The application of negative ion electrospray mass spectrometry for the sequencing of underivatized disulfide-containing proteins: insulin and lysozyme

Andreazza, Hayley J.; Bowie, John H.

doi:10.1039/c0cp00717j

Cited by 16 publications

(27 citation statements)

References 8 publications

(11 reference statements)

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“…It is known that S–S undergoes marginal cleavage under positive ion CID except with high energy or with metal ions [33]. But S–S undergoes facile cleavage under negative ion CID [3, 34]. Proton abstraction from the C α and C β of the cysteine residue causes diverse fragmentation reactions including the cleavage of disulfide bonds.…”

Section: Resultsmentioning

confidence: 99%

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Zhang

Cui

et al. 2011

J. Am. Soc. Mass Spectrom.

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Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno) phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

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Section: Resultsmentioning

confidence: 99%

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Zhang

Cui

et al. 2011

J. Am. Soc. Mass Spectrom.

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“…It should be noted that in a previous study, where the negative ion cleavages of disulfide containing proteins were investigated [30], it was shown that all four possible product ions were not evident in all cases. This indicates that some care may need to be taken in analyzing the low energy MS/ MS spectra, as all four possible cleavage products may not be observed.…”

Section: Interpretation Of Cross-linking Datamentioning

confidence: 99%

“…Historically, negative ion MS has had limited application for the study of complex systems as the MS/MS data of peptides are often dominated by side chain-induced fragmentations [31,35], whereas spectra in the positive ion mode generally show characteristic backbone cleavages well suited to peptide sequencing. However, equally important and complementary information can be obtained from both ionization modes, and many examples exist whereby negative ion MS allows for mapping of disulfide bonds in proteins with greater ease [30,33,34].…”

Section: Cross-linker Rationalementioning

confidence: 99%

“…These reagents have also shown limited use as positive ion mode CID-cleavable cross-linking reagents as linker cleavages compete with backbone fragmentation, complicating the spectra, and have been shown to be charge-state dependent [25,29]. However, in the negative ion mode, disulfide bonds within peptides undergo facile cleavage upon collisional activation [30][31][32][33][34]. The fragment anions produced are readily identifiable, and sequence information can be obtained by CID of the resultant product ions [35].…”

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confidence: 99%

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A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Calabrese

Good

Wang

et al. 2012

J. Am. Soc. Mass Spectrom.

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Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS 3 analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.

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“…Electron-based ion dissociations, such as electron-capture dissociation (ECD) [46] and electron-transfer dissociation (ETD) [47], are able to cleave disulfide bonds of protein/peptide ions, in which the cleavage sites of disulfide bonds can be either S-S or C α -S. In addition, upon collision-induced dissociation (CID) in the negative-ion mode, cleavage of disulfide bonds of protein/peptide ions is more favorable compared with backbones [48,49], which could provide unique sequence information by yielding characteristic fragment ions from both S-S and C α -S cleavages. However, in the most widely used positive-ion mode, disulfidebond cleavage is usually suppressed by backbone fragmentations upon CID, resulting in limited sequence information, especially for the regions covered by disulfide bonds.…”

Section: Coupling Of Ec-ms With Liquid-sample Desimentioning

confidence: 99%