Protein identification, characterisation and quantitation by Mass Spectrometry
Low complexity mixtures.
Samples from bands of 1D gel or spots from 2D gels are digested and peptides are analysed by LC-MS/MS on a fast gradient.
1D gel bands for in-depth analysis.
Used for analyzing simple to medium complexity mixtures (typically 50-100 proteins) as well as for searching for modified peptides (PTMs). It is also recommended for samples from species with uncomplete genome sequence information available.
Starting from gel bands or LC fractions, proteins are digested and peptides extracted. The mixture is then separated on a nano-HPLC system on-line to an electrospray mass spectrometer, which isolates and fragments as many peptides as possible during a 30- to 90-min gradient. Collections of MS/MS spectra are used for database search for protein identification.
Medium complexity mixtures; only for samples ready to inject.
Same as above but for large projects in which the customer performs sample preparation so that samples are ready to inject. Workflow and final sample quality are subject to test before acceptance.
Customized database setup (for example organism-specific) or specific quantifications (PTM, label-free or targeted quantitation).
A peptide or small protein is analysed directly by electrospray-MS to obtain its accurate molecular weight. Usually works well only for masses up to 30'000 Da. Requires concentrated and detergent-free samples.
Whenever necessary, a sample preparation step before MS can be performed by off-line liquid chromatography (reversed-phase or cation exchange) to clean up the sample. Often required for Direct Electrospray analysis.
Extraction and digestion of up to 20 mg material from cells or tissues, desalting and QC
It determine site(s) of modification of a protein of interest.
Gel-separated or liquid phase samples are digested. The eluate is analysed by nano-LC-MS/MS to identify a maximum of modified peptides. If needed, it may include a second digestion with a different protease and/or MS analysis with alternative fragmentation methods (ETD , ETHCD). Quantitative data analysis is included (up to a certain level of complexity) to compare levels of modification among samples.
An appropriate negative control should be supplied whenever possible.
More complex cases may require price supplement for special data analysis.
Low or medium complexity protein mixtures(protein compelxex or subcellulsr fractions) are separated by limited electrophoresis after which 4-6 molecular weight regions are cut and digested. Analysis is performed by LC-MS/MS on every fraction. The resulting collections of spectra are pooled for every sample before database search. Lists of identified proteins for each sample with their scores are subjected to statistical validation and aligned for comparison.
For the analysis of protein complexes, a negative control is essential for background subtraction.
Extended shotgun analysis of complex samples : same as above, but 10-12 molecular weight regions are cut and digested after separation of protein mixtures by electrophoresis.
SILAC is an accurate quantitative proteomics technique based on stable isotope labelling with amino acids in cell culture. It is mostly only applicable to mammalian cultured cells but offers great advantages for experiments in which complex purification steps are required to isolate a fraction of interest, as well as for general quantitative profiling.
The PAF offers support during the setup of SILAC labelling experiments as well as the full analytical pipeline (including data analysis). However, carrying out cell culture and the first steps in sample preparation remain responsability of the customers. Preliminary in-depth project discussion is mandatory. Contact us for more information.
A preliminary test to monitor label incorporation is carried out by migrating test samples on a SDS-PAGE gel and analyzing the trypsin digest of a band by LC-MS/MS.
The PAF can provide users with all reagents necessary to SILAC cell culture : special media (DMEM or RPMI), dialysed serum, isotope labelled amino acids. Exact prices are based on amount necessary and isotope labels chosen.
Based on SILAC quantitative technique and combined with extensive SDS-PAGE fractionation (50 slices), slice SILAC allows study of protein processing ("degradomics") by large-scale mapping of protein mobility shifts, in mammalian cultured cells. The PAF offers support during the setup of SILAC labelling experiments as well as the full analytical pipeline (including data analysis). However, carrying out cell culture and the first steps in sample preparation remain responsability of the customers. Preliminary in-depth project discussion is mandatory. Contact us for more information.
Isobaric labeling is used for multiplex relative quantitation of up to 10 samples in the same experiment. Labelling is carried out at the peptide level after protein digestion. Any type of proteomics sample (cell culture, tissue, etc. ) can be quantitated by this technique. The workflow includes data analysis. Preliminary project discussion is mandatory.
For very complex samples in SILAC or isobaric labeling experiments, additional fractionation is recommended for higher proteome coverage.
Simple separation on standard minigels for MS.
This two-dimensional separation is mostly used for preliminary tests to optimize sample preparation and migration conditions. For maximum sensitivity silver staining can be used, but is not recommended for subsequent MS analysis. For midigels, we use precast gradient gels in the second dimension, which provide very good separations despite their relatively small size.
For western blotting applications, 2D separation on a minigel is often sufficient. The service we provide includes transfer onto nitrocellulose, after which the membrane is returned to the user for detection with antibodies.