Proteomics: FAQs
What is your turn around time for sample analysis?
We strive to process all samples within 1 working week of when the samples are submitted. Samples are processed on a first come, first serve basis. Thus, limitations in instrument and personnel capacity, or instrument breakdowns can sometimes delay analysis. If your analysis will take longer than one week you will be contacted with an update on the estimated time for completion. For larger projects the timeline for completion will be discussed prior to sample submission.
How do I cut out my gel bands? How big should they be?
Gel bands should be cut into into slices of 1–2 × 5–6 × 1mm3 dimension. If a gel slice is larger, divide it accordingly into several tubes. Remember to wear clean gloves, put your gel in a clean dish, and use a new razor blade, if possible, for each band.
Can I use detergents in my sample prep protocol?
The use of detergents should be minimized as much as possible. Many detergents (IGEPAL, Triton X) cannot be removed from a sample sufficiently to allow for successful ionization in the mass spectrometer. This means that you will not get much if any data. Please see the link on the Proteomics webpage about detergents and mass spectrometry.
Can you do the digestions for me?
Yes. The cost for us to digest your samples can be found on our Proteomics Pricing webpage. We also offer training on proper digestion protocols and have an open access hood that is available for you to use. This is a cost effective alternative as we only charge the cost for consumables if you perform the digestion yourself in our facility.
Can I use colored tubes? If not, what type of tubes should I use?
You should use clear polypropylene low retention 1.7 ml microcentrifuge tubes. Colored tubes contain dyes that can leach out into solvents during digestion.
What type of instruments do you have?
The two instruments we utilize for protein/peptide analysis are a MALDI-TOF/TOF mass spectrometer and a nano LC-LTQ linear ion trap mass spectrometer.
MALDI-TOF/TOF: Matrix Assisted Laser Desorption Ionization Tandem Time-of-Flight. This instrument can be used for the analysis of peptides, lipids/carbohydrates, DNA, polymers, and intact proteins. It can measure mass up to ~200 kDa (depending on the molecule). In the low mass region between 700-3500 Da it can measure mass with high accuracy (~10 ppm = +/- 0.02-0.08 Da). This instrument is appropriate for the analysis of relatively pure samples as there is no additional separation, everything in your sample will go into the instrument at the same time.
Nano LC-LTQ linear ion trap: This instrument is primarily used for the analysis of peptides. It is coupled with nanoscale liquid chromatography which means that the peptides in your sample will be separated over time (between 40-90 min) before they enter the mass spectrometer. This allows for the analysis of complex mixtures of peptides from many proteins. This instrument is considered to be a low mass accuracy instrument and measures mass within ~500 ppm (+/- 0.5-1 Da).
Which instrument should I use? MALDI/LTQ?
MALDI-TOF/TOF: If you have a non-peptide sample. If you have proteins that have been isolated on a 1D or 2D gel and you are confident there is only 1 protein in the sample. If you need high mass accuracy.
Nano-LC-LTQ linear ion trap: If you have a mixture of proteins (i.e. more than 2 proteins).
All I see in my sample is keratin? What happened?
There is always some level of keratin contamination in all samples. This will generally be overwhelmed by the signal of the peptides in your sample. If not, it could mean two things: your sample has been contaminated with high levels of keratin sometime during the sample preparation process OR there is little to none of your protein in the sample (this is commonly seen with samples from silver stained gels). It is usually very obvious what the reason is. If you find that you are having problems with keratin contamination, please see our sample submission webpage for advice on creating a keratin free environment.
How will you send me my data?
Your data will be sent electronically. For routine samples we will send the results as a .pdf document. For larger project or complex samples your data will be sent to you in a Scaffold file. This is a software we use that allows us to share all of your data with you in a single file. To view the Scaffold file you need to download the Scaffold viewer. For more information and a tutorial on Scaffold please visit our Proteomics Educational Resources webpage. In all cases, you will receive your results and a description of the analysis sufficient for inclusion in a publication.
Will I see the entire proteome?
