Assignment of the five disulfide bridges in an α‐amylase inhibitor from wheat kernel by fast‐atom‐bombardment mass spectrometry and Edman degradation

Abstract

The assignment of the five disulfide bridges in an a-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.

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

“…The 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 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.…”

“…both containing two cysteine residues, can be linked by either one or two S-S bridges, giving rise to structures with different mass values. This aspect should be considered even if the native protein does not contain free thiol groups, as the reduction of S-S bridges can take place under FAB conditions [4,13,17]. The algorithm has been designed to consider even the presence of either homoserine or the corresponding lactone at the C-terminus of peptides derived from CNBr hydrolysis of the native protein.…”

“…For this reason, when using a personal computer, the running time for a complete search including these combination ways is reasonable for proteins containing up to 17 cysteines. In any case, this should be a very high threshold, since, up to now, the assignment of disulphide bridges by mass spectrometry has been done for proteins containing a maximum of 10 cys residues [13]. However, excluding the search for fragments containing six Cys residues, it is possible to analyze proteins containing more than 17 cysteines.…”

“…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.…”