Counting sulfhydryls and disulfide bonds in peptides and proteins using mercurial ions as an MS-tag

Abstract

Organic mercurial compounds are the most specific and sensitive reagents for reaction with the sulfhydryl groups (OSHs) in peptides and proteins because of the strong mercury-sulfur affinity. Using the monofunctional organic mercury ion RHg ϩ as a mass spectrometry (MS)-tag has the advantages of reacting with one sulfhydryl group, offering definite mass shift, and especially stable and characteristic nonradioactive isotopic distribution. Mass spectrometric analysis of derivatized sulfhydryls in peptides/proteins is thus an alternative for precisely counting the number of sulfhydryl groups and disulfide bonds (OSOSO). Here the tags used include monomethylmercury chloride, monoethylmercury chloride, and 4-(hydroxymercuri) benzoic acid. The feasibility of this strategy is demonstrated using HPLC/ESI-MS to count OSHs and OSOSO in model peptides/proteins, i.e., glutathione, phytochelatins, lysozyme and ␤-lactoglobulin, which contain increasing OSHs and various OSOSO linkages. (J Am

Discovering Mercury Protein Modifications in Whole Proteomes Using Natural Isotope Distributions Observed in Liquid Chromatography-Tandem Mass Spectrometry

“…8 10.1074/mcp.M110.004853-7 unmodified peptides or sensitivity for the modified peptides. The specificity and sensitivity of ⌬Fit on these peptides from GAPDH, and their relation to the choice of threshold, are shown by the red line in Fig.…”

“…8 10.1074/mcp.M110.004853-11 the elution profiles of individual isotope peaks to correlate before joining them into a multi-scan feature. This method should exclude those that have significantly different elutions but will not exclude those with similar elutions (see supplemental Fig.…”

“…Hg is unusual among metals in making nearly covalent bonds with sulfur that survive the LC-MS/MS process (7,8), and identifying its most vulnerable protein targets in intact cells is of interest in understanding Hg's pleiotropic toxicity. However, there are no simple preparative procedures with which to extract mercurated proteins from a cell lysate.…”

Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

“…Characterizing cysteine content by mass spectrometry has also become a popular option as a consequence of the development of wellestablished bottom-up proteomics approaches [5][6][7] often in combination with various clever cysteine-selective derivatization methods [8]. MS strategies for determination of cysteines have been based on utilization of mass tags [9] or isotopic labels [10,11], differential monitoring of ESI mass spectra after cysteine-selective reactions [12], proteolysis in isotopically heavy solvents [13], characterization by high resolution top down MS/MS [14], or utilization of selective ion/ion reactions [15,16], all of which have proven to be versatile methods for either qualitative or quantitative characterization of cysteine content of proteins and peptides. Electrochemical tagging reactions of cysteines have also been implemented via coupling an electrochemical cell in an on-line fashion to a mass spectrometer [17][18][19][20][21].…”

Se-Hg Dual-element Labeling Strategy for Selectively Recognizing Selenoprotein and Selenopeptide

“…In our previous works, quantifications of the selenoproteins and Se-containing species via determination of the endogenous Se-label with HPLC/ICP-MS and the model proteins (peptides) through determination of the exogenous Hg-tag, which was chemically labeled through the specific interaction between the -SHs in the molecules and monofunctional organic mercurials, were accomplished [14][15][16][17][18][19] . Se and Hg are an interesting pair, and one typical example of the best known biological antagonism interactions existing universally in organisms [20][21][22] .…”