Partial Reduction and Two-Step Modification of Proteins for Identification of Disulfide Bonds

Schnaible, Volker; Wefing, S.; Bücker, Anne; Wolf-Kümmeth, Sybille; Hoffmann, Daniel

doi:10.1021/ac015719j

Cited by 20 publications

(36 citation statements)

References 32 publications

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“…Hence mass spectrometry in conjunction with differential alkylation has been commonly used to differentiate between various cysteine species in a given protein (Schniable et al, 2002;Schilling et al, 2004;Ueberheide et al, 2004;Lu et al, 2005). The biotin switch assay is a differential modification method especially developed for detecting Snitrosylated proteins ( Jaffrey et al, 2001).…”

Section: Determination Of S-nitrosylation Sites In Enos Treated With mentioning

confidence: 99%

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

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S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that Snitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975=990, and 1047=1049 as being susceptible to Snitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

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Section: Determination Of S-nitrosylation Sites In Enos Treated With mentioning

confidence: 99%

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

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“…In a variation of the partial reduction and alkylation method, nascent thiols of partially reduced proteins are cyanylated with the reagent 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) at pH 8, or with 1‐cyano‐4‐dimethylamino‐pyridinium tetrafluoroborate (CDAP) at pH 3 (Wu & Watson, ; Wu, Gage, & Watson, ). Both cyanylating reagents are able to react with TCEP quantitatively, which is useful to terminate the partial reduction reaction (Schnaible, Wefing, Bucker, Wolf‐Kummeth, & Hoffmann, ). Cyanylation with CDAP is preferred, as the reaction can be carried out at low pH where disulfide scrambling is minimized.…”

Section: Commentarymentioning

confidence: 99%

“…In addition to cleavage by reducing agents, disulfide bonds can undergo spontaneous dissociation when analyzed by MALDI MS (Patterson & Katta, ) or in source reduction in ESI instruments (Cramer et al., ; Li et al., ). This observation has led to MS being used to directly analyze HPLC fractions of digested proteins for disulfide‐containing peptides without the need for chemical reduction (Qin & Chait, ; Schnaible et al., ,b). In addition to producing the constituent peptides, fragmentation of the disulfide bonds by MALDI or ESI in‐source decay also produces various combinations of disulfide‐linked peptides which can be used for successful determination of disulfide linkages.…”

Section: Commentarymentioning

confidence: 99%

“…To overcome this problem, free cysteines can be alkylated prior to protein denaturation (Yen et al., ; Zhang, Marzilli, Rouse, & Czupryn, ). For this purpose, N ‐ethylmaleimide (NEM) or other maleimide derivatives are useful alkylating agents that have been used successfully at acidic pH, from 3.0 to 6.5 (Bures et al., ; Schnaible et al., ; van den Hooven et al., ; Yen et al., ; Zhang et al., ). Alternatively, the free cysteines can be cyanylated with CDAP, which works well at pH 3.0 where disulfide scrambling is minimized (Wu & Watson, ); however, cyanylated cysteines can undergo undesired cleavage at basic pH.…”

Section: Commentarymentioning

confidence: 99%

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Experimental Assignment of Disulfide‐Bonds in Purified Proteins

Tang

Speicher

2019

CP Protein Science

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The formation of disulfide bonds in proteins is an important post‐translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified ‐SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three‐dimensional structure and contribute to the understanding of structural‐functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide‐containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.

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“…The difficult part of cyanylation is that the fragments after cleavage may not be detected due to either the variation of reaction efficiency at different locations on peptides or the chemical background interference during MALDI analysis (Wu et al 2004). The method was later modified by adding a second alkylation step with NEM and using enzyme digestion instead of chemical cleavage prior to detection with MALDI-ISD/PSD (Schnaible et al 2002a).…”

Section: Partial Reduction and Differential Alkylationmentioning

confidence: 99%

Mass spectrometry-based strategies for protein disulfide bond identification

Tsai

Chen²,

Huang

2013

Reviews in Analytical Chemistry

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Abstract:The formation of disulfide bonds is critical for stabilizing protein structures and maintaining protein functions. It is important to understand the linkages between multiple cysteine residues within a protein. In this review, the analytical approaches using mass spectrometry (MS) for disulfide linkage assignment are classified and discussed. Enzymatic digestion under appropriate conditions followed by various MS detection strategies remains the primary method for cysteine linkage analysis. In-source decay (ISD) and electron transfer dissociation (ETD) have been used to generate significant peptide signals that indicate the identities of peptides involved in disulfide bonds. In addition, chemical labeling and software algorithms were also developed to facilitate the automation of disulfide bond analysis. For proteins with complex disulfide structure, methods involving partial reduction coupled with differential alkylation were demonstrated to be useful. In the past two decades, MS has become one of the most valuable tools for protein disulfide bond analysis. It provides irreplaceable information including the peptide backbone sequences as well as the cysteine connection pattern when coupling with appropriate sample preparations. The related approaches with their unique features can be applied for different aims such as structural characterization or functional studies of proteins.

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Partial Reduction and Two-Step Modification of Proteins for Identification of Disulfide Bonds

Cited by 20 publications

References 32 publications

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Experimental Assignment of Disulfide‐Bonds in Purified Proteins

Mass spectrometry-based strategies for protein disulfide bond identification

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