Ongoing Projects

I. Mass Spectrometric Identification of Proteins from Organisms with Unknown Genomes

Anna Shevchenko, Vinzenz Link

Until recently mass spectrometry was not conducive for facile protein identification from organisms with unsequenced genomes, which represent an overwhelming majority of the biosphere. Despite the relative deficiency of available genomic sequences, the emerging interplay of mass spectrometry and bioinformatics is now significantly expanding the organismal scope of proteomics (Liska & Shevchenko, 2003).

In collaboration with bioinformaticians from European Molecular Biology Laboratory (EMBL, Heidelberg) and Harvard Medical School (Boston) we have developed software tools, which efficiently utilize mass spectrometry data for identification of proteins by homology searching. Mass Spectrometry driven BLAST (MS BLAST) (Shevchenko et al, 2002) is a generic webÆaccessible tool for sequence-similarity protein identification in a protein database. MS BLAST can utilize a large number of inaccurate, degenerate and redundant sequence proposals obtained by automated de novo interpretation of tandem mass spectra, obtained by NanoES, LC MS/MS or MALDI Q TOF MS. The alternative MultiTag method (Sunyaev et al, 2003) is based on error-tolerant alignment of multiple peptide sequence tags, and enables confident identification of homologous proteins using very short stretches of peptide sequences consisting of 2-4 amino acid residues.

We have successfully applied the methods to characterize the proteomes of organisms that are very distantly related to organisms with sequenced genome, such as the extreme halotolerant Dead Sea alga Dunaliella, the Amazon moth Cerodirphia speciosa, and the African clawed frog Xenopus laevis, as well as a variety of popular mammalian model organisms.

Selected references:
Liska, A.J. and Shevchenko, A. (2003) Proteomics 3, 19-28
Shevchenko, A et al (2001) Anal Chem 73, 1917-1926
Sunyaev, S et al (2003) Anal Chem 75, 1307-1315

MS BLAST site at EMBL server: http://dove.embl-heidelberg.de/Blast2/msblast.html

II. Deciphering Protein Complexes and Protein Interaction Networks

Anna Shevchenko, Natalie Schmalz, Henrik Thomas

In collaboration with the group of Prof. Francis Stewart (BIOTEC Technical University of Dresden, http://www.biologie.tu-dresden.de/stewart/index.htm ) we employed a combination of Tandem Affinity Purification (Rigaut G.et al, 1999) and mass spectrometry for deciphering protein complexes and protein interaction networks in budding yeast and in fission yeast. Genes of interest were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates and identified by mass spectrometry. A sequential strategy of epitope tagging, immunoaffinity isolation and mass spectrometric identification of subunits of protein complex provides multiple readout of its composition and allows semi-quantitative estimation of the stoichiometric ratio of their subunits (Seol et al, 2001).

In a recent study encompassing 38 protein baits, potentially involved in chromating remodeling, pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. On average, every fourth protein is shared between complexes of different functionality thus charting segments of a protein interaction network (Shevchenko et al, 2002).

We observed remarkable conservation of the composition of orthologous protein complexes purified from between budding and fission yeasts their proteomic environment and links to other orthologous complexes are dramatically altered (Roguev et al, 2003).

Selected references:
Seol J.H.et al. (2001) Nature Cell Biol. 3, 384-391
Shevchenko A. (2002) Mol Cell Proteomics 1, 204-212
Roguev A. (2004) Mol Cell Proteomics 3, 125-132

III. Quantitative Analysis of Gel Separated Proteins

Henrik Thomas, Natalie Schmalz

One-dimensional and two-dimensional polyacrylamide gel electrophoresis is a recognized method of separating complex protein mixtures. Although identification of gel separated proteins has become routine, their quantification of these proteins has not been explored.

Using 18O labeled peptides as internal standard, we quantified the yield of in-gel digestion of proteins by trypsin (Shevchenko A & Shevchenko, A, 2001) by MALDI TOF mass spectrometry.

Based on the kinetics of in-gel digestion, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage (Havlis et al, 2003).

Selected References:
Havlis, J., et al (2003) Anal Chem 75, 1300-1306
Shevchenko A, and Shevchenko, A. (2001) Anal Biochem 296, 279-283

IV. Quantitative Profiling of Lipids using Quadrupole Time-of-Flight Mass Spectrometry

Kim Ekroos, Christer Ejsing

We employ a hybrid quadrupole time-of-flight mass spectrometer featured with ion trapping capabilities (QSTAR Pulsar i) for quantitative profiling of total extracts of endogenous phospholipids. Simultaneous acquisition of precursor ion spectra of multiple fragment ions allowed detection of major classes of phospholipids in a single experiment. Precursor ion scanning spectra could be acquired simultaneously for acyl anions of major fatty acids in negative ion mode and identified the fatty acid moieties and their relative position at the glycerol backbone in individual lipid species.

As the method allowed us to detect multiple classes of lipids in parallel in a single mass spectrometric experiment, it is expected to be more comprehensive, accurate and robust and deliver higher throughput compared to conventional methods.

Selected references:
Ekroos, K. et al (2002) Anal Chem 74, 941-949
Ekroos et al (2003) J. Lipid Res 44, 2181-2192

V. Mass Spectrometry Service: Characterization of Gel Separated Proteins

Anna Shevchenko

Mass Spectrometry Core Facility supports researches of MPI CBG and Biopolis Dresden with mass spectrometric identification of gel separated proteins and characterization of intact proteins on a fee for service basis. For further details please contact Facility Leader Anna Shevchenko ashevche@mpi-cbg.de.; The Facility home page is currently under construction.

