Sulfotyrosine peptide

For certain structural and functional studies, in particular NMR spectroscopy studies, rather large quantities of sulfotyrosine peptides in the 10-mg range are required. Due to the low tyrosine sulfation activities of soluble TPST enzymes for peptide substrates, enzymatic peptide sulfation at this scale requires quantities of TPST enzymes that are difficult and expensive to obtain from transfected HEK293 cells. 

While successful separation of sulfotyrosine peptides has been described with standard RP-HPLC solvent systems containing 0.1% trifluoroacetic acid, we found that separation at pH 6.5 using 20 mM ammonium acetate as a buffer gave best results. Under these conditions, the potential loss of the sulfotyrosine modification caused by acid-catalyzed hydrolysis is minimized (Seibert Sakmar, 2008 Stone Payne, 2015). Furthermore, at pH 6.5... 

Analysis of sulfotyrosine peptides and locahzation of sulfotyrosine positions in the presence of multiple potential sulfation sites can be a challenging task, in particular, if multiple sulfotyrosines are present in a single peptide chain, which is the case with most N-terminal chemokine receptor peptides. While mass spectrometry analysis of protein phosphorylation on a proteomics scale is well established, this is not the case for protein tyrosine sulfation. Due to the inherent lability of the sulfotyrosine sulfoester bond, partial or complete loss of the sulfotyrosine modification is generally observed as a neutral loss of SO3 (AMr = -80 Da) under standard mass spectrometry conditions. In particular, irrespective of the desorption/ionization method employed, positive... 

In our experience, an ultra-thin layer sample preparation method (Cadene Chait, 2000) using ammonium acetate as an additive gave best results for MALDI-TOF MS of sulfotyrosine peptides (Seibert et al., 2002, 2008). 

Under optimized measurement conditions, with low laser power in linear negative ion mode, complete sulfate retention was routinely observed in the most abundant ion. However, some loss of sulfate was generally observed, giving rise to additional signals that could be mistaken for contaminating peptide species. Hence, it is crucial to use RP-HPLC-purified sulfotyrosine peptides for MALDI-TOF MS analysis. For a more detailed discussion of the characteristic loss-of-sulfate peak patterns observed in MALDI-TOF mass spectra of sulfotyrosine peptides, we refer to the following references Seibert et al. (2002, 2008) and Seibert and Sakmar (2008). 

Alternatively, sulfotyrosine peptides can be analyzed using ESI MS in negative ion mode. In our experience, using optimized measurement conditions, virtually no loss of sulfate was observed, and therefore, interpretation of negative ion mode ESI mass spectra of sulfotyrosine... 

Desalt sulfotyrosine peptide samples by binding to RP-C18 ZipTips according to the manufacturer s protocol. After washing with HPLC-grade water, elute peptides with 70% acetonitrile. 

Cormier EG, Persuh M, Thompson DA, et al. Specific interaction of CCR5 amino-terminal domain peptides containing sulfotyrosines with HIV-1 envelope glycoprotein gpl20. Proc Natl Acad Sci U S A 2000 97(ll) 5762-5767. 

Figure 21 Solid-phase synthesis of a sulfotyrosine-containing peptide using the 2-chlorotrityl resin.

With up to 1% of eukaryotic proteins potentially containing sulfotyrosine residues, sulfation of tyrosines is a common posttranslational modification whose biological impact has only just started to be elucidated (Ludeman Stone, 2014 Moore, 2003 Seibert et al., 2008). In humans and most mammals, there are two isoforms of the enzyme responsible for tyrosine sulfation these enzymes are tyrosylprotein sulfotransferase 1 and 2 (TPST-1 and TPST-2) (Moore, 2003 Seibert Sakmar, 2008). TPST-1 and TPST-2 are located in the irans-Golgi network and this limits tyrosine sulfation to secreted or membrane proteins (Moore, 2003 Seibert Sakmar, 2008). Both enzymes utilize the cosubstrate PAPS, or 3Gphosphoadenosine-5 -phosphosulfate, as the sulfate donor to catalyze the sulfation of a tyrosine s phenolic hydroxyl in a substrate protein or peptide as seen in Fig. 1 (Moore, 2003 Seibert Sakmar, 2008). 

Here, we describe approaches for utilizing recombinant TPST-1 and TPST-2 to enzymatically sulfate N-terminal chemokine receptor peptides and for the characterization of these sulfopeptides. For a more expansive introduction to TPST enzymes and proteins containing sulfotyrosine post-translational modifications, see the following references Ludeman and Stone (2014), Moore (2003, 2009), Seibert and Sakmar (2008), and Stone, Chuang, Hou, Shoham, and Zhu (2009). 

Determine the number of sulfotyrosine residues in the uncleaved (RP-HPLC-purified) peptide by mass spectrometry. If the sulfation sites are ambiguous, proceed with step 2. 

CCR5 A2-17 (Liu et al., 2014), neither study investigated sY3 in the context of an N-terminal CCR5 peptide. As one would expect, knowledge regarding the order of tyrosine sulfation by TPSTs, which in some instances appears to correlate with functional importance, along with the ability to control the location and pattern of sulfotyrosines chemical synthesis will provide the broadest picture of functional importance for individual sulfotyrosine residues. 

The classical view of sulfotyrosine is that it is a posttranslational modification that increases affinity between binding partners. We have shown this to be the case for CXCL12 and enzymaticaUy produced CXCR4 N-terminal peptides with affinity increasing with increasing sulfation... 

Cormier, E. G., Persuh, M., Thompson, D. A., Lin, S. W., Sakmar, T. P., Olson, W. C., et al. (2000). Specific interaction ofCCRS amino-terminal domain peptides containing sulfotyrosines with HIV-1 envelope glycoprotein gpl20. Proceedings of the National Academy of Sciences of the United States of America, 97(11), 5762—5767. 

Seibert, C., Sakmar, T. P. (2008). Toward a framework for sulfoproteomics Synthesis and characterization of sulfotyrosine-containing peptides. Biopolymers, 90(3), 459—477. 

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