A selection of  papers authored and co-authored by members of the CEBI

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2003

ERCC1/XPF removes the 3' overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes.

Zhu XD, Niedernhofer L, Kuster B, Mann M, Hoeijmakers JH, de Lange T.

Mol Cell. 2003 Dec;12(6):1489-98

Human telomeres are protected by TRF2. Inhibition of this telomeric protein results in partial loss of the telomeric 3' overhang and chromosome end fusions formed through nonhomologous end-joining (NHEJ). Here we report that ERCC1/XPF-deficient cells retained the telomeric overhang after TRF2 inhibition, identifying this nucleotide excision repair endonuclease as the culprit in overhang removal. Furthermore, these cells did not accumulate telomere fusions, suggesting that overhang processing is a prerequisite for NHEJ of telomeres. ERCC1/XPF was also identified as a component of the telomeric TRF2 complex. ERCC1/XPF-deficient mouse cells had a novel telomere phenotype, characterized by Telomeric DNA-containing Double Minute chromosomes (TDMs). We speculate that TDMs are formed through the recombination of telomeres with interstitial telomere-related sequences and that ERCC1/XPF functions to repress this process. Collectively, these data reveal an unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity and establish that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC1/XPF.

A novel proteomic screen for peptide-protein interactions

J Biol Chem. 2003 Dec 16 [Epub ahead of print].

Schulze WX, Mann M.

Regulated interactions between short, unstructured amino acid sequences and modular protein domains are central to cell signaling. Here we use synthetic peptides in 'active' (e.g. phosphorylated) and 'control' (e.g. non-phosphorylated) forms as baits in affinity pull-down experiments to determine such interactions by quantitative proteomics. Stable isotope labeling by amino acids in cell culture (SILAC) distinguishes specific binders directly by the isotope ratios determined by mass spectrometry (Blagoev, et al Nature Biotech, 2003, 21, 315-318). A tyrosine-phosphorylated peptide of the Epidermal Growth Factor Receptor (EGFR) specifically retrieved the SH2- and SH3 domain-containing adapter protein Grb2. A proline-rich sequence of Son of Sevenless also specifically bound Grb2, demonstrating that the screen maintains specificity with low affinity interactions. The proline-rich Sos peptide retrieved only SH3 domain containing proteins. Two of these, Pacsin 3, and Sorting Nexin 9, were confirmed by immunoprecipitation. Our data are consistent with a change in the role of Sos from Ras-dependent signaling to actin remodeling/endocytic signaling events by a proline - SH3 domain switch.

Proteomic characterization of the human centrosome by protein correlation profiling

Nature. 2003 Dec 4;426(6966):570-4.

Jens S. Andersen, Christopher J. Wilkinson, Thibault Mayor, Peter Mortensen, Erich A. Nigg & Matthias Mann

The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.

Integrated Analysis of Protein Composition, Tissue Diversity, and Gene Regulation in Mouse Mitochondria
Cell, Vol. 115, 629–640, November 26, 2003,

Vamsi K. Mootha, Jakob Bunkenborg, Jesper V. Olsen, Majbrit Hjerrild, Jacek R. Wisniewski, Erich Stahl, Marjan S. Bolouri, Heta N. Ray, Smita Sihag, Michael Kamal, Nick Patterson, Eric S. Lander, and Matthias Mann

Mitochondria are tailored to meet the metabolic and signaling needs of each cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations to produce a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. The protein expression data were largely concordant with large-scale surveys of RNA abundance and both measures indicate tissue-specific differences in organelle composition. RNA expression profiles across tissues revealed networks of mitochondrial genes that share functional and regulatory mechanisms. We also determined a larger “neighborhood” of genes whose expression is closely correlated to the mitochondrial genes. The combined analysis identifies specific genes of biological interest, such as candidates for mtDNA repair enzymes, offers new insights into the biogenesis and ancestry of mammalian mitochondria, and provides a framework for understanding the organelle's contribution to human disease.

