Please follow the procedures below when submitting your samples for analysis.
Proteins identification by mass spectrometry (LC/MS/MS)
Samples from acrylamide gel:
- Be careful to wear gloves all the time to prevent keratin contamination of your samples. Avoid dust and use clean dishes during samples preparation.
- Stain your gel with coomassie blue or sypro ruby. It's essential to apply the destain steps described in the protocols in order to eliminate the residual SDS. If you must use silver, please read this comment and contact us.
- Cut each gel band with a scalpel (it's important to remove all the extra gel arount the spot) and transfer them into eppendorfs filled with 100 µl of bi-distilled water.
- To prevent eppendorfs from being crushed during shipping, you should put them into a falcon tube or a box.
- Send your samples at room temperature but by express mail (e.g.: FEDEX or PUROLATOR) preferably at the beginning of the week to avoid leaving the samples at room temperature over the weekend.
Samples for digestion in solution:
- Ideally, we prefer to receive samples for 'in solution' digestion in a volatile buffer like formic 0.1%, Acetonitrile, ammonium bicarbonate, without detergent nor other salts, and evaporated to dry. Please contact us before submitting your sample.
For silver stained gel:
- Please make sure to use a protocol that is compatible with mass spectrometry (without glutaraldehyde). If you don't have such a protocol, we recommend you to use one of the two protocols provided in the Protocols section of this website.
- Whenever possible, it is always better to use coomassie blue or sypro ruby staining instead of silver. Our experience is that for 100 fmol of BSA deposited on a gel, more peptides will be analysed by mass spectrometry for a coomassie blue or sypro ruby stained gel than for a silver stained one. It seems that silver staining itself or the extras steps required for the destaining of the gel reduces the digestion efficiency.
Payments by account transfer (applicable to researchers of the CHU de Québec only), purchase order and credit card are accepted.
Identification of post-translational modifications
On a regular basis, we do post-translational modifications analyses, phosphorylations amongst others are in high demand. Our success rate is outstanding for enriched or partially purified proteins.
If you want to submit samples for this kind of analyses, a larger quantity of proteins to be analysed would be desirable (e.g. a coomassie blue band of medium to high intensity).
We need the sequence of the potentially modified protein in electronic format, since we perform searches directly on this sequence for modifications sites.
Moreover, if potential sites are known, we could increase our success rate by targeting these sites in a particular way in the mass spectrometer. For instance, a phosphorylated peptide might be present but in too low abundance to be detected and fragmented. By targeting the site of interest, the corresponding peptide would be fragmented preferentially if it is present. We can target about 5 to 10 sites per protein.
Relative quantification by isotopic labeling (iTRAQ)
Researchers interested in submitting samples for a iTRAQ quantitative analysis are first requested to contact the platform personnel.
For this kind of analysis, the researcher should submit from 100 to 200 µg of proteins for each studied condition. When the amount of protein available is below this threshold, for example in case of immunoprecipitation, please contact us.
Upon reception of the sample, an acetone precipitation of the proteins will be done to concentrate the proteins and eliminate buffers and detergents used for protein extraction. Proteins will then be resolubilized in the iTRAQ buffer. Proteins are then quantified by the Bradford method on a spectrophotometer and equal quantities of proteins are labeled with iTRAQ reagents. iTRAQ labelling requires that proteins be in a solvant free of primary amines (Tris, ammonium bicarbonate, etc.) and of detergents. (See below for a list of products that should be avoided or that are suggested.)
- Any proteomic analysis is a challenge due to the huge dynamic range of protein concentrations in a cellular extract. Unlike nucleic acid molecules which can be amplified, proteins are detected by the mass spectrometer according to their initial concentration in the extract. In this context, the most abundant proteins will monopolize the mass spectrometer detector at the expense of proteins of lower abundance. It is therefore important to keep in mind that the more a cellular extract is fractionated, the better are the chances to identify low abundance regulatory proteins. Analysis of crude extract will mostly yield identifications of proteins from the cytoskeleton or implicated in the intermediary metabolism.
Products that should be avoided or entirely proscribed from lysis and extraction buffers:
MRM relative or absolute quantification
Please contact us!