The presence or absence of post-translational modifications (PTMs), such as phosphorylation, on a protein are often as interesting as the presence of absence of the protein itself. The PTM ID service offers identification of PTMs on a protein from a purified protein or from a sample enriched for a specific PTM of interest.

A common way to submit purified proteins is by running your sample into an SDS-PAGE gel, to isolate the protein of interest from contaminants that may affect our ability to detect modofied peptides of interest. A STRONG COOMASSIE BAND IS REQUIRED TO GET GOOD SEQUENCE OCVERAGE OF A PROTEIN! 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 can also identify PTMs in samples that are enriched for a specific PTM of interest. We can do phosphorylation enrichment (TiO2 and Fe-NTA) or ubiquitination enrichment (Cell Signaling Technology kit #59322). Other PTMs can be enriched by the customer using kits from Cell Signaling Technology (https://www.cellsignal.com).

We do not manually identify PTMs, but instead search acquired MS data for PTMs of interest specified by the customer. We offer to identify common PTMs such as phosphorylation, methylation, acetylation, etc. We may consider unusual PTMs where literature demonstrating prior identification by MS using methods similar to ours is available.

What Does it Cost?

Standard Service: $265 per sample with tryptic digest, or $306 with an alternative enzyme. Each gel  sample submitted must consist of no more than 1 cm of gel lane. Certain PTMs may be excluded due to complexity of analysis. Some PTM samples can potentially be run on one of our lower sensitivity instruments at a reduced cost. 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 short reverse-phase LC-MS/MS method. We identify proteins from samples using Proteome Discoverer 3.0 and search for PTMs that you have specified. We provide a list of the proteins and the peptides identified, including the PTMs and their localization. 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, if necessary.
• 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 the PTMs you wish to attempt to identify.

Sequence Coverage Limitations

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

PTMs 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. In addition various modifications have very similar masses, and it can be complex or impossible to distinguish between them.

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.

Any Questions?

If you have any questions please contact ProteomicsCore@UTSouthwestern.edu before you prepare and submit your samples.