Proteomics and Genomics

The Proteomics and Genomics Facility CIB Margarita Salas is a support service of scientific and technological research for both CIB investigators, and researchers from other centers and institutions. The service  is a part of the laboratory network of the Community of Madrid. Since 2012 the laboratory is certified by AENOR according to UNE-EN ISO9001:2015 in “The molecular weight determination by mass spectrometry.”

IQNetES-0286-2009_2021-12-09. 

SERVICES OFFERED:

PROTEOMICS:

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

• Proteins separation by mono (SDS-PAGE) or two-dimensional electrophoresis in 7 cm gels and subsequent colloidal Coomassie Blue staining.
• Molecular mass determination of biological or synthetic material (MALDI-TOF).
• Protein identification by their peptide fingerprint and peptide fragmentation (MALDI-TOF/TOF).
• Identification of complete proteomes and complex protein mixtures by mass-mass liquid chromatography LTQ Orbitrap Velos or Q-Exactive.
• Identification and relative quantification of protein expression level by mass spectrometry analysis LTQ Orbitrap Velos or Q-Exactive (SILAC, iTRAQ, label-free).
• Selective monitoring of parental ions and their fragments (SIM and PRM, respectively) by Q-Exactive in targeted proteomics experiments.
• Phosphopeptides identification from protein complex mixtures.

GENOMICS:

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

• Biomolecules quantification (NanoDrop spectrophotometer).
• Chip electrophoresis of:
  • RNA, for integrity evaluation prior to microarray or rt-PCR experiments
  • DNA;
  • Proteins.
• Quantitative and qualitative analysis of nucleic acids.

• Real-time PCR.

• The Facility has the Roche Universal Probe Library collection of human genome probes.

SCIENTIFIC EQUIPMENT:

PROTEOMICS

• 2 Protean IEF cell (BIO-RAD), for first dimension of 2D gels in strips of 7 cm and different nonlinear pH 3–10 , 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.
• MALDI-TOF-TOF Autoflex III (Bruker) Mass Spectrometer, for molecular mass determination and protein identification through peptide fingerprinting and fragmentation of peptides.
• 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.

GENOMICS

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