Identification by HPLC -ESI/MS of the phosphorylated peptides and sites of wheat translation initiation factor (eIF) 4B in vitro.
Item
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Title
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Identification by HPLC -ESI/MS of the phosphorylated peptides and sites of wheat translation initiation factor (eIF) 4B in vitro.
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Identifier
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AAI3144139
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identifier
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3144139
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Creator
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Shen, Zhengming.
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Contributor
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Major Professor: Dixie J. Goss
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Date
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2004
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Chemistry, Analytical | Biology, Molecular
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Abstract
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In wheat, the translation initiation factor eIF4B is a phosphoprotein whose phosphorylation state is developmentally regulated. To understand its biological role, the sites of phosphorylation have to be determined. Recombinant eIF4B protein expressed in E. coli was purified and subsequently phosphorylated by Casein Kinase II. Generally only limited amounts of proteins are phosphorylated. Here, we report a powerful technique for isolation of phosphorylated peptides with immobilized metal affinity chromatography (Fe(III)-IMAC) and direct High Pressure Liquid Chromatography/Electrospray Ionization Mass Spectrometry (HPLC-ESI/MS) analysis of phosphorylated peptides without any additional desalting steps.;For a purified protein digested with trypsin where each peptide sequence is known in a database, the phosphorylated peptide is confirmed in mass spectroscopic analysis by an increase of 80 Da (HPO3) for each phosphorylated amino acid present compared with the calculated masses of the corresponding unphosphorylated peptide if no second unphosphorylated peptide has the same mass. Some phosphorylated peptides underwent beta-eliminate reaction. A neutral loss H3PO4 (98 Da) from phosphorylated serine or threonine forms a alpha, beta unsaturated bond and yields a residue of dehydroalanine (69 Da), or a residue of dehydroaminobutyric acid (83 Da). Thus, this 98 Da loss (or a multiple of 98 Da) also provided unequivocal identification of phosphorylated peptides containing serines and/or threonines. So far, twenty-one phosphorylation sites have been observed from a total of 74 serine and threonine residues of eIF4B protein. The phosphorylated sites can be assigned if all the serine and/or threonine residues are phosphorylated in each peptide. Ten phosphorylated sites were confirmed in deconvolution mass spectra, and seven phosphorylated sites could be possibly determined only in ion mass spectra due to their peptide ions were not transferred into deconvolution mass spectra. Further confirmation needs to be done by tandem mass spectrometry. The other four phosphorylated sites in three peptides could not be assigned to exact positions, because each peptide contains more potential phosphorylation sites than observed phosphorylated site(s). Two dominant CKII phosphorylated regions were observed, which corresponded to NH2-terminal residues 136--204 and COOH-terminal residues 395--444.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
