Contributing editor Tanuja Koppal, PhD, talks to Sunia Trauger, PhD, director of the Small Molecule Mass Spectrometry facility at Harvard University, about the recent trends in mass spectrometry. Advances in instrumentation, automation, and remote access seem to be leading the way to improved detection, faster results, and more diverse applications. While challenges in sample prep and data analysis remain issues, access to emerging informatics tools and experience handling and analyzing samples seem to mitigate some of the problems.

Q: Can you describe the goals of your facility, the instrumentation, and the types of analysis you undertake?

A: We are a core mass spectrometry (MS) facility, and our goal is to serve the needs of the scientists at Harvard University. We do mostly small-molecule analysis, but we also look at proteins, DNA, RNA, and other biological molecules. We have different types of MS instruments, such as an electrospray timeof- flight, an electrospray quadrupole time-offlight (Q-TOF), several triple quadrupole mass spectrometers, a matrix-assisted laser desorption/ ionization (MALDI)-TOF, a MALDI-TOF/ TOF, and a GC-MS. So we cover everything from volatile to nonvolatile samples using three different ionization techniques. We also serve a lot of nonprofit organizations and small local companies, although priority is given to our clients at the university.

Our main client is the chemistry department. They do a lot of chemical synthesis, and we do a lot of formula and structural confirmations for them. We work with the Harvard Medical School to develop assays to quantitate natural products. We also work to quantify endogenous and drug metabolites. We sometimes help detect the molecular weight of a purified protein by electrospray or MALDI-TOF or take on simple protein identification projects and look at post-translation modifications. We have a MALDI-TOF/TOF instrument that is connected to all the search engines and databases to help us with protein identification. There is a separate biomolecules group here at Harvard that takes on the more complex protein identification projects.

Q: What are the current challenges in mass spectrometry, and what do you need to overcome those challenges?

A: One of the emerging areas for MS applications is metabolomics, which is a lot harder to do than proteomics. In proteomics, the final step in structural characterization is taking the MS/MS data and searching against the fragments from the hypothetical protein database. In metabolomics, the small molecules could have any structure, and you need some experience to interpret the structure from the fragmentation patterns. In metabolomics there is a lot of carryover of knowledge and manual interpretation for structural characterization. Having people who have that kind of experience allows you to get a lot more out of your facility and helps you solve complex problems. People expect metabolomics to be push-button, where they walk in with the samples and we can do the metabolomics profiling data analysis to identify which peaks are different. But then the key question arises about what that peak is, and it’s not that easy to find the answers. There are tools emerging that we are trying to incorporate in our facility to assist with structural characterization.

The other important trend is that researchers now need a quick turnaround on the data for their samples. If you are doing a fee-for-service analysis and you don’t get back to them in a couple of days, they move on and go to another facility that offers them a faster turnaround. A lot of managers at core facilities do not realize that the turnaround time is key in getting people to come back to you. We have strict guidelines in place for getting results back quickly. For simple samples that involve confirmation of formula or mass, our turnaround time is twenty-four hours. For a quick protein identification, it’s typically three days. But if it’s a metabolomics sample, it can take a week or two, because there is a lot of data analysis involved.

Q: Have advances in instrumentation helped alleviate some of these challenges?

A: Automation has certainly helped in that we can leave the samples to run overnight. The other aspect that has helped in making sure that things are running smoothly is the remote access to instrumentation, from online at home. We can even do injections remotely, and the only thing we can’t do is put samples in. I can have the system set up to send me a report by e-mail at the end of each run. So if there is no e-mail, I know there is a problem. However, I would have to log in to the system to find out what the problem is. It would be nice if there were a way for it to communicate to me what went wrong. We have service contracts on all the key instruments that need to be operational all the time. We also have redundancies built in, so if one instrument breaks down we can go to another instrument to perform the same experiment.

Q: You also mentioned that you now have tools to assist you with structural characterization. What are they?

