Cross‐species identification of proteins separated by two‐dimensional gel electrophoresis using matrix‐assisted laser desorption ionisation/time‐of‐flight mass spectrometry and amino acid composition

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Abstract
Amino acid analysis and matrix‐assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two‐dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS‐PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer‐generated rankings. Results were verified using N‐terminal protein microsequencing. The combined use of amino acid composition and MALDI‐TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4–10 tryptic digest fragments). A further two parameters, estimated Mr and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.