Showing papers by "Steven P. Gygi published in 2002"

Comprehensive proteomic analysis of the human spliceosome.

Abstract: The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.

Abstract: The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information ...

Steps in Assembly of Silent Chromatin in Yeast: Sir3-Independent Binding of a Sir2/Sir4 Complex to Silencers and Role for Sir2-Dependent Deacetylation

Abstract: Transcriptional silencing at the budding yeast silent mating type (HM) loci and telomeric DNA regions requires Sir2, a conserved NAD-dependent histone deacetylase, Sir3, Sir4, histones H3 and H4, and several DNA-binding proteins Silencing at the yeast ribosomal DNA (rDNA) repeats requires a complex containing Sir2, Net1, and Cdc14 Here we show that the native Sir2/Sir4 complex is composed solely of Sir2 and Sir4 and that native Sir3 is not associated with other proteins We further show that the initial binding of the Sir2/Sir4 complex to DNA sites that nucleate silencing, accompanied by partial Sir2-dependent histone deacetylation, occurs independently of Sir3 and is likely to be the first step in assembly of silent chromatin at the HM loci and telomeres The enzymatic activity of Sir2 is not required for this initial binding, but is required for the association of silencing proteins with regions distal from nucleation sites At the rDNA repeats, we show that histone H3 and H4 tails are required for silencing and rDNA-associated H4 is hypoacetylated in a Sir2-dependent manner However, the binding of Sir2 to rDNA is independent of its histone deacetylase activity Together, these results support a stepwise model for the assembly of silent chromatin domains in Saccharomyces cerevisiae

Automation of Nanoscale Microcapillary Liquid Chromatography−Tandem Mass Spectrometry with a Vented Column

Abstract: To fully automate the sample introduction step for nanoscale microcapillary liquid chromatography−tandem mass spectrometry (LC−MS/MS) analyses, 75 μm i.d. × 14 cm capillary columns were interfaced with a commercial autosampler instrument using a novel procedure which allowed dilute peptide samples to be transferred from the AS loop injector to the nanoscale column at flow rates up to 5 μL min-1. On-column enrichment and desalting was demonstrated for large sample volumes (>40 μL) by constructing a vent 2 cm after the entrance to the packed bed of 5-μm ODS-AQ modified silica. Salts and nonretained solutes were removed via the vent, which allowed for column washing independent of the continuation of the bed into the electrospray source. Separations of test peptide mixtures demonstrated 50-nL elution peak volumes with low- to subfemtomole detection levels. In addition, a highly complex peptide mixture (outer membrane preparation from Psuedemonas aeruginosa) was efficiently separated with more than 100 protei...

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Abstract: Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK)—found in guard cells, which control stomatal pores—has been shown to regulate plasma membrane ion channels1. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins2 may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.

Absolute quantification of proteins and modified forms thereof by multistage mass spectrometry

Abstract: The invention provides reagents, kits and methods for detecting and/or quantifying proteins in complex mixtures, such as a cell lysate. The methods can be used in high throughput assays to profile cellular proteomes. In one aspect, the invention provides a peptide internal standard labeled with a stable isotope and corresponding in amino acid sequence to the amino acid sequence of a subsequence of a target polypeptide. In another aspect, the peptide internal standard is labeled at a modified amino acid residue and is used to determine the presence of, and/or quantitate the amound of a particular modified form of a protein.

Serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog.

Abstract: After i.p. infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guerin, macrophages recovered from the peritoneal cavity display classical signs of immune activation. We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guerin infection. Serpin 2a (spi2a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products. The cytokine IFN-gamma also induces spi2a expression in macrophages, and this induction is synergistic with bacterial products. We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages. The ISG15-spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15. Whereas spi2a was induced by either bacterial products or IFN-gamma, ISG15 was induced only by bacterial products. Although many protein targets have been described for ubiquitin conjugation, spi2a is the first ISG15-modified protein to be reported. Macrophage activation is accompanied by the activation of a variety of proteases. It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.

Development of a Multiplexed Microcapillary Liquid Chromatography System for High-Throughput Proteome Analysis

Abstract: Comprehensive proteome analysis requires the identification (and quantification) of the proteins in samples consisting of thousands of proteins spanning a range of abundance of several orders of magnitude. The currency of proteome analysis by mass spectrometry is the peptides generated by protein proteolysis. The high sample complexity of such samples requires a large separation capacity, which is commonly achieved by fractionation of the mixture followed by further serial separations of each fraction. The sample throughput of proteome analysis is therefore limited by the need to sequentially process large numbers of samples. We have developed a novel fourplexed microcapillary liquid chromatography system for automated, high-throughput separation of complex peptide samples. The system supports the concurrent separation of four different samples by directing identically split solvent−gradient flows into four microcapillary C18 columns. The simple design of the system achieves multiplexed separation without...

Mitochondrial aconitase modification, functional inhibition, and evidence for a supramolecular complex of the TCA cycle by the renal toxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine.

