Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting

Abstract

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.

Use of surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) to study protein expression in a rat model of cocaine withdrawal

“…Subsequent studies need to include further separation or purification, digestion by known proteases, characterization of digests by MALDI-TOF MS, comparison of results to protein databases, then verification by an independent technique, such as immunoblotting. The unique identification of low mass proteins or peptides is more difficult than that of high mass proteins (because of fewer entries in protein databases), but more information is continually becoming available (Bienvenut et al, 2002;Zhu et al, 2004). The discovery of low mass proteins or peptides is important; data obtained from SELDI-TOF MS studies are beginning to indicate that characteristic protein degradation patterns occur in many pathological states and that the biomarkers of interest are frequently fragments of protein degradation (Nomura et al, 2004;Paradis et al, 2005;Petricoin and Liotta, 2004).…”

Using Statistical Models To Identify Factors That Have a Role in Defining the Abundance of Ions Produced by Tandem MS

“…To better understand what factors influence the fragmentation mechanism, we also investigated replacing the N-terminal and C-terminal variables indicating the amino acid residues either side of the fragmentation site with a description of the essential physical and chemical properties of the residues, thus leading to a physicochemical regression model. The properties of amino acids readily available in the literature include volume of the residue in solution, the accessible surface area of the residue, two different scales of hydrophobicity (a measure of their relative affinity for water), , and a measure of how many hydrogen bonds could be formed by each residue . We decided to incorporate both hydrophobicity scales, normalized to a standard normal distribution (mean = 0, SD = 1) into the model for intensity as they measure slightly different aspects of hydropathy.…”

FindPept, a tool to identify unmatched masses in peptide mass fingerprinting protein identification

“…With increasing database sizes (SWISS-PROT release 40.11 and TrEMBL release 19.8 contain sequence data and annotation of 700 000 proteins), and therefore an increasing likelihood of false positive identifications, protein identification by peptide mass fingerprinting (PMF) alone is becoming more and more difficult [1]. It is indeed quite common for PMF tools not to be able to find matching theoretical peptides for a few of the less intense peaks that were detected and submitted to the identification process.…”