No. As great as mass spectrometers are, they are always going to be limited by the fact that they can only detect a finite number of molecules in a given amount of time. In addition, at any given point in time they can only sample molecules within ~3 orders of magnitude in concentration. A typical complex biological sample may contain hundreds or thousands of proteins spanning up to 10 orders of magnitude in concentration. The level of coverage you will obtain in your proteomics analysis will depend on your sample preparation and the level of fractionation performed prior to mass spectrometry analysis. For example, let’s assume that our mass spectrometer can only analyze 10 molecules at a time. Your sample contains 100 molecules. If you inject this all at once you will just see the top 10 (in terms of abundance). However, if you were to first fractionate this sample into 10 fractions that each contains 10 molecules and then inject them individually you will achieve much higher analysis coverage of the molecules in your sample. The level of fractionation you do will depend on your experiment (how much do you really need to see?) and your budget (every fraction is more mass spec time).
How many proteins can I identify?
In a single injection on the nano LC-LTQ linear ion trap system we can generally see anywhere from 5-200 proteins depending on your sample.
Do I still pay for the analysis if the results are not what I am expecting or are uninteresting?
Yes. Unfortunately, even unsuccessful analyses utilize mass spectrometry time and facility personnel effort. In order to keep our instruments running and pay for the highly qualified people to manage them we have to charge users fees. However, we will always do our best to help you identify and fix the problem for future analysis.
What is the lowest concentration you can detect?
This depends largely on your sample. In general for a single component sample (i.e. peptides from only one protein) we can detect down to the low femtomole to high attamole range. The more complex the sample, the harder it becomes to detect individual molecules at these low levels.
Can you analyze silver stained gels?
We can as long as you are using a mass spec compatible silver stain. However, the silver stain required additional washing steps which can lead to sample loss. Also, because silver is a non-quantitative stain it is hard to say if there is enough protein there for us to detect. In general, if you band shows up right away we can probably see it. If it takes 10-15 minutes of staining for a band to visualize, we probably won’t see anything by mass spec. A great alternative to silver stain are fluorescent stains like Sypro Ruby. These types of stains offer similar sensitivity levels but are much more mass spec friendly and they are quantitative.
Can you quantitate proteins in my samples?
Mass spectrometry is inherently a non-quantitative technique. The intensity of a given molecule is dependent on both its abundance and its ionization efficiency (remember that a molecule has to be charged for the mass spec to detect it). There are ways to obtain relative quantitative information by mass spectrometry. The primary method we utilize in our facility is a label free approach call Spectral Counting which is based on the assumption that the number of spectra that are detected for peptides from a given protein is directly related to the abundance of that protein in the sample. Additionally, we also utilize 2 dimensional electrophoresis approaches in which relative quantitation is determined from spot intensity in the gel prior to mass spectrometry analysis (for spot identification).
Should I include facility personnel in my publication?
Co-authorship is generally expected when core personnel have made significant contributions to the research in the form of consultation, experimental design, method development, data analysis and/or data interpretation. Significant can be defined as “the project would not have progressed, or progressed at a substantially slower pace, without the guidance of facility personnel”. In such cases, core personnel should have the opportunity to review and edit the appropriate sections of a manuscript before submission.
Acknowledgement of facility contributions is expected in publications that include any data generated in the facility. For example, when fee-for-service is performed with no method development and minimum effort by Core personnel.
An example of an appropriate acknowledgement is “The authors wish to thank the Proteomics and Metabolomics Facility at Colorado State University for analyzing samples”.
Please inform us when relevant publications are accepted and forward us a copy for our records. We may also post citation information on our website. This information is vital to the continued support of the facility.
Can you help me with my grant proposal?
Yes. We are very happy to provide letters of support and assistance in grant writing. For letters of support, please allow at least 2 weeks from the time of your request. Please furnish the following information: Title of grant, agency to which you are submitting the grant, your title and mailing address, and 1-3 sentences on the goals (or specific aims) of your proposal.
Do facility personnel participate as Collaborator and Co-Investigators in grant proposals?
Yes. In fact this is a crucial way for our facility and personnel to remain funded and available for your research at CSU.
We are always willing to participate in this capacity. To determine is this is a appropriate you can ask yourself the following questions about your work:
Does the proposal include a specific aim(s) that focuses on mass spectrometry?
Is method development is required?
Are there large numbers of samples are to be analyzed?
Would the aim or proposal not receive a favorable review without the support of Core personnel?
Would the project progress at a substantially slower pace without the guidance of facility personnel?