IMPORTANT NOTE: We will soon be requiring all submissions of samples to PSR to go through the new ilab system. iLab integrates sample submission, communications, billing, and will function similar to a LIMS system for the Proteomics Shared Resource. You can find the login page here. Principal Investigators at OHSU already have had their information uploaded to the system, and accounts created. All others will need to create an account in order to submit samples. You can find a help manual here, and we'll be happy to assist you as best we can as well (though we're still learning how to use the system ourselves!).
In the meantime, we are accepting samples both through iLab, and the old-fashion way as well. If you are interested in using the old-fashion method to have the Proteomics Shared Resource process your samples there is a submission form on the right. You can either fill in the form electronically and e-mail it to us; or print out the form, and drop it off with your samples. Either way it is generally recommended that you consult with us prior to your experiment to maximize your chances for success.
What follows is a description of the fields on the intake form. Feel free to read through it if you are having trouble, or would like an explanation about part of the form. You can always contact us directly with your questions as well.
Information Needed for a Successful Experiment
There are several pieces of general information we'll need to perform a proteomic analysis on your samples. To help us collect all of this information as quickly and easily as possible we've put together a sample submission form. What follows is a short walk through the information we'll need to process your samples, as well as an explanation as to why we need the different pieces of information. The information falls into 3 categories: information about you the client, the state of the samples, and questions related to the type of analysis you would like to perform.
The first portion of our sample submission form pertains to information about you the client. There are several pieces of information we'll need:
- Name, Phone number, e-mail address
This information is in case either we or our accountants need to contact you for any reason. Occasionally this will happen if questions arise about your samples, if billing information is out of date, or for us to send you a simple status update on your samples.
- Lab / PI and Department
These pieces of information are used by us and our accounting team to track who is using the core facilities and how often.
- Alias / FAID or Billing address
Most of our samples are processed with a fee-for-service type of agreement. This means that at some point we'll have to bill you for the work we perform. OHSU labs may provide an Alias and FAID for us to bill to. Our accountant will then use IBS to charge your specified account. For labs outside of OHSU we will need an address to send an invoice to. You can then generate a PO# for the billing. If you require a quote prior to setting up a PO#, please let us know and we can prepare one when you drop off your samples.
The billing information is an extremely important piece of information for us. We realize that you may not know this prior to dropping off your samples, and we will still accept samples without this information. However, unless you have a prior agreement with PSR, we will not release results until we have your billing information. We will send you a billing report prior to putting the charges through in order to give you time to review the bill and reply with and questions or concerns. We usually bill a project out 3 business days after sending you a copy if we do not hear back from you concerning the charges.
- Quoted Price & PO#
If you require a quote, or if we need a PO# from you, prior to us starting work on your project. Please let us know and we'll make sure this information gets recorded in the appropriate place on the sample submission form when you drop off your samples. We currently need a PO# from members of the Veterans Hospital prior to starting any project.
- Funding Source
We have special agreements with members of the National Cancer Institute and National Eye Institute that have resulted in a different fee structure for these clients. If your grant is funded through either of these Institutes let us know as you may qualify for a discounted price. If your project is not funded though either of these sources, please provide this information.
- Acknowledgement statement
The short statement that follows the Client Information box asks you to confirm you've read the our guidelines for sample preparation, and confirm that your samples are compatible with our mass spectrometers. In general the samples are considered mass spec compatible if they are free of the following:
- Significant particulate or insoluble material
- Radioactive or Biohazardous substances
- Salts: NaCl, KCl, Phosphates, and other non-volatile salts
- Detergents: SDS, NP-40, Triton, Tween, etc.
- Cyroprotectants: DMSO, Glycerol, etc.
- Cell debris: nucleic acids, mono- & polysaccharides, lipids, etc.
- PEG, and other polymers
The sample must also have a sufficient amount of protein present for us to generate a results set. Also, there may be other concerns that are particular to the type of analysis you wish to perform. Be sure to read the section of this document that relates to that analysis and be able to answer all the questions related to your samples. If you are concerned that your samples may cause problems with the sample digest, instruments, etc., please let us know and we can help you determine this prior to analysis.
