Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Glocker, Michael O.; Arbogast, Brian; Ml, Deinzer

doi:10.1016/s1044-0305(05)80053-4

Cited by 13 publications

(18 citation statements)

References 8 publications

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“…Digestion was carried out at pH 7.5 at 37°C but was limited to 2 h to minimize possible disulfide bond scrambling (14,15,38). MALDI-MS peptide mapping showed comparable spectra as with reduced MalS, but additional ion signals were observed that could not be addressed as tryptic peptides but as disulfide-bonded dipeptides at m/z 4515, assignable as T2-S-S-T3; at m/z 2544, assignable as dipeptide T7-S-S-T42; and at m/z 2586, assignable as dipeptide T3-S-S-T7, respectively.…”

Section: Migration Of Mals On Sds-page Undermentioning

confidence: 99%

Biochemical Characterization and Mass Spectrometric Disulfide Bond Mapping of Periplasmic α-Amylase MalS of Escherichia coli

Spiess

Happersberger

Glocker

et al. 1997

Journal of Biological Chemistry

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Section: Migration Of Mals On Sds-page Undermentioning

confidence: 99%

Biochemical Characterization and Mass Spectrometric Disulfide Bond Mapping of Periplasmic α-Amylase MalS of Escherichia coli

Spiess

Happersberger

Glocker

et al. 1997

Journal of Biological Chemistry

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“…Molecular mass analyses identify the peptides and disulfide-bonded dipeptides by assigning the observed ion signals to the corresponding calculated masses from the amino acid sequence. To confirm whether or not an observed peak in the recorded mass spectrum represents a disulfide-linked dipeptide, a chemical reduction reaction is performed afterward with the sample either in vitro (4,5,7) or on target (8 -11). Subsequent re-analyses of these samples by matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-MS) 1 should show the disappearance of the ion signals from dipeptides that are disulfide-linked and the appearance of signals corresponding to the resulting pairs of reduced peptides.…”

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confidence: 99%

Mass Spectrometry Unravels Disulfide Bond Formation as the Mechanism That Activates a Molecular Chaperone

Barbirz¹,

Jakob²,

Glocker³

2000

Journal of Biological Chemistry

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The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.Hsp33 is a newly discovered heat shock protein that functions as a highly efficient molecular chaperone (1). Hsp33 protects bacterial cells from deleterious effects caused by oxidants like H 2 O 2 showing that it plays a role in the bacterial defense system against oxidative stress. Hsp33 is distinguished from all other known chaperone proteins by the finding that Hsp33 is functionally regulated at posttranslational level, by the redox conditions of the environment (1) (reviewed in Refs. 2 and 3). Elevated H 2 O 2 concentrations induce the chaperone functions of Hsp33 (1), but the precise molecular mechanism that translates the changes of the cellular redox environment into differences in chaperone activity is not yet understood.The Hsp33 amino acid sequence contains a novel conserved motif consisting of four cysteinyl residues near the C terminus of the protein. These cysteinyl residues form a C-X-C motif and a C-Y-Z-C motif (Fig. 1), separated by 27-30 amino acid residues in Escherichia coli Hsp33 and its homologues. These four cysteinyl residues are present in all 27 known Hsp33 homologues suggesting that they play an important role in the function of Hsp33. In addition, two further cysteinyl residues, Cys 141 and Cys 239 , are present in Hsp33. Cys 239 is very poorly conserved occurring in only 4 of the 27 known Hsp33 homologues. Cys 141 is moderately conserved and is present in 10 of the 27 Hsp33 homologues known so far. We have postulated that disulfide bond formation is involved in the activation process of Hsp33. Thus, analysis of the disulfide bond status and connectivities in inactive and in active Hsp33 seemed very important to further understand the regulation of this efficient molecular chaperone.Two complementary mass spectrometry-based strategies are suitable for determining the presence and locations of disulfide bonds in proteins. Which one is superior is dependent on the proximity of the involved cysteinyl residues in the amino acid sequence. Both approaches involve cleavage of the protein by enzymatic or chemical means under conditions that minimize disulfide bond scrambling (4 -6). The first approach can be used when the proteolytically derived peptides contain zero or one cysteinyl residue. The peptide mixtures are directly analyzed by mass spectrometric peptide mapping. Molecular mass analyses identify the peptides and disulfide-bonded dipeptides by assigning the observed ion signals to the corresponding calculat...

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“…The problem of analysing reduced proteins and their digests is the (possibly uncontrolled) refolding of the protein and the recombination of cysteinyl peptides effecting "disulfide bond scrambling" [114], i.e., the creation of protein or peptide artefacts with non-native disulfide linkages. This obstacle can be circumvented by acidic quenching of the reduction, since reduced thiols are stable at low pH.…”

Section: Determination Of Disulfide Bond Positionsmentioning

confidence: 99%

“…In more recent studies, proteins were reduced under non-denaturing conditions and the reduced cysteines were probed with dithiol specific reagents, that exclusively cross-link spatially (not necessarily sequentially) vicinal thiol groups [114][115][116]. This protein chemical approach enables the spatial differentiation of cysteinyl thiol groups and provides a tool for probing protein folding and its intermediates.…”

Section: Determination Of Disulfide Bond Positionsmentioning

confidence: 99%

Characterisation of the covalent structure of proteins from biological material by MALDI mass spectrometry ‣ possibilities and limitations

Kussmann¹,

Roepstorff²

1998

Journal of Spectroscopy

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Abstract. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) has become a primary tool for the detailed characterisation of the covalent structure of proteins isolated from biological material, mainly because of its following potentials: high sensitivity and specificity, speed of analysis, appropriateness for mixture analysis, high tolerance towards contaminants, and compatibility with separation techniques, e.g., gel electrophoresis. These characteristics enable the structural analysis of proteins even if they are only available in limited amounts and/or in mixtures, and even if the protein preparations contain large amounts of salts, buffers, detergents and denaturants. Additionally, structural data can be generated within a relatively short time.Whereas X-ray crystallography and multidimensional NMR techniques can provide "absolute" structural data, i.e., a threedimensional "picture" of the protein of interest, MALDI-MS -especially in combination with selective protein chemistryyields information on particular aspects of the entire protein structure, e.g., primary structure, active site(s), binding sites, and posttranslational modifications, all of which are often of crucial interest for the understanding of the protein function. Taking into account that protein crystallography and protein NMR studies require large quantities of highly purified sample, MALDI-MS can be even more regarded as a powerful complement in protein structure analysis.This review aims at describing the state-of-the-art of MALDI-MS for characterisation of proteins from biological material by evaluating its potential and limitations.

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Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Cited by 13 publications

References 8 publications

Biochemical Characterization and Mass Spectrometric Disulfide Bond Mapping of Periplasmic α-Amylase MalS of Escherichia coli

Biochemical Characterization and Mass Spectrometric Disulfide Bond Mapping of Periplasmic α-Amylase MalS of Escherichia coli

Mass Spectrometry Unravels Disulfide Bond Formation as the Mechanism That Activates a Molecular Chaperone

Characterisation of the covalent structure of proteins from biological material by MALDI mass spectrometry ‣ possibilities and limitations

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