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

Abstract

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

CysMap and CysJoin: Database and tools for protein disulphides localisation

“…The same logic is employed to create the peptides containing two cysteines. Although this algorithm is similar to that already described [23], two main differences have to be remarked. The actual program is able to work on the whole database and any type of mass data (integer, average or monoisotopic) can be chosen by the user to create the map file.…”

“…The second goal is the unambiguous attribution of mass signals obtained from digests of a protein of known sequence to cluster peptides containing disulphide bridges. In this view, the program can be considered as an upgrade of the previous one [23], since the code has been re‐written for both Macintosh and Windows modern computers. Furthermore, average and monoisotopic mass data as well as integer variables can be utilised.…”

“…In this case, further sub‐digestions are necessary to produce useful peptides. In 1996, we developed a computer program generating all the possible pairings of Cys residues in a protein, followed by filtering the results on the basis of new data obtained after degradation and/or information on the specificity of the proteolytic agent(s) [23]. This program was developed for Macintosh computers and was able to work just on a single sequence inputted from the keyboard by the user.…”

The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viperBitis arietans

“…All theoretical fragments containing two disulphide‐bonded peptides were calculated with the program DISULPHIDE [30]using the experimental MALDI mass spectrometry data (±1 Da) as input. The primary outputs were filtered manually taking into consideration information gathered from amino acid analysis and/or N‐terminal sequencing.…”

Identification and Characterization of Disulfide Bonds in Proteins and Peptides from Tandem MS Data by Use of the MassMatrix MS/MS Search Engine

“…25,26 Tandem MS data from disulfide-linked peptides under reduction conditions can be analyzed using traditional database search programs. [27][28][29] However, high-throughput software for analysis of tandem MS data of disulfide-linked proteins and peptides under nonreducing condition are limited.…”