The Proteomics Platform is fully equiped to perform a large variety of manipulation on your samples to optimize the quality of your proteomics experiments. This includes protein extraction from various media (cell, tissue, biofluids, etc.); tryptic digestion of the proteins, either in-gel (a process optimized on our robotic station), on beads or in solution; enrichment steps, like serum depletion or phosphopeptide enrichment using TiO2; purification steps, like desalting or deglycosylation; offline peptide fractionationby high-pH reverse phase chromatography; and protein labelling by iTRAQ or TMT.
If you have any question or request regarding the protocols that are used for your samples, please contact us. For a list of upstream protocols (for example, gel staining) that are compatible with mass spectrometry, look here. For information on the cost of some specific sample preparation steps, take a look here.
A mass spectrometry ‘bottom up’ or ‘shutgun’ protein identification provides an overview of the protein content of a sample: in complex samples, thousands of proteins are routinely identified. Our typical workflow involves digesting the proteins with trypsin, then injecting the digested peptides into a capillary HPLC system linked to a high resolution and high accuracy mass spectrometer (currently, a TripleTOF 5600+ or an Orbitrap Fusion). The results are then interpreted by specialized software to provide statistically validated matches between observed spectra and identified peptides and list of proteins. Results are returned in a ‘Scaffold’ document, which allow the experimental dataset to be explored and visualized within the intuitive Scaffold Viewer from Proteome Software. This high-quality visualization software is free, runs on Windows, Mac and Linux, and provides useful export to text or Excel formats.
The Proteomics Platform has a large variety of mass spectrometers and analysis software. This allows us to use an identification workflow tailored to the specificity of your samples and your scientific hypotheses. Contact us to design a proteomics experiments for your project.
Study of post-translational modifications (PTM)
Some common post-translational modifications (PTM) can be readily observed from a protein identification experiment. These biologically important modifications include phosphorylation, methylation, acetylation, ubiquitinylation, etc. We can specifically search for any modification that has a single form (and thus a single mass) in a protein identification experiment.
However, the modification stoichiometry is often low, so if you are specifically interested in studying post-translational modifications, some steps might be needed. If you want to confirm a known modification site or verify a specific potential modification site, we recommend that you tell us so in advance and send us your protein sequence and the location of the site of interest. We will then be able to target the peptide bearing the modification during LC/MS/MS analysis. It often happens that a modified peptide does not get fragmented because it is much less abundant than its non-modified counterparts, but by targeting preferentially the modified peptide mass for fragmentation, we increase the probability of acquiring an MS/MS spectrum for the modified form of peptide. Some enrichment steps can also maximise the observation of certain type of modifications. For example, phosphopeptide enrichment with titanium dioxide beads can be provided to identify a larger amount of phosphopeptides.
Post-translational modifications that have many forms, like glycosylations, are particularly challenging for mass spectrometry. We are currently working on a protocol aimed at the identification and quantification of glycosylation. If you are interested studying glycosylation, please contact us.
Being able to compare the concentration of a given protein in samples from different conditions, or to measure the absolute quantity of a protein in a given condition, are central to many scientific investigations. Our service offers absolute or relative quantification from targeted proteomics as well as a variety of relative quantification techniques from label-free approaches (MS1 precursor intensity, data independent acquisition, SWATH) or label-based approaches (iTRAQ, TMT, SILAC).
Targeted proteomics (MRM/SRM)
Targeted proteomics is the gold standard of protein quantification, and the sole technique that can provide an ‘absolute’ measure of a protein abundance. Selected peptides that are unique to the protein of interest are specifically targeted by our most sensitive mass spectrometer (currently the QTRAP 6500). For absolute quantification, synthetic peptides containing a stable isotopically labelled amino acid are used as standards. (Note that, for each protein that we want to quantify, the choice of peptides need to be optimized to increase the sensitivity of the assay).
In traditional MRM/SRM, up to 50 proteins can be quantified in a single run. We have also optimsed PRM (Parallel Reaction Monitoring) on the Orbitrap Fusion. This technique can be an interesting alternative to MRM for targeted proteomics on a greater number of proteins.
Label-based quantification methods use isotopic labelling to distinguish the signal from samples of different conditions that are analysed simultaneously. The signals from the isotopes are then compared to indicate the ratio of a given peptide accross different conditions. The Proteomics Platform offers label-based quantification based on iTRAQ reagents from Sciex (up to 8 conditions), TMT reagents from Thermo (up to 10 conditions).
Our iTRAQ/TMT relative quantification analytical package includes: protein extraction, digestion by trypsin, peptide labeling with iTRAQ/TMT reagents, labeled peptide separation by high pH reverse phase chromatography (14 in general but can change upon sample complexity) , analysis on orbitrap Fusion mass spectrometer and, finally, identification and quantification of the proteins using state-of-the-art bioinformatics. The report delivered to the researcher details all the proteins detected and identified in the submitted extract and their relative abundance in each of the experimental conditions tested. We can provide the same service for SILAC, except that in this case, the labeling will have to be done by the lab that grows the cells.
Label-free techniques for relative protein quantification are more and more popular and they don’t require standards or isotopic labels (although they still require replicates). One way to perform label-free protein quantification is based on calculating the area under the curve of the MS1 precursor (peptide) and comparing its intensity to the intensity of the same precursor from another condition. Long chromatography runs (2 to 5 hours) can be performed for this approach. The MaxQuant software is then used for data analysis. This strategy is good for low complexity samples such as those obtained from immunoprecipitation, pull-down, etc.
Data Independent Acquisition (including SWATH) is a family of approaches that enables concurrent detection and quantification of (potentially) all analytes in the sample. It involves setting the mass spectrometer to repeatedly scan through the complete observable mass range. This strategy is good for low and medium complexity samples. Look at our article Bourassa et al. 2015 for more information.
Statistical or functional bioinformatics analysis
Any experiment performed by the Proteomics Platform includes all the bioinformatics analyses necessary to obtain validated lists of proteins and peptides from the experiment. But we can perform supplementary statistical or functional analyses to help you extract everything from your dataset and publish it in high impact journals.