Protein Identification
$250 per sample (gel band or spot) for the in-gel digestion procedure and protein identification by LC/MS/MS

Note: Cost above is reduced 20% for internal users. Above cost includes in-gel digestion using a single enzyme (trypsin) and a protein identification report from a search against the NCBI nonredundant database (which contains all protein sequences from all species). Alternatively we can search only human, bovine, rodent, ecoli, or yeast protein sequences (see check box on sample submission form). Please contact us if you need to search your data against a different protein sequence database or a specific protein sequence.

New lower volume discount pricing for >5 samples!
Volume discount:  see volume discount information below. Yearly contracts are available (please contact us).

LC/MS/MS only: $150 per sample for protein identification by LC/MS/MS of samples you have already digested.

Liquid (non-gel) samples: For protein identification of liquid samples (in-solution digest), please contact us for pricing and precautions. Contact information at bottom of this page.

Post-translational Modifications
Please call or email for the additional costs for analysis of post-translation modifications (such as phosphorylation, ubiquitination, etc.). The projects costs and timeframe will vary depending on many factors including sample amount, extent of analysis, etc.. We specialize in finding sites of phosphorylation an ubiquitination but apply the same techniques to find most any defined posttranslational modification of a protein. 

MudPIT (Multidimensional Protein Identification Technology) or multi-dimensional LC/MS/MS
MudPIT or multi-dimensional LC/MS/MS analysis is a powerful technique for identifying proteins in a complex mixture (such as a cell lysate, purified organelle fraction, or a mix of 10 proteins or more) that would overwhelm the normal reverse phase LC/MS/MS analysis. MudPIT can also be an alternative to 2D gel electrophoresis for separating complex mixtures of proteins. The MudPIT technique utilizes a single, custom-packed LC column with strong cation exchange (SCX) material packed back-to-back with the standard reversed phase (RP) material. Please call or email to discuss if your sample might benefit from this more detailed form of analysis and the costs of the MudPIT analysis.

Information for Submitting Samples

What proteomic instrumentation/methods will be used?

1. Two state-of-the-art Thermo Finnigan LTQ ion trap mass spectrometers will be used for high sensitivity, nanospray (nL/min flow rates) LC/MS/MS using 75 micron i.d. nanobore columns. All data is searched against protein sequence databases using the SEQUEST algorithm. If MALDI-TOF analysis is requested we will use our Applied Biosystems DE PRO MALDI-TOF mass spectrometer.

How much sample is needed?

1. In general, for protein identification from gel bands, any band visible by Coomassie or Sypro staining is acceptable (roughly this corresponds to a minimum peptide or protein concentration in the range pmol/µl down to fmol/µl). See note below in "Gel and staining suggestions" section about silver staining. You can also combine up to 3 duplicate gel bands (for example, the same band from parallel 1D gel lanes) in one vial to send us for analysis (this is one way to make sure you have exceeded our detection limits, especially for faint silver stain bands).

Do I need to send my protein sequence?

1. In general, we can only identify proteins in your sample whose sequences are already in a public protein sequence database. If you have proprietary sequence you will have to send it to us ahead of time (if needed, call for CDA and non-disclosure info). For protein identification projects please let us know of any atypical or significant amount of post-translational modification (except for methionine oxidation) that is expected on your protein as it must be accounted for in our search of the protein databases.

Sample Preparation:

**Keratin contamination will always be observed so extra precautions must be taken to minimize the amount of contamination!!!!!

