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Proteomics

Scientific Coordinator

Xosé R. Bustelo

+34 923 294 720

xbustelo@usal.es

Proteomics Units

Centro de Investigación del Cáncer (CSIC-USAL).

Campus Universitario Miguel de Unamuno s/n E-37007 Salamanca (Spain)

+34 923 294 720

nibarrola@usal.es

Personnel

No information is available

In this Facility we strive to provide investigators with access to the latest proteome analysis technologies.

These technologies are implemented with a broad spectrum of techniques for protein and peptide separation as well as mass spectrometry-based techniques to characterize and quantify analytes from complex biological samples.

The philosophy of this Resource is to provide full services to both internal and external customers. We provide technical advice about proteomics experimental design and carry out quality control of the processes undertaken. Through our involvement in ProteoRed and ABRF multicentric studies we contribute to the evaluation and set up of new proteomic approaches.

The facility itself and its experimental protocols have been certified by ISO9001. The Occupational Health and Safety Management System of the facility has been also certified using the OHSAS18001 system.

In this Facility we provide complete services for:

  • Protein separation by electrofocusing in IPG strips. Separation of proteins according to their pI is done in an Ettan IPGphor.

    http://www5.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~2delectrophoresis_handbook

  • Protein separation by SDS-PAGE. Separation of proteins in a SDS-acrylamide:bis-acrylamide denaturing gel according to their molecular weight.

    http://www5.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~2delectrophoresis_handbook

  • Protein separation by 2D-electroforesis. Protein separation by electrofocusing in IPG strips is followed by SDS-PAGE separation.

    http://www5.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~2delectrophoresishandbook

  • Gel staining with Coomassie. Staining of gel separatedproteins with Coomassie brilliant blue G-250.

  • Gel staining with silver. Staining of gel separated proteins with silver using a modification of Heukeshoven and Dernick protocol, Electrophoresis 6, 103-112, (1988), mass spectrometry compatible.

  • Protein or peptide fractionation by in solution IEF. Protein or peptide in solution fractionation by IEF using the 3100 OFFGEL fractionator.

    http://www.chem.agilent.com/Library/usermanuals/Public/G3100-90001_OFFGEL_UserManual_ebook.pdf

  • Protein or peptide fractionation by HPLC. Protein or peptide separation by gel filtration, ion exchange or reverse phase chromatography using HPLC 1100.

  • Enrichment of phosphopeptides by IMAC. Phosphopeptides are enriched by Fe3+ chromatography following SIMAC procedure. (Thingholm et al. Molecular & Cellular Proteomics 7:661-671,2008.).

  • Enrichment of phosphopeptides by TiO2. Phosphopeptides are enriched by TiO2.

  • In gel protein digestion. Digestion with trypsin of a protein gel spot or band. (Schevchenko et al. Anal. Chem. 1996, 68 850-858.).

  • In solution protein digestion. Digestion with trypsin of a protein sample in solution.

  • Desalting and concentration of peptide digests by C18. Low abundance or dirty tryptic peptide samples are cleaned and concentrated using reverse phase C18 columns before MS analysis.(Rappsilber J, Anal Chem. 2003 Feb 1;75(3):663-70.).

  • Peptide Mass Fingerprinting analysis by MALDI-TOF MS. Identification of a protein by peptide mass fingerprinting analysis. The masses of the tryptic peptides generated by digestion of single protein are analyzed by a MALDI-TOF mass spectrometer. The pattern of masses obtained is compared against the pattern of masses of each protein in a database of the same organism digested «in silico» with the same endoprotease. (Pappin et al. Current Biol. 1993, 3, 327-332.).

  • Protein analysis by LC-MS/MS of low, medium or high complexity protein samples. Peptides derived from digested protein mixtures will be separated by reverse phase chromatography using a nanoUPLC coupled to the mass spectrometer. Different gradient lengths are used depending on the complexity of the protein sample. Eluting peptides are directly analyzed by MS/MS with the LTQ-Orbitrap velos (Olsen et al. Mol Cell Proteomics. 2009 Dec;8(12):2759-69.

