General Information (17)
We are Located in Room 1414 of the Genome center.
You can use the map links below
If you have any questions please do not hesitate to call
The Lab number is 530-754-9474
Amino Acid Analysis and Edman Sequencing = (530) 752-7327
Brett S. Phinney 530-754-5298
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It varies, but is usually 5-10 days for bottom up protein ID, pull-downs… etc… We can be much quicker (2-3 days) if the samples are already digested. We will inform you if there are any delays.
Complicated projects (Label free quantitation for example) that require multiple replicates and extensive data analysis can take a very long time and probably should not be attempted by people off campus. It is always much better to do these types of experiments with your local proteomics facility. If you don’t have that option, give us a call, we still maybe able to help you.
Our facility rule is that someone will be in contact with you at least every three days to update you on the status of your samples. If you don’t hear from us in three days, please call and complain (loudly if you’d like)
We have been thinking about how to implement an automated system for checking the status of your samples. We haven’t had a lot of requests for this, but if it interests you, please let us know
Around 2006 the MSF was split into two parts. The Amino Acid Analysis and Edman sequencing moved over to the proteomics core in the Genome Center and the self use mass spectrometers from the MSF turned into the campus mass spectrometry facility.
The MSF and the Genome Center proteomics core are are essentially the same thing and occupy the same space in the Genome Center Building on campus. We maintain the MSF name and website because the Molecular Structure Facility (MSF) has been around a long time and a lot of people know our Edman and Amino Acid Analysis services through that name. We our proud that we are able to keep Edman and AAA operational (and even expanding) when most other facilities have closed them.
Ok, this is a great question (thanks for asking!). We have a number of quality control procedures we do before and during the analysis of your sample. Every facility will do this a bit differently, but this is how we do it. It is also evolving as new technology and software becomes available.
We practice bottom up proteomics, which means that your protein sample is digested into peptides before we analyze them by LC-MS/MS. This digestion step is crucial and if it fails, nothing will work. When we digest your sample, we also digest a known control sample. Usually this is a gel piece from a gel of a human cell lysate. If your sample looks unusual or shows signs of a failed digestion (rare, but it does happen), excess keratin contamination or polymer contamination we analyze the control digestion and compare it to our historical data of that control sample. If the control digestion has failed we will contact you and apologise profusely. We will also of course not charge you. This type of failure does happen, but it is rare.
Nano-Scale LC-MS/MS is tricky and the sensitivity and chromatography of our machines can vary (sometimes dramatically). So how do we control for this and how do we know when our machines are performing well enough to run your samples. We have two types of controls we use for this step. The first is replicate control runs of 125 fmol of a BSA digest. We buy this BSA digest from Michrom Bio-resources and analyze them right before we run your sample. Here is an example of a recent control run (July 2011). We then make 7 extracted ion chromatograms for 7 peptides from this digest that span the entire gradient. We look for such things as peak shape, intensity, retention time, etc..
We then run the same sample again and look for retention time reproducibility.
Finally we search the BSA QC run and look for the number of peptides identified, sequence coverage etc… We store all this data in an excel spreadsheet and currently have historical data going back several years for every LC-MS/MS system we own. So we know before we run your sample, how the machine is behaving and how it’s performance compares to past performance.
We also have a more complex QC (Human cell lysate) which we run every few weeks or when we have have a suspicion something is wrong. We do not run this sample daily because running such complex samples usually will degrade the machines performance after the run is complete, unlike our BSA control.
If you have any other questions please give us a call or send us an e-mail.
Last updated on July 14, 2011 by Brett S. Phinney, Ph D.
We know there are many labs you can send you samples to (for a list please see the ABRF Core Marketplace or Science Exchange) and we appreciate you considering us for your samples. What we guarantee is that we will treat your samples like they were our own and do every test and take every precaution we can reasonably take to produce the best data for your samples. We cannot guarantee that we will give you the result you are looking for however, as that has a lot to do with sample prep and variables that are outside our control. But I will guarantee that the personnel in the lab are always a phone call or e-mail or short walk away and will be happy to discuss your project and samples and will work with you to solve any problems that arise. We are always here to bounce ideas off of or discuss your data (or data you obtained from elsewhere).
Glad you asked. If you want another place to send your samples or just want a second opinion, I can recommend the following labs. They do great work. You will not be disappointed
Ok, this comes up from time to time. Essentially if you feel we contributed intellectually to your project it is appropriate for us to share authorship. If not, then no we should not be authors on your paper. Usually it’s pretty obvious what side your project falls on, but if you have any questions it’s always a good idea to talk to us about it. We don’t mind.
If you want a more detailed explanation of authorship when using a core facility please have a look at the ABRF Authorship Guidelines. These are the community standards most core facilities follow when there are questions about authorship
We operate on a first come first serve queue with the following exceptions
Samples from major users of shared instrumentation grants are prioritized over non major users for that particular instrument
On campus customers are prioritized over non campus customers
Experiments that require batching or have samples that need to be run in close proximity with each other may be prioritized or de-prioritized depending on the other samples in the queue to ensure efficient operation of the equipment
The Proteomics Core has an advisory committee that provides oversight to the facility and is the appropriate group to resolve any conflicts that might arise regarding sample prioritization of on campus customers.
Actually we think we are rather inexpensive for the quality of services we perform.