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Andrej Shevchenko research focus group leader contact projects doctoral theses publications Ongoing Projects I. Mass Spectrometric Identification of Proteins from Organisms with Unknown Genomes Anna Shevchenko, Vinzenz Link Until recently mass spectrometry was not conducive for facile protein identification from organisms with unsequenced genomes, which represent an overwhelming majority of the biosphere. Despite the relative deficiency of available genomic sequences, the emerging interplay of mass spectrometry and bioinformatics is now significantly expanding the organismal scope of proteomics (Liska & Shevchenko, 2003). In collaboration with bioinformaticians from European Molecular Biology Laboratory (EMBL, Heidelberg) and Harvard Medical School (Boston) we have developed software tools, which efficiently utilize mass spectrometry data for identification of proteins by homology searching. Mass Spectrometry driven BLAST (MS BLAST) (Shevchenko et al, 2002) is a generic webÆaccessible tool for sequence-similarity protein identification in a protein database. MS BLAST can utilize a large number of inaccurate, degenerate and redundant sequence proposals obtained by automated de novo interpretation of tandem mass spectra, obtained by NanoES, LC MS/MS or MALDI Q TOF MS. The alternative MultiTag method (Sunyaev et al, 2003) is based on error-tolerant alignment of multiple peptide sequence tags, and enables confident identification of homologous proteins using very short stretches of peptide sequences consisting of 2-4 amino acid residues. We have successfully applied the methods to characterize the proteomes of organisms that are very distantly related to organisms with sequenced genome, such as the extreme halotolerant Dead Sea alga Dunaliella, the Amazon moth Cerodirphia speciosa, and the African clawed frog Xenopus laevis, as well as a variety of popular mammalian model organisms. Selected references: Liska, A.J. and Shevchenko, A. (2003) Proteomics 3, 19-28 Shevchenko, A et al (2001) Anal Chem 73, 1917-1926 Sunyaev, S et al (2003) Anal Chem 75, 1307-1315 MS BLAST site at EMBL server: http://dove.embl-heidelberg.de/Blast2/msblast.html II. Deciphering Protein Complexes and Protein Interaction Networks Anna Shevchenko, Natalie Schmalz, Henrik Thomas In collaboration with the group of Prof. Francis Stewart (BIOTEC Technical University of Dresden, http://www.biologie.tu-dresden.de/stewart/index.htm ) we employed a combination of Tandem Affinity Purification (Rigaut G.et al, 1999) and mass spectrometry for deciphering protein complexes and protein interaction networks in budding yeast and in fission yeast. Genes of interest were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates and identified by mass spectrometry. A sequential strategy of epitope tagging, immunoaffinity isolation and mass spectrometric identification of subunits of protein complex provides multiple readout of its composition and allows semi-quantitative estimation of the stoichiometric ratio of their subunits (Seol et al, 2001). In a recent study encompassing 38 protein baits, potentially involved in chromating remodeling, pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. On average, every fourth protein is shared between complexes of different functionality thus charting segments of a protein interaction network (Shevchenko et al, 2002). We observed remarkable conservation of the composition of orthologous protein complexes purified from between budding and fission yeasts their proteomic environment and links to other orthologous complexes are dramatically altered (Roguev et al, 2003). Selected references: Seol J.H.et al. (2001) Nature Cell Biol. 3, 384-391 Shevchenko A. (2002) Mol Cell Proteomics 1, 204-212 Roguev A. (2004) Mol Cell Proteomics 3, 125-132 III. Quantitative Analysis of Gel Separated Proteins Henrik Thomas, Natalie Schmalz One-dimensional and two-dimensional polyacrylamide gel electrophoresis is a recognized method of separating complex protein mixtures. Although identification of gel separated proteins has become routine, their quantification of these proteins has not been explored. Using 18O labeled peptides as internal standard, we quantified the yield of in-gel digestion of proteins by trypsin (Shevchenko A & Shevchenko, A, 2001) by MALDI TOF mass spectrometry. Based on the kinetics of in-gel digestion, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage (Havlis et al, 2003). Selected References: Havlis, J., et al (2003) Anal Chem 75, 1300-1306 Shevchenko A, and Shevchenko, A. (2001) Anal Biochem 296, 279-283 IV. Quantitative Profiling of Lipids using Quadrupole Time-of-Flight Mass Spectrometry Kim Ekroos, Christer Ejsing We employ a hybrid quadrupole time-of-flight mass spectrometer featured with ion trapping capabilities (QSTAR Pulsar i) for quantitative profiling of total extracts of endogenous phospholipids. Simultaneous acquisition of precursor ion spectra of multiple fragment ions allowed detection of major classes of phospholipids in a single experiment. Precursor ion scanning spectra could be acquired simultaneously for acyl anions of major fatty acids in negative ion mode and identified the fatty acid moieties and their relative position at the glycerol backbone in individual lipid species. As the method allowed us to detect multiple classes of lipids in parallel in a single mass spectrometric experiment, it is expected to be more comprehensive, accurate and robust and deliver higher throughput compared to conventional methods. Selected references: Ekroos, K. et al (2002) Anal Chem 74, 941-949 Ekroos et al (2003) J. Lipid Res 44, 2181-2192 V. Mass Spectrometry Service: Characterization of Gel Separated Proteins Anna Shevchenko Mass Spectrometry Core Facility supports researches of MPI CBG and Biopolis Dresden with mass spectrometric identification of gel separated proteins and characterization of intact proteins on a fee for service basis. For further details please contact Facility Leader Anna Shevchenko ashevche@mpi-cbg.de.; The Facility home page is currently under construction.
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Andrej Shevchenko

Ongoing Projects