The molecular weight determination by mass spectrometry.

Separation of proteins through 2D gel electrophoresis.

The Proteomics and Genomics Service CIB is a support service of scientific and technological research for both CIB investigators, and researchers from other centers and institutions. The service belongs to ProteoRed, a network integrated in the Biomolecular and Bioinformatics Resources Platform (PRB2) from the Carlos III Health Institute and is a part of the laboratory network of the Community of Madrid. Since 2012 the laboratory is certified according to ISO9001 with ER-0286/2009 AENOR ISO9001 Certification code.

Satisfaction survey 2016.

PROTEOMICS

In the Service it can be performed separation, quantification, identification and characterization of proteins from prokaryotic and eukaryotic organisms by two-dimensional electrophoresis, MALDI-TOF/TOF and multidimensional liquid chromatography (LTQ Orbitrap Velos) mass spectrometries.

Current equipment:

GENOMICS

The Facility has the necessary tools for basic studies of functional genomics and expression.

  • 2 Protean IEF cell (BIO-RAD), for first dimension of 2D gels in strips of 7 and 17 cm and different pH ranges: 3-10 non linear, 3-10 linear and 4-7 linear gradients.
  • 1 Multigel Miniprotean Dodecacell (BIO-RAD), for up to 12 simultaneous electrophoresis gels of 8 x 7 cm. This equipment allows the optimization of the electrophoretic separation conditions with a smaller amount of sample and a low cost.
  • 1 Protean II Multicell (BIO-RAD), for simultaneous electrophoresis of up to 6 gels of 19 x 17 cm.
  • PD Quest software (BIO-RAD),for image analysis of two-dimensional gels.
  • Exquest Spot Cutter (BIO-RAD), for detection and automatic excision spots of gels stained with silver, Coomassie blue or fluorescent SYPRO Ruby..
  • Protein digestor INTAVIS, for automation of the entire process, ranging from the tryptic digestion "gel" to final preparation of the samples in the MALDI plate.
  • MALDI-TOF-TOF Autoflex III (Bruker) Mass Spectrometer, for molecular mass determination, protein identification through peptide fingerprinting and fragmentation of peptides in order to know their sequences.
  • Mass spectrometer LTQ Orbitrap Velos (Thermo Scientific) with a Proxeon´s EASY-nLC. LTQ Orbitrap Velos is a hybrid mass spectrometer incorporating the LTQ Velos dual cell linear trap and the Orbitrap analyzer. The LTQ Orbitrap Velos is a high performance LC-MS and MSn system, combining rapid LTQ Ion Trap data acquisition with high mass accuracy Orbitrap mass analysis. Significantly, the Orbitrap includes a fragmentation regime, higher-energy C-trap dissociation (HCD) that is complementary to the LTQ’s collision induced dissociation (CID) option. These additional, alternative fragmentation capabilities are essential for the success of the diverse projects focused on phosphoproteomics; posttranslational modification analyses and “middle-down” proteomics. The outstanding data quality and sensitivity of the LTQ Orbitrap Velos make it ideal for the analysis of complex mixtures, the identification of low level proteins, structural elucidation of metabolites in biological matrices, differential proteomics and quantitative proteomics (SILAC, iTRAQ, Label-free).
  • Mass Spectrometer Q-Exactive (ThermoScientific) with a Proxeon’s nano-UPLC Easy-LC 1000. Q-Exactive consists of a quadrupole, an ion trap (C-trap) and an Orbitrap analyzer. Because of its configuration, Q-Exactive is particularly suitable for performing "shotgun" proteomics and targeted proteomics experiments (based on SIM -single ion monitoring- and PRM -parallel reaction monitoring-). It combines the advantages of the quadrupole for mass filtering and the Orbitrap for mass resolution and accuracy. Currently Q-Exactive is the only available equipment that can operate alternatively in experiments of either qualitative massive or quantitative targeted proteomics.
  • Nanodrop ND-1000. UV-visible spectrophotometer which allows fast quantification of DNA, RNA, proteins, labeled nucleic acids and proteins, etc., consuming only 1-2 μl of sample.
  • 2 Experion automated electrophoresis on chip system (BIO-RAD). In 30 minutes and with minimum sample consumption, the system performs the electrophoretic separation and detection of proteins in the range of 10-260 kDa (10 samples per chip), RNA (12 samples per chip) and DNA (11 samples per chip), being very useful for routine integrity analyses of RNA preparations.
  • 2 Thermocyclers for real-time PCR (rt-PCR): iQ5 (BIO-RAD) and LightCycler 96 (Roche). Detection by fluorescence (SYBR Green or specific probes) allowing the multiplex analysis of up to 5 fluorophores. 96 well plates are used. Its applications include relative quantification of gene expression, allelic discrimination (SNPs), absolute quantification, viral load determination, endpoint analysis, etc.