Proteomics
The Proteomics Unit offers a service for the study of proteins to researchers at the University of Vigo and other institutions, both public and private. Proteomics is defined as the study of the proteome (set of proteins expressed by the complete genome of a cell or tissue at a given time, under precise and determined conditions), using biochemical methods of protein separation on a large scale, in order to obtain a global and integrated view of cellular processes.
The spectrum of proteomics study is divided into four major areas: expression proteomics, differential expression proteomics, interaction proteomics, structural or cellular mapping proteomics and modificomics, which focuses on the analysis of post-translational modifications. In the Proteomics Unit, both protein identification and differential expression analysis are carried out, following three methodological approaches:
1. Relative quantification of protein expression level by gel image analysis:
- Protein separation by one-dimensional electrophoresis in polyacrylamide gels, under native (NATIVE-PAGE) or denaturing (SDS-PAGE) conditions.
- Separación de protes mediante electrofores bidimensional (2-DE), na que a separación en xeles SDS-PAGE é precedida por un fraccionamiento base no punto isoeléctrico (isoelectroenfoque, IEF).
- Image revealed by means of colorimetric tinting (Coomassie Blue, Silver Nitrate) or fluorescence (Sypro, Oriol, etc).
- Image acquisition, alignment and analysis.
2. Protein identification by enzymatic digestion followed by mass spectrometry analysis and database search:
- Peptide fingerprinting (MALDI-MS) and peptide fragmentation spectra (MALDI-MS/MS) analysis. Used to identify purified proteins, bands on SDS-PAGE gels, and protein spots on 2-DE gels.
- Separation of peptides by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Used to identify proteins in samples of moderate/high complexity, in terms of protein count.
3. Identification and relative quantification of protein expression levels by LC-MS/MS mass spectrometry analysis:
- Isobaric labeling (iTRAQ and TMT). Allows the comparison of up to 10 samples in a single LC-MS/MS analysis.
- Sen marcaxe (Label-free).
Instrumental Facilities
Direct Detect™ Protein Quantification Equipment (Merck Millipore)
Infrared (IR) based spectrometer that measures amide ligands.
Electrophoresis Equipment (Bio-Rad):
- Protean IEF System
Protein separation system based on the isoelectric point in strips of 7, 10 or 17 cm in length.
- Mini-PROTEAN Tetra Cell
Protein separation system based on the relative molecular mass in 7 cm x 8 cm x 0.75 mm minicells.
- Protean II XL
Protein separation system based on their relative molecular mass in 19.5 cm x 18.4 cm x 1.5 mm gels.
ChemiDoc™ XRS+ Image Analysis Equipment (Bio-Rad)
High resolution and sensitivity CCD camera for acquiring images of colorimetry, fluorescence, chemiluminescence, etc. gels and blots.
PharosFX™ Plus (Bio-Rad) Imaxe Analysis Equipment.
Multiple lasers with diverse applications: ethidium bromide, SYPRO Ruby, Deep Purple, Alexa Fluor 488, 532, 546, and 635, Cy2, Cy3, Cy5, Pro-Q Diamond, etc.
ÄKTApurifier 10 FPLC Liquid Chromatography System (GE Healthcare)
Peptide and protein purification system with Frac-950 fraction collector, triple UV detector (190-700 nm) and P-903 pump (10 ml/min, 250 bar, 25 MPa).
Nano-UPLC Vanquish NEO (Thermo Fisher)
Automatic injector with temperature control. Gradient formation and vacuum solvent degassing system.
MALDI-TOF/TOF Autoflex III Smartbeam Mass Spectrometer (Bruker)
MALDI (Matrix-Assisted Laser Desorption/Ionization) and Time-of-Flight (TOF) analyzer. N2 laser. Two-stage gridless reflector for pulsed ion extraction.
SolariX XR Mass Spectrometer (Bruker)
Horizontal superconducting magnet with a 7 Tesla field. ESI (Electrospray Ionization), nanoESI and MALDI sources. INFINITY cyclotron ion resonance measurement cell.
LTQ-Orbitrap ELITE Mass Spectrometer (Thermo Fisher)
Hybrid system of ion trap-orbitrap mass spectrometer. Coupled to EASY-nLC 1000 UHPLC system. High resolution and fragmentation power using CID, HCD and ETD.
Applications
Biomedicine: search for biomarkers, development of diagnostic and prognostic methods, identification of target proteins for therapy, development of drugs (anticancer, antimicrobial, etc.), development of vaccines, studies of pathogenesis mechanisms, etc.
Cellular biology and biochemistry: study of metabolites, toxins, poisons, etc.
Ecology and evolutionary biology: ecological adaptation, speciation, etc.
Food technology: development, improvement, quality control and food safety.
Sample requirements
Sample types: biological fluid, protein or peptide extract in solution, protein band or spot, or a gel. The service does not accept samples of biological tissue or cells as it does not have the necessary equipment for protein extraction. Protein quantification with the Direct DetectTM equipment: recommended for samples between 0.25 and 5mg/mL of protein. A minimum volume of 2µL is required. Not compatible with high concentrations of bicarbonate buffer, EDTA, GndCl, HEPES, NaOH, Tris, Urea, glycerol, SDS, Tween, etc. It is necessary to provide the service with the sample buffer to use as a blank.
SDS-PAGE: the sample must have an approximate concentration of between 1 and 10 µg/µL.
2D-PAGE: perform a cleanup of the protein extract to remove salts from the sample and the buffers used in its preparation (PBS, RIPA, HEPES, etc.). The presence of salts is critical due to their interference with isoelectrofocusing (IEF). The limit of total ion concentration that the sample can have is 40mM. SDS is incompatible with IEF due to its ionic nature. The final extract must be delivered in electrophoresis buffer or lyophilized. The recommended amount of protein for an analytical gel (50-200µg) is lower than for a preparative gel (500µg-1.5mg).
Cutting and enzymatic digestion: cut out the protein band or spot with a clean scalpel, avoiding areas of unstained gel and protein mixtures. Carry out the entire preparation with gloves and in a laminar flow cabinet to avoid contamination with keratins, which are found in hair, skin and clothing, as they make it difficult to identify the proteins of interest.
MALDI-MS/MS analysis: avoid the presence of salts, ionic (SDS) and non-ionic (Triton X-100, Tween, etc.) detergents, non-volatile solvents (glycerol, PEG, β-mercaptoethanol, DMSO and DMF), chaotropic agents (Urea, GndCl) and other contaminants that hinder sample crystallization with the matrix, desorption or ionization. Non-interfering TFA, formic and acetic acids, HCl, DTT, NH4OH and volatile organic solvents.
LC-MS/MS analysis: use reagents of high purity for HPLC. The sample quantity will depend on the type of analysis required (consult the service personnel). If the sample contains urea, salts or other possible interferents (SDS, PEG, glycerol, TFA, etc.), it is essential to perform a thorough sample cleaning (ZipTip or equivalent).