Abstract: Metabolism of the common industrial gas tetrafluoroethylene in mammals results in the formation of S-(1,1,2,2)-tetrafluoroethyl-l-cysteine (TFEC), which can be bioactivated by a mitochondrial C-S l...

Automated capillary liquid chromatography small volume analysis system

Abstract: A system for automatically performing liquid chromatography analysis of low volume liquid chemical samples at nanosecond flow rates using an analysis column that integrates a pre-concentration trapping column and a chromatography separation column terminating at an electrospray nozzle of an online mass spectrometer. The analysis column consists of a capillary having an inside diameter of between 75 and 125 microns packed throughout with a porous bed of micron particles. A branch outlet positioned 10 to 16 centimeters upstream from the nozzle divides the analysis column into an upstream pre-concentration trap and a downstream separation column. An autosampler delivers low volume liquid samples to the upstream inlet via a two-position valve. Feed connections couple the autosampler to upstream inlet when the valve is open to inject a liquid sample into the pre-concentration trap at a maximum loading flow rate in the range from 0.5 to 50 microliters/minute. Thereafter, when the valve closes, it terminates the further injection the sample, and a concentrated portion of the sample then passes though the chromatography separation column at a much slower flow rate between 10 and 1,000 nanoliters per minute. Throughput can be doubled by coupling two such analysis columns to a single autosampler using a ten-port, two position valve. A single column can be supplied through a six port two-position valve.

Non-affinity based isotope tagged peptides and methods for using the same

Abstract: The invention provides non-affinity based isotope tagged peptides, chemistries for making these peptides, and methods for using these peptides. In one aspect, tags comprise a reactive site (RS) for reacting with a molecule on a protein to form a stable association with the peptide (e.g., a covalent bond) and an anchoring site (AS) group for reversibly or removably anchoring the tag to a solid phase such as a resin support. Anchoring may be direct or indirect (e.g., through a linker molecule). Preferably, the tag comprises a mass-altering label, such as a stable isotope, such that association of the tag with the peptide can be monitored by mass spectrometry. The reagents can be used for rapid and quantitative analysis of proteins or protein function in mixtures of proteins.

Quick quantitative analysis of protein or protein function in composition mixture

Abstract: PROBLEM TO BE SOLVED: To provide a method used in a proteome analysis for conquering the limitation peculiar to the conventional technique, and to provide a reagent. SOLUTION: An automation LC/MS/MS system comprises an auto sampler that is subjected to fluid connection to a capillary HPLC (a), an electro spray ionization triple quadrupole electrode MS/MS apparatus that is subjected to fluid connection to the capillary HPLC (b), and device control and a data analysis system that are electrically connected to the autosampler, the capillary HPLC, and an MS/MS apparatus.

The proteome: Analysis and utility

Abstract: In this manuscript we have shown that in the emerging post-genomic era, technologies that can quantitatively, globally, and automatically measure gene expression at the protein level are essential for the comprehensive analysis of biological processes and systems. We have furthermore documented the limitations of the current standard method for large-scale protein analysis with respect to the analysis of low abundance proteins and proposed a new approach to quantitative proteome analysis. We anticipate that the new ICAT strategy will provide broadly applicable means for the quantitative cataloging and comparison of expressed proteins in a variety of normal, developmental, and disease states.

Quantification absolue de proteines et de formes modifiees de proteine par spectrometrie de masse multistade

Abstract: La presente invention concerne des reactifs, des kits et des techniques de detection et/ou de quantification de proteines dans des melanges de complexes, tels qu'un lysat cellulaire. On peut utiliser ces techniques dans des dosages a haut rendement pour profiler des proteomes cellulaires. Dans un de ses aspects, cette invention concerne une norme interne de peptide marquee avec un isotope stable et correspondant dans une sequence d'amino acide a la sequence d'amino acide d'une sous sequence d'un polypeptide cible. Dans un autre aspect de cette invention, la norme interne de peptide est marquee au niveau d'un residu d'amino acide modifie et elle est utilisee pour determiner la presence d'une forme modifiee particuliere de proteine et/ou pour quantifier celle-ci.

Double inhibition de la separation de chromatides soeurs en metaphase

Abstract: L'invention concerne des molecules d'acide nucleique, appelees molecules d'acide nucleique separases, qui codent la separase, une endopeptidase modulant la separation de chromatides soeurs. L'invention concerne aussi des vecteurs d'expression recombinants contenant des molecules d'acide nucleique separases,et des cellules hotes dans lesquelles les vecteurs d'expression ont ete introduits. L'invention concerne egalement des proteines separases, des proteines hybrides, des peptides antigeniques et des anticorps diriges contre les separases. L'invention concerne en outre des methodes d'identification de modulateurs de la separase, des methodes de modulation de la separase, methodes de modulation de separation de chromatides soeurs, et des methodes de traitement de troubles associes a une separation aberrante de chromatides soeurs, tels que le cancer, le syndrome de Down, ou l'avortement spontane.