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Protein and Peptide Molecular Weight Analysis |
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Molecular weight analysis by MS-MALDI (MALDI-TOF) |
 This analysis determines very precisely the molecular weight of peptides and proteins. As regards to the peptides not bigger than 5 kDa, measured mass errors are below 50 ppm in reflector mode. Molecular masses of proteins of up to 70 KDa can be measured in linear mode with mass errors lower than 200 ppm at 10000 KDa. Although this is not the mass limit of the instrument analysis of proteins of proteins higher than 100 KDa becomes ingreasingly difficult.
Fairly complex peptide or protein mixtures can be analyzed without previous separation (The tecnique can be used to obtain proteome fingerprints which is the basis of technologies such as SELDI).
Samples must be delivered dissolved in a buffer, preferably a volatile one, with a salt concentration lower than 50 mM. |
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Molecular weight analysis by ESI-MS (Linear Ion Trap or Orbitrap).
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This analysis determines very precisely the mass of peptides and proteins of up to 60 kDa. Electrospray ionization allows the analysis of peptides with labile groups such as phospho- or nitrosylated peptides as well as of non-covalent complexes given they are stable in solution.
Samples are directly introduced in the spectrometer either by infusion of by nanospray so that the sample must be as pure as possible (>90%) to prevent interferences. A pre-purification by liquid chromatography using volatile solvents and buffers is the most effective method for that purpose.
Samples must be delivered dissolved in a buffer, preferably a volatile one and with a salt concentration lower than 10 mM.
Linear Ion Trap
This instrument provides of mass measurements with resolutions of up to 40000 in ZoomScan.
Orbitrap
The Orbitrap is a high resolution analyzer that provide resolutions of up to 100000 in full scan mode with accuracies of about 2 ppm for peptides and 10 ppm for proteins of about 10-20 KDa. |
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Protein Identification |
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Peptide Mass Fingerprinting by MS or MS+MS/MS (MALDI TOF/TOF) |
This approach is the more effcient for rapid identification of isolated proteins from sequenced organisms. The analysis can be carried starting from a liquid solution of the target protein but very often the starting material are proteins separated by gel electrophoresis.
First, the gel spots/bands with the target proteins are cut and the protein in the gel slice digested in situ with a specific enzyme, generally trypsin. Then, digestion peptides are extracted and analyzed by MALDI TOF.
MS
For the MALDI MS analysis, a fraction of the peptide digest is placed on a MALDI plate where it is mixed with the MALDI matrix (alpha-ciano-4-hidroxicinamic acid) and it is let to dry. The crystalized sample is analyzed in a MALDI-TOF ABSCIEX 4800 TOF/TOF (Applied Biosystems, Barcelona) mass spectrometer using the positive ion reflector mode. The analysis is made in automatic mode averaging the spectra obtained from 4000-5000 shots in the m/z range from 750 to 4500. Each spectrum is calibrated externally by using a standard 5-peptide mix (des-Arg1-bradykinin (Mr 904.46), Glu1-fibrinopeptide B (Mr 1570.68), angiotensin-1, (Mr 1296.69), ACTH 1-17 (Mr 2093.09), ACTH 18-39 (Mr 2465.20), ACTH 7-38 (Mr 3657.93) and, when possible, an internal calibration using the ions derived from the trypsin auto-digestion (Mr 842.5100, 1045.5642, 2011.1046, 2807.3145 y 3337.7577). The spectra obtained give information on the accurate mass of the digestion peptides present in the sample. The colection of these peptide masses constitutes a fingerprint of the original protein sequence. Proteins are identified from these peptide mass fingerprints (PMF) using bioinformatic tools that match them with those expected from the sequences in protein databases.
MS+MS/MS
Sometimes, when only a few digestion peptides are observed for a protein or when there are interferences in the MALDI spectra from other contaminating proteins, the PMF analysis can fail or produce ambiguous results or results of low confidence. For highly confident identification, the PMF analysis can be followed by an MS/MS analysis where several of the most abundant ions detected in the PMF spectra are automatically selected and fragmented (MS/MS) to obtain additional sequence data that usually provides unambigous identification of the protein.
Whenever the protein cannot be identified via PMF or PMF combined with MS/MS, the peptide extract can be desalted by using a ZipTip C18 (Millipore) and analyzed by electrospray MS/MS (See LC MS/MS identification and nESI MS/MS identification). This approach is complementary to MALDI-TOF/TOF, providing of a different ionization selectivity and often of a more efficent sequenciation procedure. ESI MS/MS is however more time consuming and have a higher economic cost, so that for routine identification and/or identification of high numbers of proteins, you still want to try MALDI TOF/TOF first.
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Protein Identification by LC-MS/MS |
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The sample is digested with trypsin (digestion protocol) and the peptide digest loaded into a capillary chromatography system coupled to the mass spectrometer through an microelectrospray interface. Peptides are separated by reverse phase using an acetonitrile gradient and directly eluted and ionized into the mass spectrometer. The mass spectrometer perform scan cycles consisting of a full scan from where it determines the mass of the peptide eluting at a given moment, followed by 5-10 consecutive MS/MS scans on the corresponding 5-10 more intense peptide signals detected. The identification of the peptides on the digest and hence of the original protein, is carried out from the MS/MS data using the Sequest search engine.
