Depth of Proteome Issues

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 (Gygi et al. (1999) Nat. Biotechnol. 17, 994 -999) 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. Molecular & Cellular Proteomics 3:625-659, 2004.One of the goals of proteomic research is to identify (correctly) and quantify as many proteins as possible in the biological system of choice (see Refs. 1 and 2 for general reviews). We chose a yeast system for this study because it is possible to get large quantities of biological material from yeast cells using fermentation, and the yeast genome encodes Ͻ6600 rather well-characterized proteins whose abundance can be estimated based on the codon adaptation index (3). At the biological level, we chose to study two yeast strains that were related by the knockout of Upf1p (the protein encoded by the UPF1 gene), a protein that is crucial to the process of nonsense-mediated mRNA decay (4, 5).1 We used the isotope-coded affinity tag (ICAT) 2 reagent approach (6) to

Blocking S -adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing

“…Samples were prepared for mass spectrometry analysis by mixing two portions of tryptic peptides extracted from gel samples and digested as described (37) (38), with an accelerating voltage of 20 kV and a 150-ns delay time. Spectra were obtained by averaging 200 acquisitions from single laser pulse (20 Hz, Voyager, N2 337-nm laser).…”

The Stability of Cα Peptide Radicals: Why Glycyl Radical Enzymes?

“…Such analyses are achieved through comparing the differences between LC/MS/MS runs of the proteolytic digests of both control and experimentally perturbed systems. Generally, quantitative proteomics involves the analysis of samples that are either subjected to stable‐isotope labeling, such as isotope‐coded affinity tag (ICAT), global internal standard strategy (GIST) and isobaric tag for relative and absolute quantification (iTRAQ),10–12 or analyzed without any labeling step, an approach commonly referred to as label‐free quantitative proteomics 8, 13, 14…”

Current chemical tagging strategies for proteome analysis by mass spectrometry

“…In addition, it contains a linker group that can be cleaved under acidic conditions, resulting in a smaller moiety being attached to the peptide, very similar to the solid-phase-derived tag. First applications have been described [67,[70][71][72][73][74][75][76][77]. In particular, Yu et al [74] addressed important issues such as MS/MS behavior, necessary clean-up steps, completeness of labeling and accuracy of the quantitation.…”

“…(a) The solid-phase ICAT [69], including a photocleavable linker region. (b) The cleavable ICAT [67,[70][71][72][73][74][75][76][77] with an acid-labile affinity tag region. Asterisks denote differentially ( 12 C/ 13 C) labeled carbon atoms.…”