Proteomics Area

Proteomics and protein analysis technologies are fundamental tools for molecular biology, biomedicine and translational research as understanding the changes in protein abundances, protein localization, protein modifications, and protein interactions, are crucial steps toward the elucidation of mechanism of action for health and disease conditions.

The proteomics area includes the mass spectrometry-based Proteomics Unit of the Center for Genomic Regulation and University Pompeu Fabra (Proteomics Platform, Proteomics-CRG) and the Proteomics facility of the COS node that offer a common catalogue of services with a unique access portal.

The Proteomics-CRG is a joint proteomics center of the CRG and the University Pompeu Fabra (UPF) that offers mass spectrometry-based proteomics analyses with strong added value to the scientific community. The Proteomics-CRG is allocated in the Parc de Recerca Biomèdica de Barcelona (PRBB) building and embedded in the Core Facilities program of the CRG, and it is an active part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed, Instituto de Salud Carlos III)”.

The Proteomics facility of the COS is focused on profiling and quantifying proteins of experiments that include chromatographic and electrophoretic systems for protein and peptide separation coupled to the state-of-the-art mass spectrometry systems. Its relevance in the sector lies in the provision of MS-imaging, intact protein analysis and surface Plasmon resonance interaction investigation, among other common proteomic analysis.

The Proteomics area of the OmicsTech can guide researchers at any project phase from the design of the experiments to the data analysis.

For further information and discussion of possible ways of collaboration with the PP-CRG or COS facilities, please contact proteomics@omicstech-icts.org

Applications

Characterization of protein-protein interactions

Characterization of protein interactors of a target protein pulled down by affinity purification, immunoprecipitation or labelling strategies. The expected outcome is a list of proteins that interact with a bait protein using statistical assessment to pinpoint bona fide interactors.

Protein identification

Identification of a protein in a gel band or overexpressed in the sample of interest. The expected outcome is a list of peptides and proteins identified in the sample of interest.

Proteome quantification

Quantification of proteins in different conditions relative to a control either without any prior protein labelling or using labelling techniques such as SILAC, TMT, etc. Sample types include whole cell extracts, exosomes, secretomes, and liquid biopsies, among others. The expected outcome is a list of quantified proteins relative to control samples, in which each protein is provided with a fold-change and an adjusted p-value.

Ultra deep proteome quantification (fractionation)

Quantification of proteins in different conditions relative to a control combining labelling techniques such as SILAC, TMT, etc. and sample fractionation. Sample types include whole cell extracts, exosomes, secretomes, and liquid biopsies, among others. The expected outcome is a list of quantified proteins relative to control samples, in which each protein is provided with a fold-change and an adjusted p-value.

Phosphoproteome quantification

Quantification of phosphopeptides in different conditions relative to a control without requiring any prior protein labelling or using labelling techniques such as SILAC, TMT, etc. The expected outcome is a list of quantified phosphopeptides relative to control samples, in which each phosphopeptide is provided with a fold-change and an adjusted p-value.

Post-translational modifications quantification of a purified protein

Quantification of specific post-translational modifications (PTMs) in purified proteins including phosphorylations, acetylations, methylations or ubiquitylations. The expected outcome is a list of quantified post-translational modified peptides relative to their control samples. Each modified peptide is provided with a fold-change and an adjusted p-value.

Post-translational modifications quantification in histones

Quantification of specific post-translational modifications (PTMs) in purified proteins including phosphorylations, acetylations, methylations or ubiquitylations. The expected outcome is a list of quantified post-translational modified peptides relative to their control samples. Each modified peptide is provided with a fold-change and an adjusted p-value.

Structural elucidation by mass spectrometry

Structural elucidation of purified protein complexes using cleavable chemical crosslinkers and mass spectrometry.

Targeted protein quantification

Targeted quantification of a set of proteins of interest in large number of samples. The expected outcome is a list of quantified proteins relative to control samples, in which each protein is provided with a fold-change and an adjusted p-value.

Bioinformatic Applications

Spectra interpretation

Spectra interpretation using commercial and open source software like Proteome Discoverer, MaxQuant, PEAKS, Spectrum Mill, Protein Scape, Skyline and Mascot.

