Method development for the analysis of the phosphotyrosine proteome
Abstract
Proteomics is a rapidly growing area in science and can be defined as the study of the protein profile of a cell. This versatile field integrates biology and chemistry with an ultimate goal of advancing medicine and health care. In particular, proteomics can be used to study signal transduction pathways, disease, and develop targets for drug design. On account of the complexity of the proteome (5,000–10,000 proteins), analysis is challenging. Typically, proteome separation is achieved via 2-D gel electrophoresis. Due to limitations with this technique, a multidimensional chromatography approach was investigated. Phosphoproteomics is a subset of proteomics investigating proteins that become phosphorylated in a cell. In particular, occurrences of tyrosine phosphorylation are important regulatory events involved in cellular function. Therefore, the phosphotyrosine proteome is an important analytical target. Methods were developed for the analysis of the phosphotyrosine proteome. An anti-phosphotyrosine antibody was used to enrich tyrosine-phosphorylated and associated proteins from DT40 B cell lysate overexpressing Syk, a nonreceptor protein-tyrosine kinase. Syk was nonspecifically activated with pervanadate treatment. Proteins were subjected to trypsin digestion, a β-elimination reaction, followed by immobilized metal affinity chromatography. Both capillary electrophoresis and reverse phase liquid chromatography approaches were examined for peptide separation. Liquid chromatography was more readily coupled to mass spectrometry. Peptides were separated and analyzed by nanoflow capillary liquid chromatography coupled to an electrospray ionization-quadrupole ion trap mass spectrometer. Tandem mass spectra were interpreted to provide sequences that were searched via online databases. Tyrosine phosphorylated peptides were also subjected to skimmer collision induced dissociation for the detection of the phosphotyrosine immonium ion. Low levels of proteins (∼40 μg) were analyzed. The method provided the enrichment of detected tyrosine levels compared to endogenous levels. In fact, 66% of the phosphorylations detected were on tyrosine residues. Analysis of immobilized metal affinity captured peptides provided 22 phosphotyrosine-containing peptides and six phosphotyrosine-containing peptides were not captured. Several previously unidentified chicken B cell proteins were identified. Proteins detected in these studies are putative substrates of Syk. Furthermore, new methodology in phosphotyrosine proteomics can provide critical biological information in signal transduction and disease mechanisms.
Degree
Ph.D.
Advisors
Borch, Purdue University.
Subject Area
Analytical chemistry
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