1. I’m interested in post-translational modifications in my protein. Can I get complete sequence coverage of my protein by mass spectrometry?
  2. Can you tell us every possible modification that is present on my protein?
  3. How much protein do I need for an intact analysis?
  4. How do I submit my gel bands for protein ID?
  5. I’m interested in quantitation and/or expression levels of my proteins. Can you quantitate my protein(s) or peptides in solution?
  6. When will the results from my sample analysis by ready?
  7. Does the Core Facility offer 1D or 2D gel electrophoresis services? Will the Facility run my protein on a gel for me?
  8. How much protein do I need in order for an in-gel protein ID to be successful?
  9. Does the Core Facility offer Mudpit analysis?
  10. Can we train on the instruments and run our own samples?
  11. How much protein do I need for a protein ID?
  12. Can I get electronic copies of my data?
  13. What is the sensitivity of the instruments for protein ID?
  14. How much protein do I need to identify a phosphorylation using IMAC purification?
  15. What is the best way to determine if my protein has any phosphorylations? Can you map the phosphorylation sites on my protein?
  16. What are the important aspects of purity for analysis of protein samples?
  17. Does the MS Facility offer protein preparations or protein isolations from cells, tissues, blood, or other biological matrices?
  18. Does the MS Facility offer analytical HPLC services for protein or other sample purification and fraction collection?
  19. Does the MS Facility offer “stat” or high-urgency sample turn-around analyses for extra cost?

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 most proteins. It depends on the ability to digest the protein well, which may be influenced by protein folding, protease cut sites, and the complexity of the sample.  Abundance, purity, protein sequence, hydrophobicity, and modifications to the protein that may inhibit digestion (such as glycosylations), etc. will affect coverage.  Analyzing the protein using different enzymes and using multiple strategies of analysis will increase the coverage.

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How much protein do I need for an intact analysis?

Generally, intact analyses require 10’s of picomoles at picomole/ul concentrations.  It depends heavily on the purity of the protein, the complexity of the sample (i.e 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.

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How do I submit my gel bands for protein ID?
See Sample Submission and Guidelines.

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I’m interested in quantitation and/or expression levels of my proteins.  Can you quantitate my protein(s) or peptides in solution?

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.

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When will the results from my sample analysis be ready?

See Sample Turn-Around Time

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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 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.

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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 after staining with a blue stain, 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.

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Does the Core Facility offer Mudpit analysis?

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.

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Can we train on the instruments and run our own samples?

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 only MS personnel.

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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. See Sample Submission and Guidelines

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Can I get electronic copies of my data?

Yes, for most types of data and results. There is no fee.  See Prices and Fees.

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What is the sensitivity of the instruments for protein ID?

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.

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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 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 (Galium or Fe) or TiO2.  Some phosphorylations can be relatively labile and difficult to analyze.

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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 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.

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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 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).

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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.

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Does the MS Facility offer analytical HPLC services for protein or other sample purification and fraction collection?

No. However, we do offer on-line HPLC with mass spectrometry for the analysis of samples.  (see available Services).

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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 the possibility of arranging for a specific time for your sample analysis.

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