Modification of cysteine residues for mass spectrometry-based proteomic analysis: facts and artifacts

Mass spectrometric proteomic analysis at the sample preparation stage involves the artificial reduction of disulfide bonds in proteins formed between cysteine residues. Such bonds, when preserved in their native state, complicate subsequent enzymatic hydrolysis and interpretation of the research results. To prevent the re-formation of the disulfide bonds, cysteine residues are protected by special groups, most often by alkylation. In this review, we consider the methods used to modify cysteine residues during sample preparation, as well as possible artifacts of this stage. Particularly, adverse reactions of the alkylating agents with other amino acid residues are described. The most common alkylating compound used to protect cysteine residues in mass spectrometric proteomic analysis is iodoacetamide. However, an analysis of the literature in this area indicates that this reagent causes more adverse reactions than other agents used, such as chloroacetamide and acrylamide. The latter can be recommended for wider use. In the review we also discuss the features of the cysteine residue modifications and their influence on the efficiency of the search for post-translational modifications and protein products of single nucleotide substitutions.

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Intra- and Intermolecular Disulfide Bonds of theGP2b Glycoprotein of Equine Arteritis Virus: Relevance forVirus Assembly andInfectivity

Journal of Virology ◽

10.1128/jvi.77.24.12996-13004.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 12996-13004 ◽

Cited By ~ 32

Author(s):

Roeland Wieringa ◽

Antoine A. F. de Vries ◽

Sabine M. Post ◽

Peter J. M. Rottier

Keyword(s):

Viral Entry ◽

Disulfide Bonds ◽

Rna Virus ◽

Cysteine Residue ◽

Envelope Proteins ◽

Equine Arteritis Virus ◽

Cysteine Residues ◽

Virus Infectivity ◽

Particle Assembly ◽

Positive Strand Rna

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP5 (previously named GL) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP2b (previously named GS), GP3, and GP4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP2b, GP3, and GP4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP2b protein in the assembly of the GP2b/GP3/GP4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP2b forms an intermolecular cystine bridge with one of the cysteines of the GP4 protein, while the cysteine residues at positions 48 and 137 of GP2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP4 protein is responsible for the covalent association of GP3 with the disulfide-linked GP2b/GP4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

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Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B

Journal of Virology ◽

10.1128/jvi.76.12.6073-6082.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 6073-6082 ◽

Cited By ~ 23

Author(s):

Matthew Lopper ◽

Teresa Compton

Keyword(s):

Human Cytomegalovirus ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Cysteine Residue ◽

Multiple Roles ◽

Glycoprotein B ◽

Cysteine Residues ◽

Amino Terminal ◽

High Degree ◽

Human Herpesviruses

ABSTRACT Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically determining structures even for highly conserved proteins.

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A novel approach for predicting protein S-glutathionylation

BMC Bioinformatics ◽

10.1186/s12859-020-03571-w ◽

2020 ◽

Vol 21 (S11) ◽

Cited By ~ 1

Author(s):

Anastasia A. Anashkina ◽

Yuri M. Poluektov ◽

Vladimir A. Dmitriev ◽

Eugene N. Kuznetsov ◽

Vladimir A. Mitkevich ◽

...

Keyword(s):

Disulfide Bonds ◽

Prediction Method ◽

Protein S ◽

Redox Status ◽

Cysteine Residue ◽

Cysteine Residues ◽

Amino Acid Residues ◽

Novel Approach ◽

Positive Dataset ◽

Unknown Structure

Abstract Background S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. Results One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. Conclusion Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.

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The Role of RodA-Conserved Cysteine Residues in the Aspergillus fumigatus Conidial Surface Organization

Journal of Fungi ◽

10.3390/jof6030151 ◽

2020 ◽

Vol 6 (3) ◽

pp. 151

Author(s):

Isabel Valsecchi ◽

Emmanuel Stephen-Victor ◽

Sarah Sze Wah Wong ◽

Anupama Karnam ◽

Margaret Sunde ◽

...

Keyword(s):

Parental Strain ◽

Disulfide Bonds ◽

Deletion Mutant ◽

Cysteine Residue ◽

Cysteine Residues ◽

Fungal Cell ◽

Differential Reactivity ◽

Alternative Approach ◽

Conidial Surface

Immune inertness of Aspergillusfumigatus conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the RODA deletion mutant (∆rodA). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆rodA conidia selectively bound to β-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆rodA was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆rodA. Ccrp-mutant conidia were less immunostimulatory compared to ∆rodA conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host–fungal interaction.

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Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽

10.1098/rsob.110010 ◽

2011 ◽

Vol 1 (3) ◽

pp. 110010 ◽

Cited By ~ 51

Author(s):

Clive Metcalfe ◽

Peter Cresswell ◽

Laura Ciaccia ◽

Benjamin Thomas ◽

A. Neil Barclay

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Cell Surface ◽

Disulfide Bonds ◽

Reduction Process ◽

Cysteine Residue ◽

Surface Proteins ◽

Protein Activity ◽

Functional Changes ◽

Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

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The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

Journal of Virology ◽

10.1128/jvi.01463-06 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5212-5224 ◽

Cited By ~ 32

Author(s):

Michael Mach ◽

Karolina Osinski ◽

Barbara Kropff ◽

Ursula Schloetzer-Schrehardt ◽

Magdalena Krzyzaniak ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Critical Role ◽

Point Mutations ◽

Cysteine Residue ◽

Cysteine Residues ◽

Type I ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal ◽

Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

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Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis

Biochemical Journal ◽

10.1042/bj20111170 ◽

2012 ◽

Vol 441 (3) ◽

pp. 823-839 ◽

Cited By ~ 16

Author(s):

Markus Lehrke ◽

Steffen Rump ◽

Torsten Heidenreich ◽

Josef Wissing ◽

Ralf R. Mendel ◽

...

Keyword(s):

Structural Model ◽

Bond Distance ◽

Aldehyde Oxidase ◽

Disulfide Bridge ◽

Cysteine Residue ◽

Molybdenum Cofactor ◽

Cysteine Residues ◽

Xanthine Oxidoreductase ◽

Cysteine Scanning Mutagenesis ◽

Molybdenum Cofactor Sulfurase

The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys430, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys430. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys206, was identified. Furthermore, the active-site Cys430 was found to be located on top of a loop structure, formed by the two flanking residues Cys428 and Cys435, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys428 and Cys435 are within disulfide bond distance and that a persulfide transfer from Cys430 to Cys206 is indeed possible.

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Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

Crop and Pasture Science ◽

10.1071/cp18036 ◽

2018 ◽

Vol 69 (9) ◽

pp. 873

Author(s):

Xin Ma ◽

Xuye Du ◽

Cunyao Bo ◽

Hongwei Wang ◽

Anfei Li ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Wheat Flour ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Dough Strength ◽

Typical Structure ◽

Dough Quality

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

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