Hystag - a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain.

Olsen JV, Andersen JR, Nielsen PA, Nielsen ML, Figeys D, Mann M, Wisniewski JR.
Mol Cell Proteomics. 2003 Nov 10 [Epub ahead of print].

A novel isotopically-labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H2N-(His)6-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO2H, which consists of four functional elements: (i) an affinity ligand (His6-tag), (ii) a tryptic cleavage site (-Arg-Ala-), (iii) Ala-9 residue that contains 4 (d4) or 0(d0) deuterium atoms, and (iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d4) or (d0) reagent. The HysTag-peptide is preserved in Lys-C digestion of proteins and allows charge based selection of cysteine containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d4 co-elute with their d0-labeled counterparts. To demonstrate effectiveness of the reagent a differential analysis of mouse forebrain vs. hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on non-solubilized membranes. The tagged peptides were isolated by Ni2+-affinity or cation-exchange chromatography. Finally the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by LC-MS/MS for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared to the ICAT reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of 13C isotope label, and allows robust purification and flexibility through the affinity-tag, which can be extended to different peptide functionalities.

Bioinformatic analysis of the nucleolus.
Biochem J. 2003 Oct 8 [Epub ahead of print].

Leung AK, Andersen JS, Mann M, Lamond AI.

The nucleolus is a plurifunctional, nuclear organelle which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome is dramatically expanded by the two recent mass spectrometry studies on isolated nucleoli from HeLa cells. Nearly 400 proteins were identified within the nucleolar proteome so far in human. Approximately 12% of the identified proteins were previously shown to be nucleolar in human cells and, as expected, nearly all of the known housekeeping proteins required for ribosome biogenesis were identified in these analyses. Surprisingly, ~30% represented either novel or uncharacterised proteins. This review focuses on how to apply the derived knowledge of this newly recognised nucleolar proteome, such as their amino acid/peptide composition and their homologies across species, to explore the function and dynamics of the nucleolus, and suggests ways to identify in silico possible functions of the novel/uncharacterised proteins and potential interaction networks within the human nucleolus, or between the nucleolus and other nuclear organelles, by drawing resources from the public domain.

Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs.

Mol Cell Biol. 2003 Oct;23(19):6982-92.

Mitchell P, Petfalski E, Houalla R, Podtelejnikov A, Mann M, Tollervey D.

Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.

Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode.

Anal Chem. 2003 Jul 1;75(13):3107-14.

Rappsilber J, Friesen WJ, Paushkin S, Dreyfuss G, Mann M.

Dimethylation at arginine residues has been shown to be central in cellular processes such as signal transduction, transcription activation, and protein sorting. The two methyl groups are either placed symmetric or asymmetric on the zeta standing nitrogen atoms of the arginine side chain. Here, we introduce a novel method that enables the localization of dimethylarginine (DMA) residues in gel-separated proteins at a level of sensitivity of better than 1 pmol and that allows one to distinguish between the isomeric symmetric and asymmetric position of the methyl groups. The method utilizes two side-chain fragments of DMA, the dimethylammonium ion (m/z 46.06) and the dimethylcarbodiimidium ion (m/z 71.06), for positive ion mode precursor ion scanning. Dimethylcarbodiimidium ions (m/z 71.06) are produced by symmetric as well as asymmetric dimethylarginine but are observed more strongly for symmetric DMA. It is utilized here in the precursor of m/z 71 scan to indicate the presence of DMA in a peptide. The dimethylammonium ion (m/z 46.06) is specific for asymmetric DMA and is utilized here in the precursor of m/z 46 scan. The positive ion mode allows for the identification of the protein by peptide sequencing and simultaneous detection and localization of the modified residues. The analysis can be conducted on any mass spectrometer capable of precursor ion scanning. However, the high resolution of a quadrupole TOF instrument is beneficial to assign the accurate charge state of the often highly charged precursors. Using the precursor of m/z 71 scan, we found FUS/TLS and Sam68 to be DMA-containing proteins. We discovered at least 20 DMA sites in FUS/TLS. In MS/MS, we observed neutral loss of dimethylamine (m/z 45.05) from which it follows that the dimethylation in FUS/TLS is asymmetric. Monitoring in parallel the fragments m/z 46.06 and 71.06 in precursor ion scans and peptide sequencing, we identified at least nine asymmetric DMA modifications in Sam68. The parallel monitoring of fragments in precursor ion scans is a versatile tool to specify the nature of protein modifications in cases where a single fragment is not conclusive.

Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.
Proc Natl Acad Sci U S A 2003 Apr 30; [epub ahead of print]

Foster LJ, De Hoog CL, Mann M.

Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts

Mass spectrometry-based proteomics.
Nature 2003 Mar 13;422(6928):198-207
Aebersold R, Mann M.

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Institute for Systems Biology, 1441 North 34th Street, Seattle, Washington 98103-8904, USA

Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.

From genomics to proteomics.
Nature 2003 Mar 13;422(6928):193-7
Tyers M, Mann M.

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Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada M5G 1X5

Proteomics is the study of the function of all expressed proteins. Tremendous progress has been made in the past few years in generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients. But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.

Phosphotyrosine mapping in oncogenic Bcr/Abl using phosphotyrosine specific immonium ion scanning.

Mol Cell Proteomics 2003 Feb 25; [epub ahead of print]

Steen H, Fernandez M, Ghaffari S, Pandey A, Mann M.

Bcr/Abl is a fusion oncoprotein that is of paramount importance in chronic myelogenous leukemia and acute lymphocytic leukemia. The tyrosine-phosphorylated fraction of p185 form of Bcr/Abl was isolated by immunoprecipitation with anti-phosphotyrosine antibodies and SDS-PAGE. The tryptic digest of the gel-separated protein was prefractionated on POROS R2/OLIGO R3 microcolumns and subjected to phosphotyrosine mapping by precursor ion scanning in positive ion mode utilizing the immonium ion of phosphotyrosine, also called phosphotyrosine specific immonium ion (PSI) scanning on a quadrupole TOF tandem mass spectrometer. In total, nine different phosphorylated tyrosine residues were unambiguously localized in twelve different precursor ions. These phosphorylation sites correspond to three previously described phosphotyrosine residues and six novel tyrosine phosphorylation sites, and most of them were not predicted by the phosphorylation motif prediction programs ProSite, NetPhos, or ScanSite. This study shows the power of PSI-scanning for sensitive phosphotyrosine mapping when limited amount of samples are available.

Proteomic analysis of post-translational modifications.
Nat Biotechnol 2003 Mar;21(3):255-61
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Mann M, Jensen ON.

Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230 Denmark. mann@bmb.sdu.dk

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.

Mass spectrometric-based approaches in quantitative proteomics.
Methods 2003 Feb;29(2):124-30

Ong SE, Foster LJ, Mann M.

Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, M 5230, Odense, Denmark

Classically, experiments aimed at studying changes in protein expression have always followed a small set of proteins. This focused approach was necessary since tools to efficiently analyze large numbers of proteins were simply not available. Large-scale quantitative proteomics promises to produce reams of data that previously would have taken decades to measure with classical methods. Mass spectrometry is already a well-established protein identification tool and recent methodological developments indicate that it can also be successfully applied to extract quantitative data of protein abundance. From the first reports 4 years ago, numerous schemes to take advantage of stable isotope nuclei incorporation in proteins and peptides have been developed. Here we review the benefits and pitfalls of some of the most commonly used protocols, focusing on a procedure now being used extensively in our laboratory, stable isotope labeling with amino acids in cell culture (SILAC). The basic theory, application, and data analysis of a SILAC experiment are discussed. The emerging nature of these techniques and the rapid pace of technological development make forecasting the directions of the field difficult but we speculate that SILAC will soon be a key tool of quantitative proteomics.

