Frequently-Asked Questions
- I’m interested in post-translational modifications in my protein. Can I get complete sequence coverage of my protein by mass spectrometry?
It is difficult to get complete coverage of any protein. It depends on the abundance, purity, protein sequence, hydrophobicity, modifications to the protein that may inhibit digestion (such as glycosylations), etc. Analyzing the protein using different enzymes and using multiple strategies of analysis will increase the coverage.
- Can you tell us every possible modification that is present on my protein?
Potentially, but it may be difficult to know that we have found every modification. Comprehensive determination of all modifications will be very time consuming, require many mass spec experiments, and may require high picomole levels of highly-purified protein. Identifying modifications will depend on several factors, such as: a) the relative amount of the protein that is modified (is it modified at very low level, <5%?), b) some modifications are very difficult to fully characterize, such as complex glycosylations, c) purity of the protein, etc.
- How much protein do I need for an intact analysis?
Generally, intact analyses require picomole/ul concentrations. It depends heavily on the purity of the protein, the complexity of the sample (in terms of numbers of different proteins present), the molecular weight of the protein. For intact protein analyses to be successful, the protein sample cannot contain any polymers or detergents (even at extremely low levels), and usually cannot have been exposed to detergents at any time during preparation. See Sample Submission and Guidelines
- How do I submit my gel bands for protein ID?
- I’m interested in expression levels of my proteins. Can you quantitate my protein(s) or peptides in solution?
We don’t routinely analyze protein samples using ICAT or ITRAQ kits to quantitate proteins. In some cases, we can quantitate peptides in your samples using synthetic heavy-isotope labeled peptide standards. Please discuss your proteomics project and what you would like to quantitate with the Facility personnel.
- When will the results from my sample analysis by ready?
- Does the Core Facility offer 1D or 2D gel electrophoresis services? Will the Facility run my protein on a gel for me?
No, we don’t offer gel services. However, we routinely do in-gel digestion in preparation for LC/MS/MS analysis and protein ID. Bring us your gel, and we take it from there.
- How much protein do I need in order for an in-gel protein ID to be successful?
Usually, if you can see any indication of the protein band on the gel, we will be able to successfully identify the protein. This may not be true for silver-stained bands, if the actual amount of protein is in the low femtomole range.
- Does the Core Facility offer Mudpit analysis?
We offer SCX fractionation off-line and subsequent nanoLC/MS/MS analysis of the SCX fractions.
- Can we train on the instruments and run our own samples?
No, only Facility personnel are permitted to operate the instruments.
- How much protein do I need for a protein ID?
There is not an exact answer to this, since it depends on several factors. Generally, if you can see it on a gel, we can ID the protein. If the protein ID is from a solution, it will depend heavily on the purity of the solution, complexity of the solutions, exposure to detergents and polymers, exposure to protease inhibitors, amount of protein, etc. In principle, the sensitivity of the LTQ-FT is in the attomole range, but the above factors can have a significant impact on the ability to successfully digest and identify peptides during nanoLC/MS/MS. Sample Submission and Guidelines
- Can I get electronic copies of my data?
- What is the sensitivity of the instruments for protein ID?
- How much protein do I need to identify a phosphorylation using IMAC purification?
Isolation of phosphopeptides by IMAC purification probably requires 10 pmoles or more phosphorylated protein. It depends on the phosphopeptide sequence, however.
TiO2 is likely a far more sensitive method for isolation of phosphopeptides. We plan to provide TiO2 phosphopeptide purification as a service soon.
- What is the best way to determine if my protein has any phosphorylations? Can you map the phosphorylation sites on my protein?
When feasible, an intact protein analysis is a good method of determining if the protein has any phosphorylations at levels greater than 5 or 10% relative to unmodificed protein. Intact analyses require picomole quantities of protein, and the protein must be very pure (see Sample Submission and Guidelines). Digests of the protein followed by nanoLC/MS/MS analyses is also effective, and MS/MS sequencing is used to map the sites of phosphorylation.
- What are the important aspects of purity for analysis of protein samples?
For analysis of intact proteins, it is especially critical that the protein sample does not contain any amount of detergents or polymers. For protein ID and analysis of digests of proteins in solution, it is important that protein sample not contain detergents, polymers, and has not been exposed to trypsin protease inhibitors (such as AEBSF), or other derivatizing reagents. (see Sample Submission and Guidelines).
- Does the MS Facility offer protein preparations or protein isolations from cells, tissues, blood, or other biological matrices?
No, the user must prepare, isolate, and adequately purify their proteins of interest prior to submitting samples for analysis.
- Does the MS Facility offer analytical HPLC services for protein or other sample purification and fraction collection?
No. However, we do offer some on-line HPLC sample analysis services (see available Services).
- Does the MS Facility offer “stat” or high-urgency sample turn-around analyses for extra cost?
No. However, if you have a labile sample that requires fast analysis, you can contact the MS Facility a week or more in advance to discuss and arrange a specific time for sample analysis.