Statistical analysis

Univariate and multivariate statistical analyses (PCA, clustering, correlations, PLSDA, …). Mass Profiler Professional (Agilent)-Generic and specific Agilent data-, Gene Spring (Agilent)-Generic and specific Agilent data-, Sieve 1.4 (Thermo)-specific for Thermo LC-MS data, Progenesis (Non Linear Dynamics) – specific gel imaging analyses.

Bacterial taxonomical identification

Bacterial taxonomic identification using MALDI-TOF protein spectra. Biotyper (Bruker)

Equipment

Orbitrap Mass Spectrometers
(Velos Pro, XL, Fusion Lumos, Eclipse)

The LTQ Orbitrap Velos Pro and XL are hybrid high-resolution mass spectrometers that consist of a linear ion trap and an orbitrap mass analyser. 

The Orbitrap Fusion Lumos is a tri-hybrid high-resolution mass spectrometer that consists of a quadrupole, an ion trap and an orbitrap mass analyser.

Orbitrap mass spectrometers deliver excellent mass resolution (FWHM) >100,000 at m/z 200 and a sub-1 ppm mass accuracy in a single compact and easy-to-use instrument. These high-resolution accurate-mass systems detect a wide range of compounds, peptides and small molecules during both targeted and untargeted analyses, without losing selectivity or sensitivity.

Triple-quadruple Mass Spectrometers
(Sciex 5500, Agilent 6490, Agilent 6470)

The QTRAP/QQQ 5500 is a high-end triple quadrupole with impressive speed, sensitivity and robustness, specially indicated for targeted projects for the quantification of peptides and small molecules.

The Agilent 6490 Triple Quadrupole mass spectrometer incorporates iFunnel technology to achieve new levels of sensitivity and dynamic range for detection of target compounds in complex matrices. The innovative design of the ion funnel reduces contamination and neutral molecules thus improving overall signal and reducing system noise.

The Agilent 6470 Triple Quadrupole is equipped with an Agilent Jet Stream ion source and a curved geometry collision cell. These innovations enable the reliable detection of target compounds at low levels in diverse matrices, while achieving wide linear dynamic range. 

MALDI-TOF/TOF Mass Spectrometers
(Bruker UltrafleXtreme)

The MALDI (Matrix Assisted Laser Desorption/Ionization) tandem mass spectrometer specially designed for automated MS and MS/MS high throughput identification of samples. Tandem Mass Spectrometry is a technique that utilizes more than one mass selective stage in a mass spectrometer.

q-TOF Mass Spectrometers
(Agilent 6550)

The Agilent 6550 Q-TOF incorporates iFunnel technology which increases ion transfer and enables higher sensitivity and lower detection levels. It provides both qualitative and quantitative data with high resolution and accurate mass.

Surface plasmon resonance

Biacore is a system for real-time biomolecular interaction analysis using a SPR (surface plasmon resonance). This technique monitors the formation and dissociation of biomolecular complexes on a sensor surface as interaction occurs. SPR is a non-invasive optical measuring technique and does not require any labelling of the interacting components.

Robots and liquid handling systems
(EXQuest spot cutter, Bravo)

Bravo is Agilent's automated liquid handling platform for processing up to 96 samples.

 

EXQuest spot cutter is a precision instrument used to reduce manual labor for high resolution two-Dimensional Gel Electrophoresis (2D-GE) and two-dimensional difference in gel electrophoresis (2D-DIGE) in Proteomic studies. Detailed image analysis of sample set expression profiles and automated recovery of proteins from gels followed by proteolytic digestion of proteins of interest for further analysis by mass spectrometry is also carried out.

 

Cryostat
(Leica CM1950)

The cryostat is an instrument that has been designed for rapid freezing and sectioning of tissue samples.

Multiplex immunoassays
(Luminex, BIO-PLEX 200)

Luminex MAGPIX system is a compact instrument for multiplexed protein quantification based on immunoassays. The system can perform up to 50 different tests in a single reaction volume and reads 96-well-plates in a reduced amount of time. The Luminex MAGPIX system is supported by a broad menu of commercially available assay kits, as well as reagents to build your own assays.