Signaling initiated by overexpression of the fibroblast growth factor receptor-1 investigated by mass spectrometry.
Mol Cell Proteomics 2003 Jan;2(1):29-36

Hinsby AM, Olsen JV, Bennett KL, Mann M.

MDS Proteomics A/S, Staermosegaardsvej 6, Odense DK-5230, Denmark, the.

Overexpression of the fibroblast growth factor receptor-1 (FGFR-1), a prototypic receptor tyrosine kinase, is a feature of several human tumors. In human 293 cells overexpression of the FGFR-1 leads to constitutive activation of the receptor with concomitant sustained high increase in the cellular level of phosphotyrosine-containing proteins. Here we use mass spectrometry to study the tyrosine-phosphorylated proteins induced by overexpression of the FGFR-1. Several well known components of FGFR-1 signaling were identified along with two novel candidates: NS-1-associated protein-1 and target of Myb 1-like protein. We subsequently applied mass spectrometry precursor ion scanning to identify 22 tyrosine phosphorylation sites distributed on six substrate proteins of the FGFR-1 or downstream tyrosine kinases. Novel in vivo tyrosine phosphorylation sites were found in the FGFR-1, phospholipase Cgamma, p90 ribosomal S6 kinase, cortactin, and NS-1-associated protein-1 as a result of sustained FGFR-1 signaling, and we propose these as functional links to downstream molecular and cellular processes.

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Nat Biotechnol 2003 Feb 10; [epub ahead of print]

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Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M.

Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions

Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics

Juri Rappsilber, Yasushi Ishihama, and Matthias Mann

Anal. Chem., 75 (3), 663 -670, 2003. 10.1021/ac026117i S0003-2700(02)06117-6 Web Release Date: December 24, 2002

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 []L/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 ug of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.

Experiences and perspectives of MALDI MS and MS/MS in proteomic research,

Juri Rappsilber, Marc Moniatte, Michael L. Nielsen, Alexandre V. Podtelejnikov and Matthias Mann

International Journal of Mass Spectrometry DOI: 10.1016/S1387-3806(02)00976-4 PII: S1387-3806(02)00976-4 In Press, Corrected Proof, Available online 25 October 2002 

The simplicity, robustness, and sensitivity of MALDI have made it a fundamental technique of proteomics. Here we report on our experience in high throughput protein identification by MALDI peptide mass fingerprinting. A streamlined system has been developed that encompasses spot excision, digestion and sample/matrix preparation, data acquisition, database searching and logging of the results. Despite achieving robust and high throughput identification we find that MALDI peptide mass fingerprinting alone is not sufficiently selective for current demands in proteomic research. However, the combination of MALDI with tandem mass spectrometry such as joining a quadrupole with a TOF analyser pairs highly parallel analysis with high selectivity. Thus MALDI will remain an important technique in the modern proteomic laboratory.

Properties of ¹³C-Substituted Arginine in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)
Shao-En Ong, Irina Kratchmarova, and Matthias Mann
J Proteome Res. 2003 Mar-Apr;2(2):173-81 Web Release Date: 13-Dec-2002; (Article) DOI: 10.1021/pr0255708

We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.

Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics.

Proc Natl Acad Sci U S A 2003 Jan 21;100(2):605-10

Mootha VK, Lepage P, Miller K, Bunkenborg J, Reich M, Hjerrild M, Delmonte T, Villeneuve A, Sladek R, Xu F, Mitchell GA, Morin C, Mann M, Hudson TJ, Robinson B, Rioux JD, Lander ES.

