FAQs

Service Questions

Custom Research Projects

SDS-PAGE and Staining Gels

General Questions

PTM: Post-translational Modifications

Sample Preparation

 

Service Questions

What is the turnaround time for receiving results for mass spectrometry analysis?
For routine protein identifications via MALDI or nanospray LC-MS analysis:

• Maximum time: 10 working days after receipt of sample and completed drop-off form
• Common turnaround: one week
• Sometimes: 3-4 days
I want the best possible results. Will your staff cut out a protein band from my SDS-PAGE gel?
Yes, we prefer that you bring the gel and let us cut out your band. Our experienced staff produce excellent results. We also need to know the level of staining to allow us to guarantee results.

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Custom Research Projects

Can I collaborate with you? I have a great idea for a novel mass spectrometry research project.
Yes, please contact Mark Platt to arrange a consultation. Also see our home page for custom research and contract services.
What fees are involved for custom research projects?
Cost structures need to be negotiated before the work begins. Our consultation time is not charged to you. However, you must cover the cost of any routine services, as they are defined in the Service Listing page or pay for instrument time, material/reagent costs and technician time spent on the project.

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SDS-PAGE and Staining Gels

What protein staining method do you recommend for SDS-PAGE gels used for mass spectrometry results?
Commassee blue stained gels are best. Strongly staining bands give superior results. Silver staining works well, but follow our Silver Staining Protocol and be cautious about selecting weak bands for mass spec analysis.
Can I cut bands out of my own gel and give you a gel slice?
Yes, but follow instructions provided by the core and use LoBind Eppendorf tubes provided by the core. Observe caution with selecting and cutting out weak staining bands. Consult with the directors if you cut a band out from a complex protein sample.
I have a Silver stained gel, but I did not use your recommended staining method. Can we still use this for mass spectrometry analysis?
Please be conservative with your resources and our time. It might be best to repeat the gel and use our Silver Staining for Mass Spec Protocol. Other routine Silver Staining protocols modify proteins, particularly if they are recycled (reduced and restained). Our staining protocols are optimized for mass spectrometry applications. Of course we can test your gel to see if it works, but we will not guarantee good results.

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General Questions

I need some basic help with understanding mass spectrometry experiments and interpreting the MS data report of my sample. Who can I talk to?
All questions are welcome, particularly at the planning stages, before samples are prepared and analyzed. Please contact Mark Platt at any time.

For general questions about:
• Sample handing: see the staff, Kevin Welle or Jennifer Hryhorenko
• Protein identification via MALDI or LC-MS: see the staff, Kevin Welle or Jennifer Hryhorenko
• Questions that include any basic or complex topics: see the director, Mark Platt

For planning experiments for:
• Your first time with sample preparation: see Mark Platt
• Routine sample preparation: see the staff, Kevin Welle or Jennifer Hryhorenko
• Post-translational modifications: see the director, Mark Platt
• Complex protein samples: see the director, Mark Platt
• Contract / collaborative research services: see the director, Mark Platt

For interpretation of data for:
• Initial questions about data format: see the staff, Kevin or Katie
• Follow-up questions about significance of the data analysis: see the directors

For collaborative research:
• See the Director: Mark Platt
(See the Contact Us page to locate us.)
How do I get started using mass spectrometry-driven proteomics? (I am kind of new to this.)
This requires that you carefully define your research question, aim, and/or hypothesis. In general, the following
applications show many common starting points:

Typical applications:

1) Molecular Weight scan of intact proteins to analyze:
a. Sample complexity
b. Shifts in molecular weight due to alternative transcriptional processing
c. Post-translational processing and/or modification
d. Site-specific proteolysis

2) Identification of an unknown protein:
a. MALDI-TOF/TOF is for pure proteins available in high yield (>300 ng),
b. LC-MS/MS (LTQ or LCQ) for weaker bands or mixtures of proteins
c. LC-robot-spotter-MALDI for complex samples

