Overview

Proteomics is the large-scale analysis of protein expression patterns. Along with functional genomics, proteomics is now an established tool for investigators to better understand the molecular events underlying many biological processes. The Proteomics Laboratory within the UC/Genome Research Institute is fully equipped with protein separation and mass spectrometry equipment and the trained personnel to perform comparative analyses of complex protein mixtures and the identification of differentially-expressed proteins. Proteomics laboratory personnel also offer guidance to the research community regarding the feasibility of a given approach and/or advice on proteomics experimental design to enhance the probability of success. Please contact the lab director to set up a consultation.

Research expertise in the Proteomics Laboratory is divided into two subcategories:

The electrophoresis section assists investigators in evaluating protein expression changes that occur in a biological system, such as during disease development and progression, stress or drug exposure or during normal cell and tissue development. The identification of changing protein expression patterns can provide important information about pathways involved in these processes and can lead to the identification of new protein markers for the diagnosis or the treatment of diseases.

The mass spectrometry section provides the identification of differentially-expressed proteins and the characterization of protein modifications (e.g. phosphorylation, glycosylation, ubiquitin modification).  Furthermore the laboratory has developed quantitative proteomics methods using isotope tagging methods in which mass spectrometry can be used to both identify the proteins and quantify the changes.  

Services

The Proteomics Laboratory provides services to investigators in the analysis and identification of complex protein mixtures using Two-Dimensional Gel Electrophoresis and Mass Spectrometry. For protein identification and characterization, the laboratory is equipped with four mass spectrometers, offering a set of complementary technologies to address the different needs of the investigator. The laboratory offers the following services:

• 2-D gel electrophoresis, protein staining, and analysis of differentially-expressed proteins (including DIGE and ProQ staining).
• Assistance to investigators in the preparation of cell or tissue samples.
• A complete suite of standard protein electrophoresis services including:
• small-format and large-format denaturing electrophoresis.
• native one-dimensional gel electrophoresis.
• Complete set of Protein Mass Spectrometry services:
• Identification of post-translational protein modifications.
• Identification of protein complexes from pull-down experiments
• Mass confirmation of purified proteins.
• Sequence confirmation of synthetic peptides.

Cost for Services

Services can be provided as a simple fee-for-service or via project collaborations with direct support for core investigators within grants. Please contact Ken Greis to discuss your project and core fees.  Many of the frequently asked questions (FAQs) are addressed below.  Current fee structures for standard services are available on the UC Office of Research and Graduate Education Proteomics Website.

Contacts

Proteomics Laboratory FAQ’s

1. How do I submit a sample for proteomics analysis?

In order to ensure the best chance of success for all samples submitted to proteomics lab for analysis, all new customers should contact the proteomics director for a consultation prior to submitting samples.  Please read the other FAQ’s below since these are intended to address many of the common issues.  During the consultation, the director will ask a number of question to be sure that the analyses requested are both appropriate to meet the investigators needs and available within the proteomics research community.  If the request is outside the scope of the laboratory capability, the director will recommend other options for the investigator.  The goal of this consultation is provide the investigator with realistic expectation of what can be done, the time it will take, and the cost of the analyses.

2. How much protein do I need to identify it by mass spectrometry?

We can identify a protein by trypsin digestion and mass spectrometry from as little as a few fmoles of a purified protein.  From a 1D gel, considering the losses that one takes in the digestion and recovery of peptides from the gel, we can often identify most proteins that are distinctly visible with a MS-compatible silver stain.  For 2D gels this is a bit trickier since the protein gets concentrated into such a small spot that sometimes we get a nicely detected protein spot that just does not liberate sufficient peptides after digestions for identification.  The truth is that it is very dependent on the structure and sequence of the protein.

3. I have heard that protein identification by MS could not be done on silver-stained gels, is this true?

The answer is No.  As indicated above, we routinely identify protein from silver-stained gels; however, there are a couple precautions that need to be followed.  First, one must use an MS-compatible silver-stain.  There are a number of commercial staining kits marketed as MS-compatible and to our knowledge they all work.  You can still make your own reagents; you just have to eliminate steps that include glutaraldehyde fixation because glutaraldehyde crosslinks the proteins and makes it very difficult to recover peptides from the gel.  A second concern is that one must take extreme care to avoid handling the gels, reagents and/or stains without gloves for fear of introducing keratin contamination (see FAQ 7) which can mask the detection of low level proteins.

4. I have a protein that gives a nice band by Western blot, can you identify the protein?

Like many questions, the answer here depends on a number of factors.  First, one must keep in mind that detection with antibodies and chemiluminescence reagents only require a few hundred molecules (of course depending on the quality and specificity of your antibody). To detect proteins at the low femtomole range by MS requires about 100,000,000 molecules.  Thus a protein readily detected by Western blot may not be in sufficient quantities to identify by MS.  Furthermore, antibody detection is typically used on complex protein mixtures because there is no need to purify the protein of interest prior to Western blot to get good signals.  Unfortunately if the target protein is buried under a number of more abundant proteins at the same mobility position on the gel, then these more abundant proteins may mask the MS-detection and identification of the protein of interest.  Having said all this, we and others have been most successful at identifying proteins that interact with antibodies by using immobilized antibodies to enrich for your protein of interest (see FAQ 5 for details).  When done correctly this has two advantages; first, one selectively enriches for the protein(s) of interest, and secondly, the protein(s) can be eluted from the immobilized antibody without releasing the Ig heavy and light chains thus minimizing interference on the gel and mass spectrometer.

