Ask the Expert: How to Overcome Challenges with Mass Spectrometry

David Hachey, Ph.D., talks to Tanuja Koppal, contributing editor to Lab Manager Magazine, about his role and responsibilities as the director of both the mass spectrometry (MS) and proteomics core laboratories at Vanderbilt University. He elaborates on how the two facilities are run and the types of services they provide. He also offers valuable insights into the common challenges users are faced with when using mass spectrometers and in running an open-access core facility.

Click here to see his webcast on Overcoming the Challenges with Mass Spectrometry!

Q: Are there many differences between the mass spectrometry and proteomics core labs that you oversee?

A: There is a significant difference between the two, although they both use only mass spectrometers. The small molecule MS core is run as an open-access instrument facility, while the proteomics core is not. Proteomics involves a relatively sophisticated and technically challenging series of measurements, and casual users like graduate students don’t necessarily gain the skills to use the instruments efficiently. Hence, it is operated as a closed-access facility where users prepare samples in their own lab and we process the samples, perform the analysis and then share the results with the investigator. The two labs also differ significantly in terms of their MS capabilities. In the small molecule core we depend mainly on triple quadrupoles and ion trap MS instruments and have limited access to high resolution MS. We also use only conventional HPLC and do not use nano-flow HPLC. However, in the proteomics core we exclusively use nanoflow HPLC and the primary workhorse there is Thermo Scientific’s LTQ ion trap and Orbitrap instruments. We also have a MALDI-TOF-TOF instrument. Nano-flow HPLC is quite tricky and again, casual users lack the expertise to use the demanding microfluidics that is involved and to detect problems. They are welcome to do limited proteomics in the small molecule core but femto or attomole level detection of proteomic samples must be done in the proteomics core.

Q: Are there different sets of challenges due to the different instruments and applications being pursued in the two labs?

A: Absolutely. In the small molecule core we work mainly with graduate students and post-doctoral fellows who often do not have a lot of technical experience working with mass spectrometers and hence, they don’t work very efficiently. We do have a training program to bring them up to speed in terms of what is required to run the instrument and to optimize for a particular assay and we later also help them interpret their results. The downside is that we face more wear and tear on the instruments and they need to be cleaned more frequently, since students often tend to be less critical of sample preparation. Students also don’t immediately recognize when there is a problem and hence they are less efficient. But I accept that as a part of the training mission at the University.

Q: What does your in-house training cover?

A: Training courses are given approximately once a month, as well as on an ad hoc basis, as new students come in. The first half of each course consists of two to three hours of lectures covering both theory and operation. The second half is a hands-on tutorial covering instrument setup, operation and shutdown. The facility personnel then work with the user on a one-on-one basis to cover their specific experiment and application. We also review the data with them during the early stages so the user can learn to interpret it correctly. We have two or three people available every day to help out if any problems arise. We are open from eight AM to six PM and during late evenings and weekends we provide access to users, but only those who are technically proficient. Only in certain cases, when we don’t have the time or the technical expertise, we recommend that the user go to a vendor to get trained on a specific class of instruments.

Q: Any advice in terms of selection of instruments or vendors?

A: We have standardized based on a single vendor in order to have a single software platform. The students struggle a bit if the software systems are not user-friendly and tend to get confused if they have to move across instruments using different operating systems. We have done the same for the proteomics core in order to build a continuous workflow from sample prep to the bioinformatics analysis and found it much simpler to have a single data file structure to work with. If you have the luxury to work with a single vendor then life is definitely easier for you. We also have no service contracts on any of the instruments. We have our own in-house engineer, so we do our own servicing and routine maintenance. We have had good success with Thermo Scientific and I am sure the other vendors are equally qualified. They walk you through the diagnosis of a problem without charge, which is quite a cost savings.

Source: Data collected from a survey of mass spectrometry service facilities conducted in 2000.

Q: What are some of the common problems you encounter?

A: The big one is, “I can’t see my molecule!” Well, how do you know it is present in the sample? Have you run a calibration curve with standards and blanks? There are a number of standard protocols you use in diagnosing a problem. Sometimes it’s an instrument problem. For instance, the heated capillary in the ion source becomes clogged and has to be cleaned or the HPLC columns go bad. Sometimes the user cannot recognize the problem and we have to go through the experiment step-by-step to determine what is going wrong.

Q: How has the field evolved in the last decade?

A: When I joined Vanderbilt in 1998 the main emphasis was on gas chromatography/mass spectrometry (GC-MS) measurements of small molecules and that was 65 to 70 percent of the workload. The other 25 percent was liquid chromatography-MS (LCMS) and about 10 percent was matrix-assisted laser desorption/ionization (MALDI). Today, probably 85 percent of our work depends on LC-MS. The main advantage to using LC-MS is that it has solved a lot of problems with sample prep and is less demanding than GC-MS. We now have a total of 10 mass spectrometers in the small molecule core lab—four triple quadrupoles, three ion trap instruments, one MALDI, one GC-MS and one high resolution Q-TOF (time-of-flight). So if I were to set up a lab today I would probably get a triple quadrupole or an ion trap, a high resolution instrument like a Q-TOF or Orbitrap and finally a GC-MS. But then again, this depends very much on the user base. Our work is mostly quantitative and in the biomedical area, which is reflected in our choice of instruments.

Q: How have your own responsibilities evolved over the years?

A: Initially the biggest challenge was changing the culture at Vanderbilt. We wanted to make the core an open-access facility and we realized that in order to do so we needed a training program in place. Over the past 10 to 12 years we have trained nearly 600 to 700 people and the expertise has now been disseminated into individual laboratories. These investigators are now taking on more responsibility for training incoming students in their lab in the various techniques. So we have been fairly successful on that front. The challenge we now face is to keep our extensive operations funded with a mix of funding from individual investigators, center grants, and from the University.

David L. Hachey, Ph.D., is the professor of Chemistry, Biochemistry and Pharmacology at Vanderbilt University in Nashville, Tennessee. He has been the director of the mass spectrometry core laboratory at Vanderbilt since 1998 and in 2007 he assumed the responsibility as director of proteomics core as well. The service labs provide instrumentation and analytical expertise to Vanderbilt’s research community. They also offer an active MS training program to teach new users how to design experiments, do the sample preparation, operate the instrument and interpret the results. The MS core lab is used by about 175 investigators each year from ~75 research groups at the University. As the director, Dr. Hachey is responsible for establishing policies and procedures for the equitable use and maintenance of the facility, preparing grants for obtaining funds to support the facility and in assisting the development of new analytical methods.

If you missed the Ask The Expert webinar “How to Overcome the Challenges with Mass Spectrometry”, originally broadcast on Wednesday June 9, 2010, Click here to watch the archived video.