- Sample Turn-around Time
- Sample Submission
- Data Analysis
- Sample Preparation
I. Sample Turn-around Time
As we are a Shared Services Facility, samples are submitted to us for analysis by researchers
throughout the University of Maryland, Baltimore, in addition to other universities. Accordingly,
there is a queue for sample processing. We process samples in the order in which they are received and we do our best to ensure the timely analysis of all samples. Please allow for an average turn-around time of 15 business days from the time your sample is brought to us. For the feasibility of processing "rush orders" (**Rush orders are charged double the regular price.**), please speak with us prior to submitting your sample.
II. Sample Submission
Please place all orders using the University of Maryland, Baltimore's BIORESCO
freezer program website: http://cf.umaryland.edu/freezer/.
After logging in, select your requested service(s) from the "Proteomics Services"
link in the "CORE Services" box on the right-hand side of the page.
When you stop by our Facility (Bressler Research Building, Rm. 7-050) to drop
off your sample, please be prepared to briefly discuss with us all details pertaining
to how your sample is prepared. The quality of our proteomic analysis of your
sample is greatly affected by the amount of information about your sample that
you provide; the more details you can provide about your sample preparation, the
better. Refer to the section on "Sample preparation" for more guidelines.
Please note that the Proteomics Shared Services Facility currently does not have enough capacity
and throughput to accept the following samples: tissues (both human and animal) and unprocessed biological
fluids such as plasma, urine, and saliva.
III. Data Analysis
We will contact you once we have finished analyzing your sample to discuss the proteomic data with you.
IV. Sample Preparation
As a Shared Services Facility we offer proteomics services and the methodologies
listed here are only used as a general guideline. However, by adhering to the
following guidelines for sample preparation, you will greatly enhance the quality
of the data from the proteomic analysis of your sample. For certain experiments,
additional method development may be required prior to analysis by mass spectrometry.
A. Avoiding Keratin Contamination
One problem common to both 1D or 2D-gel/MS and LC/MS methods is keratin contamination.
Keratins are naturally occurring structural proteins and more often arrive in
the sample through the environment rather than from natural abundance. Fingerprints,
hair, dead skin flakes, wool clothing, dust and latex gloves are common source
of contaminating keratins. If keratins are present in concentrations greater
than that of the protein of interest, their abundance will overwhelm the analytical
capacity of an LC/MS system and obscure the proteins of interest. This is particularly
problematic when performing data dependent mass spectrometry, as the peptides
from the more abundant keratins will be selected for MS/MS analysis, providing
little or no information about the actual proteins of interest. However, in
low concentrations, compared to the protein of interest, keratins are not a
problem at all.
Listed below are few hints that will help you to minimize keratin contamination:
- 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. DTT, ß-mercaptoethanol and buffers are common
sources of keratin.
- ALWAYS USE NON-LATEX GLOVES and wear lab coats.
- Wash all glass plates thoroughly with 70% ethanol prior to casting an SDS-PAGE
- After completing gel electrophoresis step, disassemble the glass plates
in a laminar flow hood.
- De-stain the gel in a clean container that has been rinsed thoroughly with
70% ethanol or methanol/acetonitrile.
- For in-gel digests, excise each gel band of interest using a new, sterile
razor blade or scalpel. Put each gel band into a clean microcentrifuge tube.
The entire in-gel digestion experiment must be carried out in a laminar flow
hood or another clean work environment. It is best to keep all pipette tips,
tube racks, microcentrifuge tubes and other supplies needed for in-gel digestion
inside of the laminar flow hood. If a set of pipettes specifically designated
for in-gel digestion is not available, wipe off pipettes with 70% ethanol
prior to use.
B. Gel Electrophoresis and staining
Always wear gloves, use a clean gel running apparatus and solutions, and run
your gel in a very clean environment. This will help to minimize possible contamination
of your protein samples with keratin (see "A. Avoiding Keratin Contamination").
