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Abstract
Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.
Highlights
Because of their labile nature, SNO-modifications can be difficult to study with traditional biochemical techniques
Common critiques include the use of ascorbate as the specific reducing agent which is suspected of reducing disulfide bonds or other oxidiative modifications and the lack of a permanent label at the modified cysteine residue that is detectable by MS analysis which can lead to ambiguity in site identifications
Comparison of cysteine reactive tandem mass tag (cysTMT) Reagent to Biotin-HPDP in Biotin Switch Assay—The suitability of the cysTMT reagent for SNO detection was initially assessed by comparison with the widely used biotin-HPDP
Summary
Because of their labile nature, SNO-modifications can be difficult to study with traditional biochemical techniques. CysTMT6 is a thiol reactive version of tandem mass tag that has been established for multiplex mass spectrometry analysis [23] This new reagent fulfills the requirements for a biotin switch label and offers some distinct advantages, including a permanent mass tag and the fragmentation of up to 6 isotopically balanced reporter ions between 126 –131 Da permitting multiplex quantification. Using this technique we demonstrate specific detection of SNO-modified sites and quantify the response of individual cysteine residues to GSNO treatment by mapping the continuum of protein thiol-reactivity to SNO-modification
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