Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 × 103, 44 × 103, 38 × 103, 11 × 103 and 6.5 × 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 × 103 copies μm−2. For relative quantitation, we compared wild-type cells to gas1Δ cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins

AbstractThe coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen

Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803

BACKGROUND: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product. RESULTS: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level. CONCLUSION: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Quantitation of newly synthesized proteins by pulse labeling with azidohomoalanine

Measuring protein synthesis and degradation rates on a proteomic scale is an important step toward modeling the kinetics in complicated cellular response networks. A gel-free method, able to quantify changes in the formation of new proteins on a 15 min timescale, compatible with mass spectrometry is described. The methionine analogue, azidohomoalanine (azhal), is used to label newly formed proteins during a short pulse-labeling period following an environmental switch in Escherichia coli. Following digestion a selective reaction against azhal-containing peptides is applied to enrich these peptides by diagonal chromatography. This technique enables quantitation of hundreds of newly synthesized proteins and provides insight into immediate changes in newly synthesized proteins on a proteomic scale after an environmental perturbatio

Iron restriction-induced adaptations in the wall proteome of Candida albicans

The opportunistic fungal pathogen Candida albicans has developed various ways to overcome iron restriction in a mammalian host. Using different surface proteins, among them membrane- and wall-localized GPI-proteins, it can exploit iron from host hemoglobin, ferritin, and transferrin. Culturing C. albicans in rich medium supplemented with the ferrous iron chelator bathophenanthroline disulfonic acid or in the minimal medium yeast nitrogen base resulted in a strong decrease of the iron content of the cells. Mass spectrometric analysis of the changes in the wall proteome of C. albicans upon iron restriction showed a strong increase in the levels of the GPI-modified adhesin Als3, which also serves as a ferritin receptor, and of the GPI-modified, CFEM domain-containing proteins Csa1, Pga7, Pga10, and Rbt5. The wall levels of the GPI-modified proteins Hyr1, the adhesin Als4, and the copper- and zinc-containing superoxide dismutase Sod4 also strongly increased, whereas the levels of Tos1 (a non-GPI protein) and the GPI-modified adhesin Als2 strongly decreased. Strikingly, peptides derived from the CFEM domain of the heme-binding proteins Csa1, Pga10 and Rbt5 were capable of forming iron adduct ions during mass spectrometric analysis, consistent with a key role of this domain in heme binding

Detection of mutant protein in complex biological samples: glucocerebrosidase mutations in Gaucher's disease

We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI-ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher's disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher's patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher's disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody-antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage approximately 60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedure

Endgroup analysis of polyethylene-glycol polymers by matrix-assisted laser-desorption ionization fourier-transform ion-cyclotron resonance mass-spectrometry

Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) by external injection of matrix-assisted laser desorbed and ionized (MALDI) polymers offers good possibilities for characterization of low molecular weight homopolymers (MW range up to 10 kDa). The molecular masses of the molecular weight distribution (MWD) components of underivatized and derivatized (dimethyl, dipropyl, dibutyl and diacetyl) polyethylene glycol (PEG) 1000 and 4000 were measured by MALDI-FTICR-MS. These measurements have been performed using a commercial FTICR spectrometer with a home-built external ion source. MALDI of the samples with a 2,5-dihydroxybenzoic acid matrix in a 1000:1 matrix-to-analyte molar ratio produces sodiated molecules in a sufficient yield to trap the ions in the ICR cell. The masses of the molecular weight distribution of PEG components were measured in broad-band mode with a mass accuracy of &lt; 5 ppm in the mass range around 1000 u and within 40 ppm accuracy around 4000 u. From these measurements, the endgroup mass of the polymer was determined by correlation of the measured component mass with the degree of polymerization. The masses of the PEG endgroups have been determined within a deviation of 3-10 millimass units for the PEG1000 derivatives and 10-100 millimass units for the PEG4000 derivatives, thus confirming the identity of the distal parts of the model compounds