  • Relative quantitation of protein samples by label free and SILAC based mass spectrometry analysis.

  • Bioinformatics analysis.

    • Identification of posttranslational modifications.

    • Differential proteomics.
    • De novo sequencing.
    • Protein and Peptide Molecular Weight Analysis.
    • Molecular weight analysis of purified proteins or peptides by MALDI-TOF.
  • Analysis of protein interaction by SPR. Interaction analysis of proteins by BIACORE X.

  • Quantization of nucleotides by HPLC.

  • Protein Microarrays.

  • VTT protein expression .

  • > 9000 human proteins already cloned

  • In solution protein digestion. Digestion with trypsin of a protein sample in solution.

  • Desalting and concentration of peptide digests by C18. Low abundance or dirty tryptic peptide samples are cleaned and concentrated using reverse phase C18 columns before MS analysis.(Rappsilber J, Anal Chem. 2003 Feb 1;75(3):663-70.).

  • Peptide Mass Fingerprinting analysis by MALDI-TOF MS. Identification of a protein by peptide mass fingerprinting analysis. The masses of the tryptic peptides generated by digestion of single protein are analyzed by a MALDI-TOF mass spectrometer. The pattern of masses obtained is compared against the pattern of masses of each protein in a database of the same organism digested «in silico» with the same endoprotease. (Pappin et al. Current Biol. 1993, 3, 327-332.).

  • Protein analysis by LC-MS/MS of low, medium or high complexity protein samples. Peptides derived from digested protein mixtures will be separated by reverse phase chromatography using a nanoUPLC coupled to the mass spectrometer. Different gradient lengths are used depending on the complexity of the protein sample. Eluting peptides are directly analyzed by MS/MS with the LTQ-Orbitrap velos (Olsen et al. Mol Cell Proteomics. 2009 Dec;8(12):2759-69.

  • Relative quantitation of protein samples by label free and SILAC based mass spectrometry analysis.

  • Bioinformatics analysis.

    • Identification of posttranslational modifications.
    • Differential proteomics.
    • De novo sequencing.
    • Protein and Peptide Molecular Weight Analysis.
    • Molecular weight analysis of purified proteins or peptides by MALDI-TOF.
  • Analysis of protein interaction by SPR. Interaction analysis of proteins by BIACORE X.

  • Quantization of nucleotides by HPLC.

  • Protein Microarrays.

  • IVTT protein expression .

  • > 9000 human proteins already cloned

Equipment

  1. Electrophoresis 2D
    • Ettan IPGphor (Amersham,, GE Healthcare).
    • Ettan Dalt-6 Electrophoresis system (Amersham).
    • Hoefer miniVE electrophoresis (Amersham).
    • Hoefer SE 600 Ruby (Amersham).
  2. Image acquisition
    • Escaner Epson perfection 1640SU (Proteineersp, Bruker).
    • Escaner FLA-3000 Series, (Filtros: Y520, O580, R675Laser: 473nm, 633nm) (Fujifilm). Spot picking and sample digestionRobots.
    • Proteineersp, SPOTPICKER (Bruker).
    • Proteineerdp, DIGESTOR (Bruker).
  3. HPLC
    • HPLC1100 (Agilent).
    • Surveyor LC pump (ThermoFinnigan) coupled with the LCQ-DECA XP.
    • NanoAcquity UPLC (Waters) coupled with the LTQ Orbitrap velos.
  4. In solution IEF
    • 3100 OFF GEL fractionator (Agilent).
  5. Mass Spectrometers
    • LCQ-DECA XP (ThermoFinnigan).
    • LTQ-Orbitrap velos with ETD (ThermoScientific).
  6. Protein interaction
    • Biacore X (Biacore, GE Healthcare).
  7. Protein Microarrays
    • Ultra-Marathon Microarray Printer (Arrayjet).
    • Scanner Sensovation.
    • Array Processor M2.