1. Wear gloves at all times during sample preparation. Wash outside of gloves with water before using them to handle your samples and vials. 
2. Thoroughly wash anything that will come into contact with your sample (i.e. gel apparatus, staining trays, gel excising implements, gel storage equipment, and Eppendorf vials) to remove keratins and contaminants. Eppendorf vials should be washed with HPLC grade Acetonitrile (or use a high grade methanol if no Acetonitrile is available) and then HPLC grade water. Sample vials should be free of plasticizers and contaminants that can compromise high sensitivity mass spectrometric analysis. Anything that touches the gel or sample is a possible source of contamination. Avoid storing gel in saran wrap or similar material and instead use new, cleaned plastic or glass gel trays.
3. Avoid using molecular weight cutoff filters (ex. Centricon filters) and if they must be used, thoroughly wash them. We find these are often a source of synthetic polymer contamination in samples.
4. Call to discuss your project before sending blood, plasma, or radioactive samples. There should be no particulates in the samples as they can easily clog the 75 micron columns we use.
5. Avoid using surfactants and detergents such as CHAPS, DMSO, Triton-X, etc.. If these must be used please contact us to discuss acceptable concentrations. Minimize the salt and buffer concentration in the sample.
6. Vials: For liquid samples use the smallest vial necessary, (as low as to the 0.5-0.6ml vials). For gel samples, use a washed 1.5 ml Eppendorf tube (the vial you send us will be used for the in-gel digestion procedure so please make sure it is clean). Don't use vials with rubber o-rings or gasket. Avoid colored vials and avoid using paraflim to seal vials.
7. Vial label: each of the vials that contain your samples must be labeled with the your name, sample name (choose something unique but short and different than sample name of previously submitted samples), and date. Please label both top and side of vial.
8. After the gel has been run, non-specific dust contamination can still be introduced so keep samples covered or protected as much as possible.
9. All samples must be shipped with completed sample submission forms (see link above to download form).

Gel and staining suggestions

1. Use standard SDS-PAGE or 2D gels. Maximize the amount of protein on as minimal amount of gel as possible. A concentrated gel band works a lot better than a large diffuse gel band.
2. Gel thickness of 1mm is preferred. Gradient, denaturing and native gels are acceptable.
3. Stain with Coomassie Blue using standard conditions. Stain only until your bands are visible. If the amount of your protein is too low for coomassie stains, use silver or Sypro stains. Generally we recommend Sypro (Ruby) stains over silver stains due to higher compatibility with mass spectrometry Sypro stains require a UV source to visualize gel spots or bands. If silver stain must be used, we require you use the Invitrogen Silverquest stain kit (see link below) as we find other silver stains incompatible. See links below for more information on stain kits. Silver stained gel spots or bands have to be totally destained as the silver has been reported to inhibit trypsin digestion of proteins.

How to cut gel bands and prepare samples to be sent to us

1. Destain gels as much as you can.
2. Excise band as precise as possible. If there is any diffuse stained edges to the band, omit it and excise only the clearly stained band. Do not mince up the gel into very small pieces (too easily lost in pipetting steps of the in-gel digestion procedure)
3. Excise a band from a blank area of gel, about the same size as your sample gel bands (cut in the step above), and send it to us to run as a gel blank. You will not be charged for analyzing this gel blank. Add this as a sample on the sample submission form and label it 'gel blank.'
4. Place each gel slice into a washed, plain 1.5 ml Eppendorf tube. Do not use any tubes with o-rings or gaskets.
5. Wash the gel slice with 50% HPLC Grade Acetonitrile/Water at least 2 times for 10 min with occasional vortex mixing. If you don't have HPLC grade solvents, use a new bottle of the highest purity solvent you have for the washes. Discard the wash solution. Use plastic pipette tips to remove the solution as the gel slice tends to stick to glass Pasteur pipets. This step helps wash out any residual detergent or salt.
6. Rinse the Eppendorf cap off with the 50% HPLC Grade Acetonitrile/Water solution and close the cap tightly. No additional solution is needed to cover the gel band. Do not parafilm the tube. The gel band will remain moist and can be stored in a -20 degree freezer or shipped to us. Gel slice can be stored frozen for at least 6 months without degradation. The gel slices should be sent frozen on dry ice unless you can get them to us within two days. Put your samples in a bag instead of loose in the dry ice as vial tops can come loose. If you can get the sample to us in two days or less than just put the vials (with the gel slices in them) into a 50ml conical tube with padding (this helps as delivery companies have been known to make vials lids pop open in shipment) and send in a FedEx package. The idea is just to make sure they arrive to us wet, not dry, and don't sit for more than two days at room temperature. For local users, please try to drop off your samples in the early morning as this is when the in-gel digestion procedure for the day begins. For samples in microtiter plates, cover with just aluminum foil (do not use stick plate seal film).