A: For metabolomic samples there is XCMS, a data analysis tool developed at the Scripps Center for Metabolomics, which allows you to do comparative analysis. For instance, if you do multiple LC-MS runs and want to find the differences between the thousands of peaks obtained, this program allows you to align the peaks and find the differences. Another advantage is that this program is linked to METLIN (Metabolite and Tandem MS Database), where you can get an idea based on the accurate mass of the metabolite what compound it might correspond to. It’s not a perfect fit, but it’s a start to give you a clue as to what the compound might be. You have to do more experiments and have chemical standards incorporated to definitely identify the structure of the compound and confirm it. There are other databases that are coming up online for analysis, and the MS software itself is getting easier. It can automate some of the intelligent analysis and give you an idea of what the chemical formula is by eliminating certain possibilities based on the fragmentation pattern.

Q: Does the MS software integrate well into the laboratory informatics management system or LIMS that you have set up?

A: We have a LIMS set up to integrate the MS data, but we have to upload the data manually. It helps us automatically track the samples and sends an e-mail to the users when their results are ready. People don’t like paper reports anymore. We have automatic e-mailing and a PDF of all the reports available online, so people don’t have to come in here to get their data.

Q: Do you provide any MS training at your facility?

A: We have two or three MS instruments that have open access, and students and postdoctoral fellows receive training on how to use them. Most manufacturers provide an interface for open-access instruments, and it masks all the complicated parts of the instruments. You just have to load a method, give it a sample name, and then hit start. The samples are then run under this user interface with everything being controlled automatically. We do discuss sample preparation techniques with our users for all our instruments. We individualize the sample prep depending on the nature of the samples and the experiment, and it’s handled on a case-by-case basis.

Q: Is there an MS instrument out in the market that you wish you had in your facility?

A: My background is in Fourier transform ion cyclotron resonance mass spectrometry (FTICR- MS), which is the highest-resolution mass spectrometer that is out there. These instruments have a new cell that can give you resolution in millions more easily than ever was possible before. Ultrahigh resolution can provide additional information about individual atoms, such as the number of nitrogen and sulfur atoms, and having high mass accuracy gives you more confidence in your formula assignment. You can also do top-down proteomics with these instruments. The other instrument that we hope to get next year is a high-resolution GC/Q-TOF for complex volatile organic samples. We now have a lowresolution GC instrument, and moving to the high-res instrument will give us better mass accuracy and the ability to take on different types of analysis.

Q: What would you say MS users are demanding now and will demand in the near future?

A: Users are demanding comparative analysis of MS data, like we are doing with XCMS. They are looking for high turnaround of sample analysis and better incorporation into LIMS so results can be obtained and accessed faster. Everybody wants things done cheaper, but these MS instruments and the service contracts are incredibly expensive. Sometimes the cost of operating the instrument is greater than what we get in fees. So finding ways to do things efficiently, building redundancies in instruments to cover downtime, and manufacturers offering more targeted service contracts for extreme failures will help going forward in terms of reducing the cost of analysis per sample. The next frontier will be super-high-resolution MS that people are not asking for now but is something they will need.

Sunia A. Trauger, PhD, is the director of the Small Molecule Mass Spectrometry facility at the FAS Center for Systems Biology at Harvard University. She has more than 23 years of experience in the mass spectrometry field. After completing her graduate research at Purdue University in the field of high-resolution mass spectrometry, she worked at Bruker Daltonics for seven years as a product manager and a senior applications scientist. More recently, she was the associate director of the Center for Mass Spectrometry at the Scripps Research Institute in La Jolla, CA. Her research interests include FT-ICR mass spectrometry, tandem mass spectrometry, proteomics, and metabolomics. She oversees the operation of the mass spectrometry facility, consults on experimental design, and collaborates on mass spectrometry-based investigations with researchers in the community. The Small Molecule Mass Spectrometry facility provides services for molecular formula confirmation (accurate mass measurement), structural elucidation (MS-MS), and quantitation of small molecules. In addition, it assists with the mass analysis of a wide variety of non-proteomics samples, including metabolites, medium-sized proteins, and oligonucleotides.