Mass spectrometry (MS) has become one of the most employed methods in biological research for identification of a wide variety of molecules such as proteins, oligosaccharides, lipids, etc., by determining their mass (molecular weight) and their fragmentation pattern. MS interfaced with HPLC (high performance liquid chromatography) or gas chromatography (GC) can be used to examine molecules with high specificity.
Individual laboratories that do not have the resources to perform MS must use an MS facility. This is the situation in my laboratory; we specialize in neuro-electrophysiology, but have developed an interest in proteomics for a specific project. To perform our MS analysis, we chose the Mass Spectrometry Laboratory of Molecular Medicine Resource Centre hosted in the Medical Science Building at the University of Toronto (http://www.medresearch.utoronto.ca/d_home.html). This facility provides MS services to University of Toronto investigators as well as other academic and industrial clients.
Choosing an MS facility is not a trivial task as most of the time the sample to be analyzed is the result of months of work and sometimes obtained from sources that are not easily available, such as biopsies of rare conditions. There are a number of aspects that must be taken into consideration when choosing an MS facility.
One important factor in choosing a facility is proximity to the lab; this is due to the fact that samples must be shipped under specific temperature conditions. This factor is extremely important for labs that are located in smaller scientific centers without a close MS facility. Bigger scientific centers usually have a few MS facilities; for example, in Toronto there are a number of MS facilities: (http://www.sickkids.ca/APTC/section.asp?s=Mass+Spectrometry&sID=1446, http://www.sunnybrook.ca/research/services/proteomics, http://www.utoronto.ca/emililab/emili.html, http://www.utoronto.ca/crnd/index.htm, http://www.chem.utoronto.ca/peoples/mass.php).
Other factors to be considered when choosing an MS facility are the diversity of services offered, the facility’s experience and performance, the overall quality of service and the cost. The University of Toronto facility has three mass spectrometers (QTRAP, Voyager and API III) that allow for a variety of MS services and procedures, such as ESI, MALDI, LC/MS, MS/MS, MS3. Analyses include protein identification from the enzymatic digestion products of proteins; structural elucidation of peptides, proteins, oligonucleotides; and oligosaccharide sequencing by MS/MS/PSP. They also identify post-translational, chemical and mutational modifications of proteins and nucleotides.
In our case, we were interested in performing basic protein identification from the neuromuscular junction before and after low-frequency depression by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Due to the costly fee ($160.00/sample for NanoLC MS/MS Protein ID and peptide sequence, and $40.00/peptide for peptide sequence) and the larger amounts of protein required (very laborious and scarce in our case), we decided not to perform these additional analyses. Even more costly and laborious would have been the identification of phosphorylation sites for the identified proteins. When dealing with non-mammalian systems (as in our case), it is important to check their experience and performance with similar types of samples.
As sample preparation is the crucial step for protein identification, check the recommendations and requirements of the facility before beginning your sample preparation. It is very important to consult both the website and the person performing the analysis as some procedures require specific solvents and reagents. In my case, the information provided was very useful. The facility website contained pertinent recommendations about sample preparation according to the analysis that was to be performed including the minimum sample concentration required, the recommended buffer, as well as a list of websites for database searches. Some MS facilities also offer in-gel tryptic digestion (the client provides a standard band of interest excised from SDS-PAGE gel and they perform the digestion and extraction of the peptide), but I found the price extremely high ($100.00/gel band digestion at the facility I used). To maximize the protein concentration, they offered sample clean-up on a C18 column at $20.00/sample.
The facility’s internet site contains downloadable, easy-to-complete sample submission and procedure request forms. Using these forms, the researcher can specify various parameters (either using the recommended ones or not) as well as the purification method and solutions to be used. Researchers can also specify what steps should be performed after identification and what data is required.
Communication with the facility personnel is also important. There is no single universal MS technique available that is applicable to all samples. Conversely, several different techniques may give similar results for a given sample. Therefore, I highly recommend seeking advice for which techniques should be used. In my case, as I have limited experience with MS, the facility staff chose, designed, and executed the MS experiments and modified the experimental design in response to results. For molecular weight determination by MS/MS, the client receives a copy of the spectrum and/or a chromatogram and base interpretation. For protein identification, they provide a hard copy of database research results and a website link. I received the MS/MS data on the same day that I send the probes to be analyzed. The data provided included mass fingerprinting chromatograms showing spectra peaks; lists of peak m/z values were provided as a spread sheet. The data could be easily exported into MS Excel and then the Excel files could be imported into MASCOT Search program (Matrix Science Ltd, London). We used MASCOT for peptide analysis against all available proteins from the following databases: MSDB (comprehensive, non-identical protein database), NCB (non-redundant) or Swiss Prot. Three proteins were identified out of 10 samples. We were pleased with these results as the samples were from the very first trial in an organism from which very few proteins have been sequenced. I then followed up with physiological experiments.
A very important factor for beginners to take into consideration is whether the facility provides follow-up and instruction on the methodologies used. Some facilities have tutorials on their website (http://massspec.unm.edu/ms.htm). Websites may also include lists of their publications, information on upcoming events, conferences and meetings (http://www.rzuser.uni-heidelberg.de/~bl5/), as well as other special information such as “Watch a MALDI spectrum grow”, “Watch real-time acquisition of an FD mass spectrum”, “Get MALDI and FAB matrix spectra as PDF”, “Find parameters on MALDI spectral plots explained” (http://www.rzuser.uni-heidelberg.de/~bl5/). I found that this information helped with the interpretation of my results.
Another important factor is the cost. As incentives, some facilities, including the one in my institution, will run promotions for frequent users. The price is also reduced in the event of poor results due to insufficient peptide generation. Researchers can be trained by the staff to use the facility, thus, lowering the cost. However, I recommend spending the time to master the technique only if you intend to routinely perform MS analyses. If you choose to learn and master the technique, renting the facility can save time and lower the cost significantly. Due to the fact that I did not need to extensively use this technique and because it takes such a long time to master it, I chose to let them perform the MS analysis.
It is important to note that the facility’s input may be so essential for the successful conclusion of a researcher’s study that the user-facility interaction becomes collaborative. Therefore, it is wise to determine, from the beginning, the nature of the researcher-facility relationship that one desires.
Dr. Lorelei Silverman
HSF Postdoctoral Fellow
University of Toronto
Department of Physiology