- Run standard SDS-PAGE or 2D gels. 1 mm gel is preferred. Native, gradient
and denaturing gels are also acceptable.
- 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.
- In general, for protein identification from gel bands, any band clearly
visible by Coomassie or Sypro staining is acceptable. For low abundant proteins
(for example, those requiring visualization by Silver stain), in order to
ensure your protein is present at a detection limit that is compatible with
our instrumentation (at least femtomole level), we recommend combining multiple
gel bands from running the sample in multiple lanes of a gel.
- We prefer staining methods compatible with subsequent tryptic digestion
and mass spectrometric analysis. It is important to ensure that the
proteins are not covalently modified or irreversibly fixed in the gel during
staining. Please avoid glutaraldehyde-based crosslinking and staining reagents.
Otherwise, it may not be possible to obtain any mass spectrometry data.
- We prefer that you stain your gel using Coomassie blue stain (Bio-Safe)
from Bio-Rad, cat# 161-0786 or from Sigma (EZ-Blue gel stain), cat # G1041).
Stain the gel under standard conditions and in a keratin free environment.
Stain only until your bands are visible. Prolonged staining can result in
an over-estimate of the amount of protein present based on visual inspection.
If your protein is not visible using Coomassie stain, you can use a mass spectrometry-compatible
Silver stain or Sypro Ruby stain. Sypro stains require a UV source to visualize
gel spots or bands. If silver stain must be used, we recommend using the SilverQuest
Silver stain kit from Invitrogen (cat# LC6070) or the mass spectrometry compatible
silver staining protocol developed by Blum et al. (Electrophoresis, 8:93-99,
1987) and Schevchenko, A. et al. (Anal. Chem., 68:850-858, 1996). Silver stained
gel spots or bands need to be completely destained as residual silver ions
have been reported to inhibit trypsin digestion of proteins.
- De-stain gels thoroughly. A clear gel background is a must. Any excess
stain must be removed; otherwise residual stain artifacts may interfere with
our mass spectrometry data acquisition. If you use silver stained gels, wash
them extensively with ultra-pure water to remove any excess silver ions and
C. Excising Gel Bands
We offer protein band/spot excision services. However, if you prefer to excise
your protein band(s)/spot(s) of interest yourself, please adhere to the following
- Cut the band or spot as precisely as possible; minimize any blank gel region
around your band/spot of interest. Excise each gel band/spot of interest using
a new/sterile blade.
- Place each gel slice into a washed (with methanol or acetonitrile), plain
1.5 mL microcentrifuge tube. Do not use any tubes with o-rings or gaskets.
- Wash the gel slice with 50% HPLC Grade Acetonitrile/Water at least 2 times
for 10 min with occasional vortex mixing. Discard the wash solution. Use plastic
pipette tips to remove the solution, as gel slices tend to stick to glass
Pasteur pipettes. Washing the gel slice helps to remove any residual detergent
or salt which will interfere with mass spectrometry analysis.
- Rinse the microcentrifuge tube cap 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 wrap the tube with parafilm the tube - parafilm is a
common source of keratin contamination. The gel band will remain moist and
can be stored in a -20ºC freezer until you are ready to bring your sample
D. Preparation of Protein Samples in Solution
We prefer that you prepare your samples in a volatile buffer that can be removed
by either lyophilization or vacuum centrifugation. Whenever possible, please
dissolve your sample in ammonium bicarbonate, acetonitrile or a water-based
buffer. Please avoid including metal ions in your buffer. If buffer exchange
of your sample is required following sample submission, additional charges will
be applied. If detergent is needed in order to keep your protein in solution,
please use those detergents that are compatible with mass spectrometry analysis,
which are typically detergents with a high crucial micelle concentration. (The
formation of detergent micelles creates problems for mass spectrometry analysis
such as signal suppression and peak broadening). Please note that SDS and Triton
X-100 are generally NOT compatible with mass spec analysis so please discuss
possible alternatives with the Proteomics Shared Services Facility staff.
This page was last updated on:
January 21, 2010.