5. What about protein identification from Immuno-precipitations?

IPs, like western blots, can be a bit tricky, but can be effectively coupled with mass spectrometry to identify the target protein and other proteins that interact with the target protein.  The key here is that you must remember that the most abundant protein in an IP will be your antibody, thus when you run the resulting pull-down on a gel, you will get huge bands for the Ig heavy and light chains that often mask the protein(s) of interest.  This is a more difficult challenge for polyclonal antiserum since you not only have your antibody of interest, but also all other antibodies in the serum.  In all cases, the best success is achieved by immuno-enriching your Ig of interest then immobilizing it on a resin.  That way you can capture and elute your protein(s) of interest without liberating the interfering heavy and light chains.  We have identified interacting protein in a number of biological systems for several investigators using these methods.  One final note here; Ig purification, generation of the immobilized column and collection of the enriched fractions are all functions carried out in the investigators laboratory.  The proteomics lab personnel generally take over just before or after running the 1D gel.  Consult the Proteomics Director for additional details.

6. I think my protein has modifications (phosphorylation, glycosylation, ubiquitination, etc) can you identify all the modification sites?

Mass spectrometry is an ideal tool for mapping sites of post-translational modifications (PTM); however, the success will depend on the type of modification, the amount of protein available, the purity of the protein, the sequence of the protein, and the stoichiometry of the modification.  I’ll use phosphorylation as an example since it represents the majority of inquiries.  Let’s say we have a protein that we know is phosphorylated (e.g. antibody reactivity (+/-) phosphatase treatment) and we would like to know what sites are modified.  First, we will need enough protein (generally low µg amounts) of sufficient purity to digest and recover peptides containing potential sites of modification.  This is often the most difficult task when working with low level regulatory proteins.  Secondly, the phosphorylation at a given site of the protein must be of sufficient abundance to be detected.  In other words, if our limit of detection for peptides is 5 fmole and your stoichiometry of phosphorylation at a given site is 10% occupancy, then we would need a minimum of 50 fmoles of total peptide to even have a change to identity the phosphopeptide or site of phosphorylation.  Adding to this challenge is the fact the phosphopeptides are generally more difficult to detect and sequence compare to un-modified peptides due to ionization efficiency issues, thus you may need to have 20-50X more material to detect and sequence a phosphopeptide at a 10% occupancy rate compared than its un-modified counter part.  Lastly, this may be obvious, but I get this question all the time, “You found two sites of phosphorylation, so can I report that they are the only sites modified under my conditions?”  The answer here is a resounding NO!  The mass spectrometer can only positively confirm a modification, it cannot completely rule out other modifications that go undetected.  If the sequence of the protein is such that a site of modification is liberated as a tryptic peptide that is outside the mass range of the instrument (generally below 600Da or above about 4000Da) then it will go undetected.  Furthermore, an undetected site of modification may just have a stoichiometry that is below our current detection limits. Thus we can only report what is detected in the mass spectrometry.  Having said all this, we have successfully identified various sites of modification including phosphorylation.  We have a number of methods to enrich for phosphopeptides to increase our changes of success, but we may have to start with µg amounts of a fairly pure protein.

7. I got my results back and some of the protein identification said “keratin contamination,” what does this mean?

Human keratin is a ubiquitous contaminant that originates from sloughed off skin (a major component of common dust—kind of gross by true).  It can be inadvertently introduced from contaminated reagent, stains, handling gels without gloves or reuse of reagents.  One speck of dust when digested with trypsin can release peptides from a whole series of keratin-family proteins that so overwhelm the spectrum that peptides from other proteins may not be detected.  In the proteomics lab, we take special precautions to minimize keratin contamination, thus we prefer to have investigators submit their entire gel and we will process the protein spots or bands in our lab.  These precautions are particularly important for silver-stained gels since the protein of interest is typically near the lower limit of detection to start with.  Even with these precautions in place, keratin contamination is still turns up from time-to-time and is reported as such.

8. Why did I get multiple proteins identified from one band or spot on the gel and can I tell which one is more abundant?

It is not uncommon to get multiple proteins identified from a single band from a 1D gel.  Even with the resolving power on 2D gels, we often detect more than one protein in these samples.  This occurs both because of the sensitivity of the mass spectrometer and the fact that sometimes more than one protein has the same or very similar migration properties.  The good news is that in most cases, we can provide a pretty good idea about which protein is most abundant in the sample based on the signal intensity of the peptides from each of the proteins.  While this is far from an exact measurement (since extraction efficiencies of peptides from the gel and ionization efficiencies in the mass spectrometer can confound the issue) in most cases we have sufficient data to inform the investigator which of the proteins identified accounted for the majority of the material detected in the gel by the staining protocol.