The information required for us to successfully process your samples and return good quality data depends on the physical state of the sample when you drop it off, and the analysis you've requested.
There are a couple of pieces of general information we'll need to categorize and process your samples.
- Number of samples
Simply the number of different samples you are submitting. We'll use this information to help track your project and calculate your bill.
Genus and Species of the organism the proteins are from. If there are multiple species involved in the experiment (such as an experiment looking at interactions between viral and human proteins) please let us know all of the species involved. This information helps us refine our database searches and improves the quality of the results generated. If your protein of interest is not in our database you may have to provide an amino acid sequence for us to search on.
It is important that each sample submitted has a unique name. Ideally this name is short, not more than 8-10 characters, so that it can be written on the side of the micro-centrifuge tubes. Names as simple as 1, 2, 3… or A, B, C… are greatly appreciated.
- Preferred run order
We take precautions to run control samples before experimental samples, and gel samples in ascending molecular weight; provided we know this information ahead of time. This is done to minimize confusing results due to carry-over on our LC column. If there is a particular order you'd like to see your samples run in, please list the order in the space provided.
This section is located at the bottom of the form. We ask that you take a couple of minutes to describe your project and let us know what you hope to accomplish. We have a couple of reason for asking you to do this.
- First it will help us evaluate whether or not the procedures we are doing are in-line with your experimental goals. We may even be able to suggest a way to address the same question for less time and money.
- Secondly, this helps us with our sample preparation. At certain times your experiment may require us to pay closer attention to certain details. For example, when looking for a change in protein abundance in a co-IP experiment, it helps if we take extra steps to assure we are loading the same amount of material onto the column.
- Finally this information helps us keep your project straight. This is especially important when you, or members of your lab, are doing multiple projects with us. We received over 500 different samples, from about 40 different labs last year, and without some further information we may get projects confused. Having a project summary in front of us helps us greatly with this.
When dropping off a sample which is already in a SDS-page gel there are a couple pieces of information that will help us process your sample.
Image of the gel
The image of the gel is used to confirm that we will be able to get results based on the intensity of the gel bands. Also we attempt to run the bands in order of decreasing molecular weight, and an image helps greatly in planning the run order. We can take an image of an intact gel when you submit the samples if you prefer.
Extraction procedures vary based on the staining method.
Liquid / Lyophilized Samples
While there are relatively few pieces of information we'll need when receiving a gel sample, there are more potential problems when we receive a liquid or lyophilized sample. Here's a list of what we'll need to know.
- Physical state – Digested peptides, un-digested proteins, crude cell-lysate, tissue sample, etc.
While an SDS-page sample is almost always undigested proteins in the gel matrix, this is not true of liquid/lyophilized samples. We would hate to attempt to run your samples into a gel to purify them, only to find out you did the trypsin digest in your lab before-hand!
- All ingredients in the current or most recent solution
This information is important as it gives us the means to assess whether or not your sample will digest properly, is ready for the mass spectrometer, is free of contaminants, etc. Also remember that reagents can be carried over from previous steps in the procedure. As a rule of thumb if you haven't taken steps to remove a reagent, it is probably still present in your samples. If you are in doubt feel free to give us a copy of your procedures. We can take a look at your methods and try to determine if there is a reagent that we'll need to remove prior to processing your samples.
- Concentration / Volume of the sample or amount of protein present.
We need to know the amount of protein present in order to process your sample. If you give us a liquid sample, we'll need to know the volume and the concentration of protein in the solution. If you give us a lyophilized sample we'll need to know the number of micrograms of protein present.
There are several reasons for this: ratios of enzyme to protein are important in ensuring proper and complete digestions of your samples, there is an upper and a lower limit to the amount of protein we can load onto our LC system and return good results, and finally it is extremely important to load equal amounts of protein when doing spectral counting comparisons. If for some reason you do not know how much protein is present let us know, and we can perform a BCA protein assay to determine this.
The different analyses all have different concerns that relate to them.