Identifying the genes responsible for human diseases requires combining information about gene position with clues about biological function. The recent availability of whole-genome data sets of RNA and protein expression provides powerful new sources of functional insight. Here we illustrate how such data sets can expedite disease-gene discovery, by using them to identify the gene causing Leigh syndrome, French-Canadian type (LSFC, Online Mendelian Inheritance in Man no. 220111), a human cytochrome c oxidase deficiency that maps to chromosome 2p16-21. Using four public RNA expression data sets, we assigned to all human genes a "score" reflecting their similarity in RNA-expression profiles to known mitochondrial genes. Using a large survey of organellar proteomics, we similarly classified human genes according to the likelihood of their protein product being associated with the mitochondrion. By intersecting this information with the relevant genomic region, we identified a single clear candidate gene, LRPPRC. Resequencing identified two mutations on two independent haplotypes, providing definitive genetic proof that LRPPRC indeed causes LSFC. LRPPRC encodes an mRNA-binding protein likely involved with mtDNA transcript processing, suggesting an additional mechanism of mitochondrial pathophysiology. Similar strategies to integrate diverse genomic information can be applied likewise to other disease pathways and will become increasingly powerful with the growing wealth of diverse, functional genomics data.

Analysis of Bromotryptophan and Hydroxyproline Modifications by High-Resolution, High-Accuracy Precursor Ion Scanning Utilizing Fragment Ions with Mass-Deficient Mass Tags

Anal Chem. 2002 Dec 15;74(24):6230-6.

Hanno Steen and Matthias Mann

Protein modifications are often detected by precursor ion scanning. When quadrupole TOF mass spectrometers are used for precursor ion scanning with high-resolution, high-accuracy fragment ion selection, "reporter" ions are required to have a unique mass within ±0.04 Da or less instead of ±0.5 Da on triple quadrupole mass spectrometers, the traditional instrument used for precursor ion scanning. Thus, characteristic fragment ions can be utilized even if other fragment ions have the same nominal mass as long as the characteristic fragment ions are slightly mass deficient as compared to the other fragments, i.e., when they have an inherent mass-deficient mass tag. Here, the immonium ions of bromotryptophan and hydroxyproline are described as two fragment ions characteristic for tryptophan-brominated and proline-hydroxylated peptides, respectively. The "reporter" ion of trytophan-brominated peptides is highly mass deficient due to the presence of bromine, thereby allowing the selective detection of these species and the distinction from other dipeptidic a-, b-, and y-fragment ions by high-resolution, high-accuracy precursor ion scanning. This strategy also enables the differentiation between precursors giving rise to the oxygen-containing immonium ion of hydroxyproline and precursors of the immonium ions of near-ubiquitous leucine/isoleucine. Both immonium ions have the same nominal mass of 86 Da, but the exact masses differ by less than 0.04 Da. High-resolution, high-accuracy precursor ion scanning enabled the identification of proline-hydroxylated and tryptophan-brominated species and the directed analysis of species carrying these modifications in a highly complex Conus textile conotoxin mixture. This lead to the characterization of one novel C. textile conotoxin containing a bromotryptophan residue and one novel C. textile conotoxin carrying two hydroxyproline residues.

Computational and experimental analysis reveals a novel Src family kinase in the C. elegans genome.

Bioinformatics 2003 Jan 22;19(2):169-72

Pandey A, Peri S, Thacker C, Whipple CA, Collins JJ, Mann M.

MOTIVATION: The complete genomes of a number of organisms have already been sequenced. However, the vast majority of annotated genes are derived by gene prediction methods. It is important to not only validate the predicted coding regions but also to identify genes that may have been missed by these programs. METHODS: We searched the entire C.elegans genomic sequence database maintained by the Sanger Center using human c-Src sequence in a TBLASN search. We have confirmed one of the predicted regions by isolation of a cDNA and carried out a phylogenetic analysis of Src kinase family members in the worm, fly and several vertebrate species. RESULTS: Our analysis identified a novel tyrosine kinase in the C.elegans genome that contains functional features typical of the Src family kinases that we have designated as Src-1. The open reading frame contains a conserved N-terminal myristoylation site and a tyrosine residue within the C-terminus that is crucial for regulating the activity of Src kinases. Our phylogenetic analysis of Src family members from C. elegans, Drosophila and other higher organisms revealed a relationship among Src kinases from C. elegans and Drosophila.

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Last modified by Schandorff, February 2007