For a list of other facilities around the world that offer similar services please see the ABRF core Marketplace or Science Exchange
One Option to be aware of is our Mass Spec only rate. Essentially you send us the prepared sample, we inject it and give you back the raw data. The cost is really rather affordable
Glad you asked, We know budgets are tights (ours is too 🙂 ) so we try to facilitate the most science we can using the least amount of your precious funds. Along these lines we have the mass spec only rate. This is essentially just the raw mass spectrometry file from your sample. You would need to process it and analyze it yourself. If you have experience doing this type of analysis this can save you a lot of money. If you don’t have this type of experience, don’t worry, we can teach you (through our free monthly classes) or our paid yearly summer courses. Please see this link for more details on the classes
Sample Preparation (1)
Detergents usually show up as a bell shaped curve with each peak having the same mass difference from each other. Detergent peaks are usually so abundant that they overhsadow any peptide . Some detergent we can get rid of, some we cannot. Please contact us if you have any detergent in your sample.
Other things to watch out for
- large amounts of Urea (> 6M)
Protein ID (9)
This used to be a problem when we had a large variety of instrumentation. Nowadays we use Q-Exactives for or identification and most of our Quantiation. For Targeted proteomics applications we have the TSA Vantage triple quadrupole. We have found that out Q-exactives can do about 70-80% of what the TSQ can provide. The TSQ has a faster scan speed and it’s response is probably more linear than the Q-Exactives, although this is at the expense of greater method development
Results are usually returned with some sort of probability estimate (depending on the software we use) and usually a false discovery rate (FDR). You can have a look at our Scaffold Quick Start Guide 4.0 for some more info. To make a long story short, we cannot guarantee the results you get are 100% correct. No one really can with this type of data unfortunately. The best advice is to please call before you spend a lot of money making an antibody or designing expensive experiments if you are not really familiar with this type of data.
Here is an excellent paper that explains multiple testing correction, FDR’s, p-values and q-values
We do a large amount of work to decrease the Keratin contamination in our laboratory, having said that it is possible it is coming from us, but more often than not it comes from the submitted samples. Not to worry, most of the time Keratin can be ignored if it is not at a large level. We can work with you to decrease Keratin contamination on future samples.
One misconception about proteomics (at least bottom up proteomics) is that we can identify your protein sequence directly from the data we generate from our mass spectrometers. Although this is technically possible using something called de novo sequencing, in practice it still does not work very well.
What we really do is match MS/MS patterns we generate in the mass spectrometer with theoretical MS/MS pattern from existing sequences. Usually these sequences are stored in a FASTA formatted text file. Sometimes people refer to this as a database, which it really isn’t.
So where do we get this FASTA file? There are a few good places to look
There are a few others too
Another option is to generate your own transcriptome if you cannot find any available protein sequences.
Here is a good article that can get you started
I’ll add more to this section hopefully soon
Please contact me if you can’t find a good database 530-754-5298. Usually we can find if one exists.
Yes, Please see
A lot of these come from the GPM data set of the week, so please have a look. You can also download the raw data in most cases
- Deep proteome and transcriptome mapping of a human cancer cell line 2011
- A comprehensive map of the human urinary proteome
- Proteome and transcriptome profiles of a Her2/Neu-driven mouse model of breast cancer
- A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease.
- A protein epitope signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.
Quantitative Proteomics (1)
So the best way to answer this question is to ask what is the variability in your system. This can be both technical and biological. Technical is a little more feasible to measure, but in practice it is really hard to measure either one of them . When it all comes down to it, we really have no idea how many replicates you need to do for your particular sample. 1,3 5, 7 ??? You need at least three to do a simple T-test, but beyond that it normally comes down to time and money. How much time and money do you want to invest and how much sample do you have. Based on past experience between 4-6 replicates is a good starting place. Usually we like to do a preliminary experiment to see how variability looks between conditions. After we do a preliminary experiment we can do a power analysis in MSStats to get an idea of how many replicates you need to see a certain fold change. Just a warning MSStats usually says you need far more replicates than you have time or money to acquire.
Here are some other resources on this topic
Chris Colangelo and I also hosted a variability workshop at ASMS in 2014. The slides can be found here
Phosphorylation Detection and Quantitation (2)
Unfortunately this is quite complicated and there is no clear consensus on if it is better to enrich or which enrichment technique is best. So the answer is “Quite Possibly” sometimes this works and sometimes it doesn’t. Not very satisfying I know…
I’ve seen cases where not enriching can generate better data and I have also seen cases where enriching phosphopeptdies will lead to an identification of a phosphorylated peptide that was missed without enriching.
Here is a recent paper That compares several phosphopeptie enrichment techniques.
The Association of Biomolecular Resource Facilities (ABRF) recently did a study on identifying phosphoylatied peptides. You can see the results here
It’s quite interesting, so it’s worth a read.
In fact I do (although this is no where near complete and getting a bit old now) I will try to move all these to a mendely group soon
This does a nice job of Comparing the various enrichment techniques for phosphopeptides
This paper does a great job of explaining the many complexities of phosphopeptdie analysis
Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways
This paper does a great job in explaining why phosphoylation is so important.
ABRF (AKA A “BARF” ) is the Association of Biomolecular Resource Facilities. It is a great organization that has a dedicated and passionate membership. You can often the answer your looking for by asking their listserv (it’s not technically a listserv…but no one really cares…) or joint one of their research groups
Financial and Billing (1)
Although we would love not to charge you…
In order to pay us, you need to open an account by clicking on this link
and then clicking on create an account down at the bottom
You can then put in all your financial contact information
If you want to pay by credit card (do not put in your credit card number) just put in a note about wanting to pay this way. You will receive a link with your invoice with instruction on how to pay using a creed card.
If you are from UC Davis, please put in your DAFIS #
If you are from another UC campus, we need your full Account strings
Please see this web site for the campus format you need to follow
UC Davis Genome Center