MS proteomics is a powerful tool for the analysis of protein samples, but there are some limits to the techniques we employ in the Proteomics Core. Knowing the limitations of the services we provide can help to ensure your experiment is a success, and you do not incur costs for unsuccessful analyses.
Generally the instrumentation we use for service work can easily identify Coomassie-stained bands, will identify visible silver-stained bands (subject to various caveats), but is not as sensitive as western blot. We identify proteins by digesting them into peptides with trypsin, and then observing peptides in the mass spectrometer. Depending on their length and sequence proteins will produce different numbers of tryptic peptides, and they will not all be easily visible in the mass-spectrometer. Therefore, the detection limit for a protein is variable per protein. Speak to us before submitting a sample if you are working with less material than a strong silver-stain band, to check whether it is likely we can successfully work with your sample.
Complex Samples Can Require Fractionation
Our instrumentation is capable of identifying over 4000 proteins in a single 60 minute run from a single sample. However, very complex samples may contain even more proteins that would not be identified. Also, as the number of proteins identified increases the sequence coverage of each protein tends to decrease – this may or may not be important to your experiment. To identify thousands of proteins in extremely complex samples, or achieve good sequence coverage for hundred or thousands of proteins requires fractionation and submission of several samples. Speak to us before submitting complex samples.
We only accept samples in-gel, and usually digest proteins with trypsin. This is a robust procedure but some difficult proteins may not run into a gel as expected, or are difficult to recover from the gel. In addition if a protein doesn’t produce tryptic peptides observable in the mass-spectrometer we won’t be able to identify the protein. We can use alternative digestion enzymes to combat the latter problem, and will work with you to maximize the chances of a successful result when you are working with a difficult protein.
Quantitation Requires Planning & Repeat Experiments!
Many customers now want to quantify differences between samples, rather than simply identifying the proteins in them. Successful protein quantitation depends on a good experimental design, with biological and technical replicates, to deliver useful results. We encourage anyone considering a quantitative proteomics experiment to discuss their plans with us before generating the first sample. A lot of time and money can be saved by considering experimental design carefully, ensuring that the techniques we use can deliver the kind of results that are neccessary.
Contaminants Can Mask Interesting Proteins
Modern mass-spectrometers can identify thousands of proteins in a single run, where the LC system efficiently resolves the proteins so that they elute in extremely tight peaks during the gradient. Unfortunately if a sample is contaminated with very large amounts of keratin or other proteins, or detergents and some other chemicals, the resolution of the LC system can be affected, seriously reducing the number of identifications the mass-spectrometer can provide. Please ensure you follow our sample guidelines carefully to avoid contamination.
Post-translational Modifications Can Be Hard
MS Proteomics is commonly used to study post-translational modification such as phosphorylation on proteins, but this is not trivial. PTMs can affect the way peptides fly in the mass-spectrometer, and this along with other reasons can make them difficult to identify. We can only identify a PTM in the region of a protein observed by MS. Multiple enzyme digests can be required to cover a large amount of the protein sequence. MS/MS spectra that show the sequence of a peptide are often incomplete, and it is not possible to confirm the exact location of an identified modification. In addition various modifications have very similar masses, and it is complex or impossible to distinguish between them.
Thorough PTM mapping of a protein is time consuming, and identification of PTMs in complex samples even more-so. We will work with customers to maximize the results of PTM experiments, within the confines of staff-time that are present in a core facility.