3) Mapping phosphorylation or methylation sites:
a. MALDI-TOF/TOF is for preliminary analysis,
b. LC-MS/MS (LTQ or LCQ) can be used in combination w/ MALDI for higher coverage of the protein
c. LC-robot-spotter-MALDI for higher coverage of target protein
d. Purification of phosphopeptides enhances mapping results

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PTM: Post-translational Modifications

What kind of post-translational modifications does your facility detect as a service?
• Phosphorylation of: serine, threonine, tyrosine (Methods 60, 70,80, 90, 200, 210)
• Methylation: lysine
• Acetylation: N-terminal, lysine
What are the concerns about mapping a phosphorylation site in my protein? (It is a large protein ~1000 aa.)
You will have two major concerns:
1) Coverage: Can the mass spectrometry technique(s) deliver high sequence coverage. With higher coverage, there is a greater statistical chance to detect and identify the peptide containing a modification group.
2) % occupancy of the modification site: What percent of the protein preparation is modified? If it is low, then the
chance of detecting the modified peptide decreases. If the occupancy is high (>30%), then this works in the favor of
identifying a modification site.
How can coverage be increased for mapping phosphorylation or methylation sites? (I understand that high coverage is essential for increasing the chance of mapping a PTM site.)
First, note that, due to its unique sequences, every protein has a different potential to be mapped at high sequence coverage.

Coverage for all proteins increases by:
1) Starting with a higher amount of protein (>5 μg of protein is desirable) for MS analysis
2) Making sure that the protein is pure
3) Using multiple MS instruments/techniques for analysis will increase coverage (See Method 200 in our service list.)
3) Enriching the phosphopeptides using a TiO2 column (Method 210)
Will glycosylation sites inhibit the identification of my protein? (I think that my protein is glycosylated.)
If purity and concentration of the protein is high, then in most cases MS-based protein identification is still favorable.
Attached carbohydrate (CHO) chains, however, can impair MS-based identification of specific peptides through the following mechanism:
1) CHO chains can block trypsin cleavage
2) The variable mass of attached CHO chains can reduce glycopeptide detection.

Options: Cleave N-linked CHO chains with N-glycanase (PNGaseF) prior to gel loading and MS analysis
I want a preliminary analysis for potential phosphorylation sites in my protein. What is your lowest cost service? What are the limitations? What are your suggestions?
A lowest-cost preliminary analysis is Method 60 (on the Services page), which uses MALDI-TOF/TOF on a pure protein from a gel or a highly pure preparations. Limitations: The coverage of the protein will be low, if the protein is very large, or if it is impure, or if it is in low yield (<500 ng).

Suggestions: For a preliminary, low cost analysis, provide only a high quality sample (this would be a single band on a gel that is in the 1 μg range) and run service method #60. Otherwise, meet with the director to discuss more thorough service offerings that are designed for higher sensitivity and coverage.

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Sample Preparation

How much protein do I need in my sample?
To determine the identity of a single protein:
  • Aim for 100 ng of a pure protein. This is ideal and guarantees best results, or
  • A good rule of thumb is that it should be detectable as a stained band on a Coomassie  blue-stained gel
  • Less than 1 ng amounts of protein are also detectable, but less than ideal, particularly in a complex sample

To map post-translational modification sites in a single protein:
• 5 μg of protein allows the core to improve coverage of the protein sequence, or
• It should be a very strong band on a Coomassie blue-stained gel
• It needs to be a pure protein

For a PF-2D biomarker screen:
• 5 mg of protein for each sample
My protein preparation is not very pure. Can I use it for mass spectrometry analysis?
Purify needs to be well defined. An impure sample will not yield good results, as contaminating proteins will compete for detection. The user needs to talk with the directors or staff and review the goals of the MS analysis/service.

For protein identification:
• Run it on SDS-PAGE. If it is unique band and not part of a smear or complex banding pattern, then it can be cut from the gel.
• Attempt to purify it: Can you enrich it substantially on a column chromatography run?
• Immunoprecipitation: Can you enrich it via a pull-down method?

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