If the proteins being analyzed do not appear in the database then a sequencing de novo is run with PEAKS software.
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Protein Identification by 2D LC-MS/MS |
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The multidimensional liquid chromatography (MDLC) is an efficient alternative to the gel electrophoresis methodology used for the proteome component analysis. Using this technique, complex mixtures of proteins and peptides can be separated through different consecutive chromatographic phases. The most used technique nowadays in proteomics is carrying out a first separation by ion exchange and the subsequent re-analysis of the fragments obtained through the separation in reverse phase. The advantage of this technique compared to the 2DE gels is the high reproducibility, the possibility of automation and the capacity to identify any kind of protein regardless of their molecular weight or isoelectric point features. This technique has helped developing the shotgun proteomics method that is used to identify proteins massively. These methods apply an enzymatic digestion of the protein extracts that, unlike the gel separation, is done previously to the separation phase.
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Protein Identification by nESI-MS/MS (off-line) |
| The nanospray offline analysis sample is loaded into an emmitter that is then mounted into the mass spectrometer's source. Sample flow is generated by the electrostatic attraction of the liquid inside the emitter, which has been changed by high voltage, or by the gentle pressurization of the source. In combination with the analysis in an ion-trap mass spectrometer, this method allows the analysiss of samples with very low concentration due to the accumulation process in the ion-trap. |
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Characterization and protein modifications |
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Identification of Post-Translational Modifications |
The analysis of phosphorylation, nitrosylations and other PTMs is made using mass spectrometry approaches.
The identification of phosphorylation-site involves enriching phosphorylated peptides using IMAC or TiO2 followed by mass spectrometry. The enrichment is critical since phosphorylated proteins often represent 1-2% of the total protein population. This analysis can be done with purified phosphorylated proteins or with protein mixtures.
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De novo Secuencing |
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| De Novo protein sequencing is the process by which the amino acid sequence of a peptide is derived without prior knowledge of the DNA or protein sequence. This differs from sequence database identification, where the protein/DNA sequence is already known. De Novo protein sequencing are performed by MS/MS (see protein identification and PEAKS sections). The MS/MS spectra are interpreted using the PEAKS program. |
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Electrophoresis |
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1D Electrophoresis IEF |
2DE is a separation technique that allows the analysis of complex protein mixtures generally extracted from cells, tissues, fluids or other biological samples. Through this technique, proteins are separated according to their isoelectric point in the first dimension (Isoelectrofocusing, IEF) and according to their molecular size in the second dimension (gel electrophoresis in denaturant conditions SDS-PAGE).
The Isoelectrofocusing runs in polyacrylamide strips of fixed gradients of 7, 18 or 24cm long and with different pl rates (from 3 to 11 in different rates). In order to do that an IEF IPGPhor (GE Healthcare) system is used.
This type of analysis is also available in self-service mode. Users will prepare their own gels working with the materials and reagents available in the proteomics laboratory and following the protocols established. |
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1D Electrophoresis SDS-PAGE |
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This type of analysis is also available in self-service mode. Users will prepare their own gels working with the materials and reagents available in the proteomics laboratory and following the protocols established.
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2D Electrophoresis |
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2DE is a separation technique that allows the analysis of complex protein mixtures generally extracted from cells, tissues, fluids or other biological samples. Through this technique, proteins are separated according to their isoelectric point in the first dimension (Isoelectro focusing, IEF) and according to their molecular size in the second dimension (gel electrophoresis in denaturant conditions SDS-PAGE). The Isoelectro focusing runs in polyacrylamide strips of fixed gradients of 7, 18 or 24cm long and with different pl rates (from 3 to 11 in different rates). In order to do that an IEF IPGPhor (GE Healthcare) system is used. Once they are separated in the gel, proteins have to be made visible using, generally a staining method. The most commonly used is the visible stain (silver, Coomassie brilliant blue, zinc-imidazole negative stain) and the fluorescent stain (SYPRO). Other visualization methodology as the radioactive marking or DIGE (Differential in Gel Electrophoresis) imply the sample marking before the separation and the afterwards visualization with radioactivity detectors or fluorescence scanners. The Isoelectro focusing runs in polyacrylamide strips of fixed gradients of 7, 18 or 24cm long and with different pl rates (from 3 to 11 in different rates). In order to do that an IEF IPGPhor (GE Healthcare) system is used. The separation SDS-PAGE is made by using the electrophoresis GE healthcare system EttanDalt Six. Gels are polymerized in the laboratory by using low fluorescence glass.
This type of analysis is also available in self-service mode. Users will prepare their own gels working with the materials and reagents available in the proteomics laboratory and following the protocols established.
