How do I submit a sample? If you are a first time user of the Proteomics Core Facility we strongly encourage you to meet with a staff member prior to sample submission to discuss your research goals and sample preparation techniques. The quality of the MS data depends on the quality of the sample. We have a website, www.protbase.org, for sample submission. Visit the site, open an account and follow the directions for submitting samples. Please give as much information about the samples as possible. Print out the form and bring it, along with your samples, to room 106 in BIO5. If the sample is a gel band please include an image of the gel.
How much protein do I need to submit for protein identification? Our most sensitive mass spectrometers can detect 50 fmol, for a 50K D protein that corresponds to 2.5 ng.
How pure does the sample have to be? For identification of a single protein the sample should be as pure as possible. Purity may be judged by the presence of a single Coomassie stained gel band on an overloaded gel. There are many ways to clean up samples, please consult a staff member for advice on your particular situation.
What buffer should my protein be in? Ideally water or a volatile solution that can be removed by evaporation. Salts, detergents, chaotropes, and protease inhibitors may interfere with MS analysis. Many clean up procedures are available and can be suggested by a staff member.
Can you tell me the N-terminal sequence of my protein? It depends. Not every peptide in a given protein is identified in the MS. Generally, LC-MS/MS identifies 15-45% of the protein sequence through peptides. The N-terminal peptide may or may not be among the peptides sequencd. In most cases it is better to send the protein to a facility that specializes in protein N-terminal sequencing if this is what you specifically need.
Can you identify a protein from a gel band? If the gel band is stained with Coomassie blue we can usually identify the protein. Silver and fluorescence-based stains are 10-fold more sensitive than Coomassie blue, so we may or may not be able to identify the protein. Coomassie or fluorescence-based stained gel bands do not have to be destained; cut out the band, put it in an eppi tube, and keep in the freezer until you are ready to submit it for analysis. If the gel was stained with silver, the band should be destained the same day the gel was stained. A protocol for destaining silver bands is at our main website, http://proteomics.arizona.edu/protocols.html. Do not use a silver staining protocol that calls for glutaraldehyde. Also, we recommend using pre-cast gels. If you do cast your own gels, let them polymerize overnight then run current through the gel for 15-30 min before loading the samples. These steps will minimize unpolymerized acrylamide that can interfere with MS analysis.
Which gel stains are compatible with MS analysis? Coomassie blue, silver, Deep Purple, SYPRO Ruby, (total protein stains), Pro-Q Diamond (phosphoproteins), and Pro-Q Emerald (glycoproteins).
Can you identify a protein on a Western blot? The sensitivity of chemiluminescence is at the attomole level which is significantly greater than our most sensitive MS. If the immunoreactive region also has a band on a Coomassie blue stained gel, we can probably identify the protein. However, keep in mind that a gel band may contain more than one protein, thus the identified protein may or may not be the immunoreactive protein. A 2D SDS-PAGE gel that further separates proteins may be helpful (isoelectric focusing followed by SDS-PAGE).
Can you identify post-translational modifications? We have experience in identifying protein drug adducts, phosphorylation, glycosylation (simple and limited type), acetylation, oxidation, glutathionylation, and methylation
How can I tell which proteins are up or down-regulated in my biological sample? We use a technique called DIGE (differential in-gel electrophoresis) to discover expression changes between two samples. Each sample is labeled with a different fluorescent dye, the samples are mixed together, and the proteins are separated by 2D SDS-PAGE. Because the samples are run on the same gel, the problem of gel variability is eliminated. Image analysis indicates the presence or absence of a particular protein, and signal intensity indicates concentration. The spots are analyzed using DeCyder, a software program. Spots can be cut out and the proteins identified by LC MS/MS.
Note that we are currently working on label-free protein quantitation using a WATERS Q-TOF Premier mass spectrometer and nanoLC.
Can you analyze serum or plasma samples? Yes, we often analyze biological fluids. Serum and plasma have several proteins, for example, serum albumin, that are in such high concentration that lower abundant proteins are masked in the analysis. To avoid this problem we recommend immunoaffinity chromatography to remove the abundant proteins prior to analysis. We offer this procedure as one of our services.
Can you analyze radioactive samples? No, our lab is not certified for radioisotope work.
How do you identify proteins? We treat the protein with a protease, typically trypsin, and then analyze the digest with nano-LC MS/MS. See below for a description of the analysis.
What is nano-LC MS/MS? Nano refers to the flow rate (nano liters per min), LC is liquid chromatography, usually C18 HPLC. MS/MS, also called tandem MS/MS, uses a trap to capture ions after the first MS. The ions are fragmented in the trap and the masses of the pieces are measured. Peptides fragment in predictable ways and the data are analyzed by complex computer algorithms which match the fragments to the protein they came from.
What is MudPIT analysis? Multidimensional Protein Identification Technology (or LC LC MS/MS), developed by the Yates group, uses two chromatography columns, typically strong cation exchange and reverse phase (C18) to separate complex mixtures of proteins before introduction into the MS. The sample binds to the SCX and fractions are eluted off with increasing salt concentrations (salt bumps). The eluates flow directly onto the C18 column where the peptides are fractionated by their hydrophobicities. The next step in the technique is nano- LC MS/MS, see above for a description. For example, a eukaryotic cell lysate can be analyzed by this technique and the less abundant proteins will be detected as well as the abundant proteins.
Why is keratin a problem? Keratin proteins from skin and hair are ubiquitous. If the analyte of interest is in low abundance and care is not taken, the MS analysis will detect only keratins. To minimize keratin contamination, use clean equipment, wear gloves and a lab coat, and tie back you hair. Changing gloves frequently is also helpful. Never touch a gel with bare hands and keep gel exposure to the atmosphere as low as possible.
What is the turnaround time for analysis? Turnaround time is dependent on the work queue and complexity of the work request. Generally, for a simple protein identification from a protein in solution, the turnaround time is a few days to a week depending on drop-off time. More involved experiments such as DIGE can take up to 1 month, including and LC-MS/MS and protein database results. LC-LC-MS/MS can take up to two weeks including searching the data.
How much do the services cost? Prices are determined by the UA and are subject to change.
How will I get my results? You will get an email informing you that your results are ready at our website, www.protbase.org. If you are a first time user, we recommend you meet with a staff member to discuss the data. We will explain the scoring and what constitutes a good spectrum.
Do I get special pricing as an UACC member? No. Univesity of Arizona policies do not allow for differential pricing.
What benefit do I get as an UACC member? UACC members get priority of samples over non-UACC users.
Is training available? Yes. We offer hands on training of graduate students and post-doctoral fellows when a high need for proteomics usage can be demonstrated. Please contact the Director of the service for more information.
|MS, all analyses||George Tsaprailisfirstname.lastname@example.org|
|MS,all analyses||Linda Breciemail@example.com|
|SDS-PAGE, Purification, sample prep, MS||Cynthia Davidfirstname.lastname@example.org|
|Smal molecule HPLC and MS||Yelena Feinsteinemail@example.com|