title: Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins.
abstract: A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.
authors.affiliation: Xzillion GmbH, Industriepark Höchst, Building G865a, 65929 Frankfurt am Main, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Kuhn
authors.forename: Andrew
authors.initials: A
authors.lastname: Thompson
authors.forename: Thorsten
authors.initials: T
authors.lastname: Prinz
authors.forename: Jörg
authors.initials: J
authors.lastname: Müller
authors.forename: Christian
authors.initials: C
authors.lastname: Baumann
authors.forename: Günter
authors.initials: G
authors.lastname: Schmidt
authors.forename: Thomas
authors.initials: T
authors.lastname: Neumann
authors.forename: Christian
authors.initials: C
authors.lastname: Hamon
mesh_terms: D000595:Amino Acid Sequence; D003488:Cyanogen Bromide / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015394:Molecular Structure; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D003488:Cyanogen Bromide