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.

Do you accept radioactive or biologically active samples?

No. We cannot accept radioactive samples of any kind and we are only a BSL-1 level lab (BSL-1 is appropriate for working with microorganisms that are not known to cause disease in healthy human humans). That means we can no accept blood, serum, or any fluid with active biological components. Generally sample run on a gel are dead but please contact us if there is any question.

Do you accept samples from commercial (non-academic) or foreign organizations?

Yes, we accept sample from academics, pharmaceutical companies, biotech companies, and anyone needing proteomics analysis from anywhere in the world. We can arrange for CDA’s and NDA’s if needed (our university has lawyers to handle CDA and NDA's. Contact us to make arrangements for these agreements.)

Where do I send my samples?

Proteomics Analysis
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).

How should I pack my samples so they arrive intact?

Please follow these simple packing instructions.

May I send my samples without dry ice?

As long as the gel band or spots stay moist it is safe for at least four days at room temperature. Just put a small amount of HPLC grade water (or the cleanest grade water you have) in each tube. Use just enough water so it keeps the gel pieces moist but not so much that the gel pieces are floating in the water (there is a small chance proteins may diffuse out of gel). Cap the vials well but avoid using parafilm (it can cause contamination) to seal the vials. See above item for a convenient way to send the vials and keep them protected.

Must I wear gloves when handling the sample vials?

We wash the outside of the vials before the digestion procedure so once the vials are sealed you don’t need to wear gloves when handling the vials. When the vials are open we suggest you wear gloves to minimize the possibility of keratin contamination.

Where do I get the sample submission forms?

Download the Protein ID / PTM / MudPIT form
Download the MALDI-TOF form

What payment methods do you accept?

Credit card, purchasing card (pcard), or purchase order.

What reports will I receive?

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 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. 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.


6. Your samples will be kept frozen for a maximum of 6 months in case further analysis of remaining sample is necessary.

Can you send me the raw data files from my analysis?

We send you a complete report listing the peptides matched to the proteins, protein sequence coverage maps, and all the necessary information for publications, grants, etc.. We can also give you the DTA and OUT files but it is not our practice to send out the raw data files from the mass spectrometer due to interpretation and proprietary issues. Also, part of the analysis fee we charge is for expert analysis of the data so needing the raw data files for re-analyzing or reinterpreting the data seems redundant to the work we are doing for you. If you need MS/MS (fragmentation) spectra for paper figures, then please contact us to make arrangements. There is an additional cost as it takes time to produce these figures.

Can I get MS/MS fragmentation figures for my paper to show how the peptide, protein, or posttranslational modification was identified?

If you need these kind of figures for papers or grants then please contact us to make arrangements. There is an additional cost as it takes time to produce these figures.

How will my samples be processed and analyzed?

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 and the Proteomics Browser software.

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 many individual protein sequences, different protein sequences databases, or posttranslational modifications can I search for each sample?

The analysis fees are based on the number of LC/MS/MS analysis runs and the number of reports sent. The costs stated on this website are for the analysis and a single report. The sending of the reports triggers the billing. Each additional LC/MS/MS run costs $150, each additional report costs $100, and the hourly data analysis charge is $150/hour. As a courtesy we will generally search one additional user-supplied protein sequence or protein sequence database and include it in the initial report. After the initial report is sent the additional report will be incurred for any requests for additional reports using new protein sequences, changes to species of protein sequence to search, confirmation of particular peptide sequences is needed, or targeting of particular peptides, then we have to charge.

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 that are behind a hardware firewall.

Who do I contact if I have sample preparation, analysis, or questions on my report?

Contact (617) 636-2407 or tucf-proteomics@tufts.edu (note that email is often responded to more quickly)

What experimental text should I include in my papers, grants, etc.?

Below is the experimental text we usually ask people to include. This text is appropriate for any protein identification project but not necessarily for posttranslational modification projects though (contact tucf-proteomics@tufts.edu to discuss what to write for posttranslational modification projects):

“Excised bands were subjected to in-gel reduction, alkylation, and enzymatic digestion (Roche Applied Science, Indianapolis, IN) in a HEPA-filtered hood to reduce keratin background. LC/MS/MS analysis was performed on the in-gel digest extracts using Agilent (Santa Clara, CA) 1100 binary pump directly coupled to a mass spectrometer. 2-8ul of sample was injected on column using a LC Packings (Sunnyvale, CA) FAMOS autosampler. Nanobore electrospray columns were constructed from 360 um o.d., 75 um i.d. fused silica capillary with the column tip tapered to a 10 um opening (New Objective, Woburn, MA). The columns were packed with 200 Å 5 um C18 beads (Michrom BioResources. Auburn, CA.), a reverse-phase packing material, to a length of 10 cm. The flow through the column was split precolumn to achieve a flow rate of 320 nL/min. The mobile phase used for gradient elution consisted of (A) 0.3% acetic acid 99.7 % water and (B) 0.3% acetic acid 99.7 % Acetonitrile. Tandem mass spectra (LC/MS/MS) were acquired on a Thermo LTQ ion trap mass spectrometer (Thermo Corp., San Jose, CA). Needle voltage was set to 3 kV, isolation width was 3 Da, relative collision energy was 30%, and dynamic exclusion was used to excluded recurring ions. Ion signals above a predetermined threshold automatically triggered the instrument to switch from MS to MS/MS mode for generating fragmentation spectra. The MS/MS spectra were searched against the NCBI nonredundant protein sequence database using the SEQUEST computer algorithm (1) to produce a list of proteins identified in each sample.
1) Yates, J. R. D., Eng, J. K., McCormack, A. L., and Schieltz, D. (1995) Anal. Chem. 67, 1426-143”

Can you suggest some papers to read that detail the proteomic methods used to analyze my samples?

Suggested reading:

The protein microscope: incorporating mass spectrometry into cell biology, Nat Methods, 2007 Oct, 4(10), 783-4.

Proteomics from the clinical perspective: many hopes and much debate, Nat Methods, 2007 Oct, 4(10):785-6.

Proteomics in 2005-2006: Developments, Applications and Challenges, Anal. Chem., 2007, 79, 4325-4344

Mass spectrometry and proteomics, Current Opinion in Chemical Biology, 2000, 4, 489–494

Proteomic tools for quantitation by mass spectrometry, Mass Spectrometry Reviews, 2003, 22, 182–194

Mapping protein post-translational modifications with mass spectrometry, Nat Methods, Oct. 2007, 4 (10), 798-806