Determination of interchain crosslinkages in insulin B-chain dimers by fast atom bombardment mass spectrometry

Toren, Paul; Smith, David L.; Chance, Ronald E.; Hoffman, J.

doi:10.1016/0003-2697(88)90287-4

Cited by 12 publications

(2 citation statements)

References 14 publications

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“…The occurrence of such a dimer during A-chain/B-chain reconstitution procedures in the preparation of recombinant human insulin has recently been independently reported [11].…”

Section: Discussionmentioning

confidence: 96%

Protein folding/refolding analysis by mass spectrometry. Scrambling of disulphide bridges in insulin

Morris

Pucci

Panico

et al. 1990

Biochemical Journal

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In this paper we present a protocol that allows a dynamic analysis of disulphide-bridge formation, based on freezing the intermediates by acid/acetone precipitation, followed by digestion with pepsin and direct fast-atom-bombardment mass-spectrometric analysis. A rapid definition of the exact nature of disulphide bridges formed can be obtained via a definitive assignment of disulphide-linked peptides according to their unique mass values. With the use of an appropriate thiol concentration, scrambling of the native disulphide bonds in bovine insulin occurs, and the process is catalysed by protein disulphide-isomerase (EC 5.3.4.1). The disruption of native and the formation of new disulphide bonds can be monitored as described above, and interestingly B-chain dimers containing Cys-B7-Cys-B7 and Cys-B7-Cys-B19 bonds are detected.

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“…The occurrence of such a dimer during A-chain/B-chain reconstitution procedures in the preparation of recombinant human insulin has recently been independently reported [11].…”

Section: Discussionmentioning

confidence: 96%

Protein folding/refolding analysis by mass spectrometry. Scrambling of disulphide bridges in insulin

Morris

Pucci

Panico

et al. 1990

Biochemical Journal

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“…The specific algorithms described in this paper have been designed for the use of integer variables such as the nominal FAB-MS MH + data, since, up to now, these are the most used data for the assignment of protein disulphide bridges [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. However, the average mass data usually provided by conventional instruments equipped for electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI) can also be processed by the method.…”

Section: Required Datamentioning

confidence: 99%

Assignment of protein disulphides by a computer method using mass spectrometric data

Caporale¹,

Sepe²,

Caruso³

et al. 1996

FEBS Letters

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We designed a computer program for the assignment of protein disulphides using mass spectrometric data. All the theoretical linear peptides containing from one to three cysteines are generated on the basis of the protein sequence. Their combination ways are determined in order to create all the possible disulphide-bridged fragments containing from two to six cysteines and to calculate their molecular weight. One, two and three S-S bridges per linked fragment were considered, taking into account the possibility that the fragments contain unabridged residues. The mass data obtained from the spectral analysis of peptide mixtures of the digested protein are then associated to the fitting structures of disulphide-bridged peptides, giving rise to the primary output. This output can then be screened by using information on the specificity of the proteolytic agent(s) used and/or any further mass data provided by Edman degradation and/or carboxypeptidase treatment of the peptide mixtures. The need for such a computer aid is discussed and examples of application are given.Key words: Disulfide bridge; Protein structure; Computer method; Mass spectrometry and the number of fragments, a single mass signal can be associated to many clusters of bridged peptides, with the ambiguity remaining still unsolved even after one step of chemical and/or enzymatic degradation [13].This paper describes the design and application of a computer program analyzing the possible solutions and filtering the results on the basis of new data obtained after degradation and/or information on the specificity of the proteolytic agent(s). We also suggest that such a computer aid can be indispensable in many cases for the unambiguous assignment of disulphide bridges. Materials and methodsThe mass data and experimental procedures of the two proteins used in the application examples have already been reported [13,15]. Programs were written using Microsoft QuickBASIC (version 1.00b) and implemented on an Apple Macintosh LC 475 computer. The operative system was System 7.1. The compiled applications are compatible with all Apple Macintosh computers. Results and discussion

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Peptide Sequencing by Mass Spectrometry

Stults¹

1990

Methods of Biochemical Analysis

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Determination of interchain crosslinkages in insulin B-chain dimers by fast atom bombardment mass spectrometry

Cited by 12 publications

References 14 publications

Protein folding/refolding analysis by mass spectrometry. Scrambling of disulphide bridges in insulin

Protein folding/refolding analysis by mass spectrometry. Scrambling of disulphide bridges in insulin

Assignment of protein disulphides by a computer method using mass spectrometric data

Peptide Sequencing by Mass Spectrometry

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