Glycosylation can be a very challenging PTM to analyze by mass spectrometry, in large part because of the multiple different sequences a glycan can have. Additionally, the glycan on a peptide can easily fall off in the mass spectrometer, making sequencing and localization challenging. However, with the Oribtrap Fusion Lumos and its capabilities to perform electron transfer dissociation (ETD) along with higher-energy collisional dissociation (HCD), we can identify a glycopeptide along with sequencing the glycan in a single run.
A strong coomassie band is required to get good sequence coverage of the proteins. If we don’t see a region of sequence we cannot identify PTMs on that sequence. The standard protocol uses tryptic digest, but this will not give complete coverage of a protein. It may be neccessary to digest with alternative enzymes to gain optimum coverage of the protein, and maximize the chances of identifying a PTM. It is possible to somewhat predict sequence coverage – contact us! We do not manually identify PTMs, but instead search acquired MS data for PTMs of interest specified by the customer.
What Does it Cost?
Standard Service: $357 per gel band with tryptic digest. Each sample submitted must consist of no more than 1 cm of gel lane. Contact the core for more information.
What We Do
We run your gel band samples on our Orbitrap Fusion Lumos mass-spectrometry platform, using a standard reverse-phase LC-MS/MS method including ETD fragmentation. We identify proteins from samples using Proteome Discoverer 2.4 and Byonic software and search for glycosylation. We are available to discuss results with you.
What You Must Do
To submit a sample for PTM ID you must:
- Contact us if you are concerned about sequence coverage, or want to look for PTMs at a certain location in your protein.
- Resolve your protein of interest on an SDS PAGE gel.
- Stain your gel with Coomassie Blue.
- Ensure the band of interest is a strongly stained band.
- Cut out the band of interest following our guidance carefully.
- Ensure your band is no larger than 1 cm of gel. The closer you can cut around a band the better.
- Slice the band into 1mm cubes – this is very important to maximize recovery of material from the gel during sample processing.
- Place the sliced band into an Eppendorf 1.5ml tube, which has been rinsed with 50% organic solvent and millipure water.
- Follow the general guidelines for sample submission.
- Ensure you provide information regarding what you wish to attempt to identify.
We can only identify a PTM in the region of a protein observed by MS. The mass spectrometer can only see peptides with length between approx. 7 and 25 amino acids, that have a sequence that allows them to ionize well. Tryptic digest typically results in as little as 20% coverage, or as much as 80% coverage for a protein from a strong coomassie band. Coverage is dependent on the sequence of the protein – does it produce good tryptic peptides? Multiple enzyme digests can be required to cover a large amount of the protein sequence, or even a moderate amount of sequence for difficult proteins.
It is always beneficial to consult the core before submitting a PTM sample. Is complete coverage necessary? Are we likely to observe PTMs in a particular region of interest? These kinds of questions should be asked early to avoid unnecessary submissions and expense.
Other Warnings & Limitations
Glycosylation can affect the way peptides fly in the mass-spectrometer, and this along with other reasons can make them difficult to identify.
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, especially for O-linked glycans.
Thorough PTM mapping of a protein is time consuming. 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.
If you have any questions please contact ProteomicsCore@UTSouthwestern.edu before you prepare and submit your samples.