General questions
See Sample Turn-Around Time
No. We don’t offer gel services. However, we routinely perform in-gel digestion in preparation for LC/MS/MS analysis and protein ID experiments.
Bring us your gel, and we process it, analyze the proteins by LC/MS/MS, perform a Mascot database search, and send you a report on the identified proteins.
Yes, but only on the Voyager DE-STR Maldi/ToF instrument. Users are required to go through a training period and they must supply their own plates, matrices, and supplies. Only one user per lab is allowed for training on the Voyager instrument. Users are not allowed to operate any other instruments at the MS Core; instruments are operated by MS personnel only.
Yes, for most types of data and results. There is no fee. See Prices and Fees.
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 the possibility of arranging for a specific time for your sample analysis.
Analysis of proteins
It is difficult to get complete coverage of most proteins. There are many things that affect the sequence coverage: the ability to digest the protein well, abundance, purity, protein sequence, hydrophobicity, and modifications to the protein that may inhibit digestion (such as glycosylation), etc. The ability to digest the protein is influenced by protein folding, protease cut sites, and the complexity/purity of the sample. Analyzing the protein using different enzymes and using multiple strategies of analysis will increase the coverage.
Generally, intact analyses require 10’s of picomoles at picomole/μl concentrations. It depends heavily on the purity of the protein, the complexity of the sample (e.g., numbers of different proteins present), and 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 the sample cannot be exposed to detergents at any time during preparation. See Sample Submission and Guidelines.
See Sample Submission and Guidelines.
We can analyze protein samples prepared by SILAC, ICAT, ITRAQ, or other isotope-labeling strategies. We also have great software for processing quantitation data (ProteoIQ; NuSep). In addition, we can quantitate peptides and proteins in your samples using heavy-isotope labeled synthetic peptide standards. We are happy to discuss your proteomics quantitation project.
No. We don’t offer gel services. However, we routinely perform in-gel digestion in preparation for LC/MS/MS analysis and protein ID experiments.
Bring us your gel, and we process it, analyze the proteins by LC/MS/MS, perform a Mascot database search, and send you a report on the identified proteins.
Usually, if you can see any indication of the protein band on the gel after staining with a blue stain (e.g., Coomassie), we will be able to successfully identify the proteins. This may not be true for silver-stained bands, if the actual amount of protein is in the low femtomole range. However, we are usually successful in identifying proteins from silver-stained gel bands.
We offer SCX fractionation off-line and subsequent nanoLC/MS/MS analysis of the individual SCX fractions. This is actually a better method compared to on-line 2D HPLC.
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. See Sample Submission and Guidelines
We have demonstrated 50 attomole sensitivity for protein ID on the LTQ-FT instrument. This was done with a 1pmole digest of BSA protein standard, then an aliquot of 50 attomoles was injected on column. 8 to 10 peptides were identified in the Mascot database search. See Instrumentation and Sample Submission and Guidelines for more details.
Isolation of phosphopeptides by IMAC purification probably requires 10 pmoles or more of phosphorylated protein. It depends on the phosphopeptide sequence, however. TiO2 is an alternative and very sensitive method for isolation of phosphopeptides. We can enrich protein digests for phosphopeptides using either IMAC (Ga or Fe) or TiO2. Some phosphorylations can be relatively labile and difficult to analyze.
When feasible, an intact protein analysis is a good method of determining if the protein has any phosphorylations at levels greater than perhaps 5 or 10% relative to unmodified protein. Intact analyses require picomole quantities of protein, and the protein must be very pure (see Sample Submission and Guidelines). A digest of the protein followed by nanoLC/MS/MS analyses is also effective method for identification of phosphorylations. MS/MS sequencing is used to map the sites of phosphorylation.
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 based on a digest of the proteins in a solution sample, it is also 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).
No. The user must prepare, isolate, and adequately purify their proteins of interest prior to submitting samples for analysis.
No. However, we do offer on-line HPLC with mass spectrometry for the analysis of samples. (see available Services).
Metabolomics
Yes, we have developed an in-house metabolomics library in all the RP +ve , -ve mode, HILIC +ve and -ve modes.