Proteomic Techniques for Functional Identification of Bacterial Adhesins

摘要

Proteomics includes the identification, characterization and quantification of proteins including isoforms, polymorphism and modifications, and also definition of all protein-protein interactions and intracellular signaling that occurs in a cell under a given time and condition. Proteomics can be described as a multi-disciplinary research activity in which separation science, mass spectrometry (MS) and bioinformatics play crucial roles. During comparative proteomic investigations of cell lines or microbial strains, lectins are frequently revealed as important functional modulators of a biological response. In the case of bacteria, surface lectins expressed at the microbial surface provide a means of attachment to host cell surface carbohydrates, the first essential step for colonization and infection. When the microbes' carbohydrate affinities are known, a functional proteomic approach to lectin identification can be employed, thus matching protein identity to carbohydrate ligand. We have previously employed a carbohydrate-containing cross-linking probe to select bacterial surface adhesins for trypsin digestion, MALDI-TOF MS and identification against genome sequence. That strategy was successful in the identification of the low-abundant Lewis b (Le~(b))-binding adhesin of Helicobacter pylori (H. pylori) [1] as well as the identification of a sialic acid-binding adhesin from the same organism [2]. Protein identification was obtained through the enrichment of approximately 300 femtomoles of adhesin from solubilized cells. We present an overview of techniques used in proteomics and describe a functional proteomic approach for identification of a lactoferrin-binding protein of H. pylori.