title: Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.
abstract: As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.
authors.affiliation: Discovery Proteomics and Small Molecule Research Center, Applied Biosystems, Framingham, MA 01701, USA. parkerkc@apliedbiosystems.com
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Parker
authors.forename: Dale
authors.initials: D
authors.lastname: Patterson
authors.forename: Brian
authors.initials: B
authors.lastname: Williamson
authors.forename: Jason
authors.initials: J
authors.lastname: Marchese
authors.forename: Armin
authors.initials: A
authors.lastname: Graber
authors.forename: Feng
authors.initials: F
authors.lastname: He
authors.forename: Allan
authors.initials: A
authors.lastname: Jacobson
authors.forename: Peter
authors.initials: P
authors.lastname: Juhasz
authors.forename: Stephen
authors.initials: S
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000096:biosynthesis*; D015180:Electrophoresis, Gel, Two-Dimensional; D048088:Informatics; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D010455:Peptides; D020543:Proteome; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010455:Peptides; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; C074151:NAM7 protein, S cerevisiae; D020365:RNA Helicases