What address do I send my samples to?

1. Tufts Medical School
   Dept. of Physiology/ST-808
   136 Harrison Ave.
   Boston, MA 02111
   
   Phone: 617-636-2422
   Fax: 617-636-6737

All samples must be shipped with completed sample submission forms (see link above to download form).

Phosphorylation and other post-translational modification projects

1. Please call (617-636-2407) to discuss post-translational modification projects beforehand and pricing. Often the post-translational project will be broken into two steps: protein identification and then determination of modification(s).
2. We will need your protein sequence sent to us by email ahead of time for post-translational projects (we prefer you send the sequence before samples are submitted). Include any tags, cleavages in the sequence, known sequence variations, GST-fusions, etc.
3. Any project involving a non-standard modification (not phosphorylation, oxidation, etc.) should be discussed with us before sending samples. A form may be sent to you where you can provide details of the modification.

How will my samples be processed and run?

1. Samples sent for in-gel digestion will go through our protocol for in-gel digestion with proteomics-grade trypsin in a clean environment to minimize the keratin contamination. Keratin can not be completely avoided but can be minimized.
2. 20-40% of your samples are loaded on a reverse phase column and electrosprayed into the mass spectrometer. The mass spectrometer is set to fragment the most intense ions in successive scans of the instrument during the liquid chromatography (LC) run.
3. The resulting spectra are searched against a database of known protein sequences using the SEQUEST algorithm

What will I get back (reports)?

1. In-gel digestions are scheduled for Wednesdays of each week. If you send in your sample before Wednesday morning at 9:00am, they will be in-gel digested an analyzed by LC/MS/MS that Wednesday. A protein identification report will be sent to you within a week (by the following Wednesday). Samples arriving after Wednesday morning will be analyzed the following Wednesday with the report being sent a week after that.
2. You should receive a report in about 1 week but allow extra time for phosphorylation and non-standard projects. Please contact us by email (email) if you don't get your report or get a status update after a week after you have submitted your samples.
3. The report will be emailed to you in Adobe Acrobat (*.pdf) format (see below for location of free Acrobat reader software) and should be viewed in the Acrobat Reader. If you need the report in another format please contact us.
4. The report will consist of a list of protein identifications with values to indicate the number of peptides matched to each protein, the sequence of the protein, and the confidence in protein identification.
5. A plus sign next to a protein identification indicates there are other protein that matched the same set of peptides. A "M*" indicates oxidized methionine. 
6. Keratin is a common contaminant and will often be found the protein identification list even though many of the common keratins are automatically removed from the results. Be aware other proteins such as Hornerin are related to keratin.
7. Your samples will be kept frozen for a maximum of 6 months in case further analysis of remaining sample is necessary.

What about confidentiality (CDA) or non-disclosure agreements (NDA)?

1. Our university has a team of lawyers to handle CDA and NDA's. Contact us to make arrangements for these agreements.
2. Customer data is always kept confidential on password protected computers, behind a hardware firewall.
   

-Note that the typical 1 week turnaround on protein identifications may not always be possible for 20 or more samples (but our two mass spec's usually can handle large number of samples)
-Yearly contracts are available (please contact us).


Useful links

1. A Proteomics Glossary of Terms
2. Association of Biological Resource Facilities (ABRF)
3. American Society for Mass Spectrometry (ASMS)
4. Sypro stains - Invitrogen web site
5. Sypro stains - Sigma web site
6. Invitrogen Silverquest Silver Staining Kit
7. Free Adobe Acrobat reader software

Questions? contact 617-636-2407 or tucf-proteomics@tufts.edu

Jon P. DeGnore, PhD
Research Assistant Professor
Director of Proteomics and Mass Spectrometry
Tufts Medical School
Dept. of Physiology
Room ST-806
Tufts University
Boston, MA 02111
Phone: 617-636-2407
Fax:   617-636-6737