MS/MS analyses: Protein Identification, PTM ID, and Relative Quantitative Analysis
- Unique protein sequence
We cannot identify proteins that are not in the database. If the protein sequence you are hoping to identify/characterize is not in the most recent release of the swiss-prot tremble database, you will need to provide us with the sequence. This is often the case for proteins that have been intentionally modified in some way from their native form.
- Length of LC-MS/MS Analysis
The length of time spent analyzing your sample has important implications on the data that will be produced. For an extremely complex sample, the more time spent on analysis, the more protein identifications we will obtain. For simpler samples a longer LC-MS/MS experiment is not useful in obtaining more results, and in some cases may even reduce the quality of the results. Also, we charge per hour for time on the mass spectrometer, so choosing a longer run time can get very expensive. What follows is a rough guide line for choosing an analysis time:
- 60 minutes
Good for a single gel band, or other samples in which you are only expecting find one or two proteins.
- 90 minutes
Moderately complex samples in which you expect to see a couple of dozen different proteins.
- 140 minutes
Suitable for Co-IP and similar experiments, and good for complex mixture analysis. We can identify up to about 100-200 different proteins in this timeframe, and it is a good choice for most analyses.
- 240 minutes
A lengthy LC gradient intended for highly complex samples, but ones you don't want to do multi-dimensional chromatography for. It isn't unusual to identify upwards of 400 different proteins from a complex mixture using this method.
- 2D LC: 7 x 140 minutes
This procedure is done as an inline mudpit experiment on the LC system by attaching a SCX column after the sample injection loop. Samples are bound to the SCX column and eluted in a series of salt bumps. Each salt bump moves a portion of the samples from the SCX column to the C18 column, where we perform a 140 minute LC-MS/MS analysis. Generally we will produce 6-7 salt fractions, though this amount can be changed upon request.
This procedure is good for analyzing complex mixtures originating from crude cell lysates, tissue samples, serum samples, and other experiments where there is an extremely large amount of protein material present. It is a slightly smaller scale than our off-line mudpit, and can identify upwards of 1,000 proteins from a sufficiently complex mixture.
- 2D LC: 30 x 140 minutes
This is the longest type of analysis we offer. Samples are fractionated using our off-line SCX LC system. One minute fractions are taken over the course of about 70 minutes. These fractions are then combined to generate, depending on the amount of protein present, about 25-45 samples for the mass spectrometer.
This type of analysis is usually reserved for characterizing extremely complex samples, looking at proteome wide changes for biomarker discovery, etc. The analysis takes up to a week of time to run on the mass spectrometer alone, and requires large amounts of time devoted to data analysis and interpretation.
Generally this type of experiment costs several thousand dollars to attempt, but we can generate several thousand protein identifications from a single experiment if all goes well. If you are considering attempting this kind of analysis it is essential that you meet with us prior to sample drop-off, to make sure the large amount of resources required for this kind of experiment are well spent.
- Post Translational Modification Table
This table is for use with PTM ID and Localization experiments. There is space for listing the modification sought, as well as the location and mass shift of the modification. If you do not know this information we can help you acquire it. Please note that some modifications (sumolyation, glycosolation, etc.) cannot be found with the software we have here.
Also it is important to realize that PTM identification and localization is a very difficult process with a high rate of failure. The software tools which we use to find the PTMs are not as robust as those for protein identification, so all potential PTM sites have to be manually validated. This greatly increases the time it takes to analyze the data. Also some modifications cause peptides to ionize poorly in the mass spectrometer. This decreases the signal to the point where we may not see the modified site.
Whole Protein Mass Determination
- Protein Purity
Information relating to the purity, concentration and amount of material present are of particular concern in doing whole mass reconstruction. An impure protein yields multiple sets of overlapping ion series that can quickly become impossible for our software to reconstruct. Also a heavily glycoslated or otherwise modified protein can cause similar problems.
- Approximate mass
We need a mass range to restrain the reconstruction software we use. A range such as about 25kDa, or between 30kDa & 40kDa is usually sufficient.
- Expected sequence
If you would like us to confirm that the protein is present in the expected form we will need the sequence and/or the expected mass value.