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Differential Proteomics
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2-D DIGE |
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2D-DIGE (Two Dimensional Difference In Gel Electrophoresis) is a gel-based approach for comparative proteomics using fluorescent tags. Distinct fluorescent (Cy 3, 5 and 2) are used to label samples and a universal internal standard prior to 1st/2nd dimension electrophoresis. An automated software program (Progenesis) is used to detect, quantify and annotate differentially expressed proteins.
The analysis include: experimental design, protein extraction and sample preparation, quantitation of proteins, 2D-DIGE analysis and image analysis in an automatic mode (the evaluation and validation of the statistic data will be done always by the customer with our advising).
This type of analysis is also available in self-service mode.
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iTRAQ or TMT Labeling |
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Isotopic labeling (iTRAQ/TMT) is a liquid-based proteomics technique allowing quantitative comparison between 4-6 samples using mass spectrometry. This approach can provide both protein identification and protein ratios in one experiment.
After tryptic digestion, peptides for each sample are labeled with the different reagents. Our approach includes strong cation exchange prior to LC-MS/MS analysis on a high resolution mass spectrometer ( LTQ-Orbitrap).
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Quantitative Targeted Mass Spectrometry |
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We use quantitative mass spectrometry of proteins and peptides by selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) assays for biomarker validation and quantification of protein expression and absolute quantification of proteins and their isoforms.
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Staining |
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Silver Gel Staining |
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Conventional method for general, sensitive visualization of proteins separated by gel electrophoresis previous to their identification by MS. It has been frecuently used for quantitative purposes. Still for precise gel-based quantitative proteomics studies, the DIGE approach is recommended.
This is the classic Silver staining method with some modifications to make it "MS-compatible". Among other changes/modifications, in MS-compatible protocols, glutaraldehyde is replaced by acetic acid. Conventional methods use glutaraldehyde as a fixer that binds proteins onto the gel that has previously been treated with silver salt. This procedure reduces the amount of peptides that can be extracted from the gel after in-gel digestion for MS identification.
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Coomassie Gel Staining. |
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Conventional method for general, fast visualization of proteins separated by gel electrophoresis previous to their identification by MS. It is less sensitive than silver staining and less frecuently used for quantitative purposes.
Coomassie brilliant blue is an organic dye that forms strong, non-covalent complexes with proteins by combination of Van der Waals forces and its interaction with the basic amino groups of aminoacids like arginine, lysine and histidine, as well as with tyrosine. The incorporation of the dye is proportional with the protein quantity, according to the Beer-Lambert law. The number of possible strong union places between the proteins and the dye varies along with the proteins and it correlates with the content of basic aa in the protein rather than with the total mass of protein.
There are two available brillian blue dyes named G and R, respectively. This two dyes have different Color Index numbers and solubilities (being G is less soluble in alcohol and water than R). Staining with conventional coomassie allows to detect from 30 to 100ng of protein but sensibility can be improved by using high temperatures while staining and destaining, or by using colloidal Coomassie brilliant blue. In this protocol the stain is added to the gel dissolved in methanol/water/acetic acid in a 50/40/10 proportion, it has to be incubated for a period of 5-20 minutes and at the end the gel is destained with methanol/water/ AcOH in a 10/80/10 proportion for a period of 1-24 hours. The colloidal coomassie brilliant blue method has a detection range of 8-50ng of protein and it takes an average of 1 to 2 days to be completed. In that case, a previous fixing step is made with methanol/water/acOH in a 40/50/10 proportion for an hour or even overnight. Afterwards they are washed with water and they are let to incubate overnight with a coomassie solution (ammonium sulphate, phosphoric acid). Water is used to destain and the process can be accelerated by using temperatures that range from 45 to 55 grades. This is a quite popular method because it is very simple and it is compatible with the mass spectrometry. It is frequently used to stain preparative gels.
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Bioinformatics tools |
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PEAKS |
| PEAK (Bioinformatics Solutions) is a software package for de novo analysis of MS/MS spectra This software allows the combination from the data obtained with de novo analysis and the one obtained by the database search function (feature also included in the software). Additionally, it is possible to combine these results with the ones obtained from external search engines. Analysis de novo can be applied to both individual spectra as well as the files resulting from an LC/MS/MS. This software runs in a dedicated computer and it is able to run an analysis of an 80 minute long chromatogram obtained (approximately 1500 spectrums MS/MS) in about 20 minutes. |
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Other Services |
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Gel scanning |
| A GS800 densitometer from BioRad is available for the digitalization of stained gels with visible dyes. |
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Protein Separation by HPLC |
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Several high-resolution liquid chromatographs are available and working from 2 mil/min to 1 µl/min connected to UV detectors.
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Determination of Protein Epitopes. |
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This analysis is directed to the proteomic characterization of the protein epitope bound to a monoclonal antibody. The method is developed for the determination of lineal epitopes of proteins with a molecular weight lower than 45 KDa. For biggest proteins or in the case of conformational epitopes the procedure has to be evaluated.
For this study the customer has to provide the necessary amount of protein and antibody (between 0.3-1 mg each).
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