is the sulfation analysis of extracellular leucine-rich repeat proteins when different proteolytic enzymes, MALDI and ESI, positive and negative ionization were used .
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