LPAT researchers Ming Zhou (left) and Athar Masood set up equipment in the new mass spectrometry laboratory at the ATRF.
By Andrew Stephen, Timothy Veenstra, and Gordon Whiteley, Guest Writers, and Ken Michaels, Staff Writer
The Laboratory of Proteomics and Analytical Technologies (LPAT), Antibody Characterization Laboratory (ACL), and Protein Chemistry Laboratory (PCL), previously located on different floors or in different buildings, are now together on the first floor of C wing in the ATRF.
Considering that the primary focus of all three laboratories is on protein characterization, their incorporation into a single, large space makes perfect sense. This integration affords scientists in these laboratories the opportunity to interact and share knowledge more readily than when they were in separate locations. As most scientists will attest, the ability to readily exchange ideas leads to more efficient methods for solving existing problems while seeding fertile ground for the development of novel studies. In the area of technology innovation, several LPAT and PCL staff members are currently working together on projects, and this collective effort is expected to increase as lab members become more familiar with each other’s areas of expertise.
NIH’s Highest Concentration of MS Expertise
The primary analytical technology within this collective laboratory is mass spectrometry (MS). The mass spectrometers are in a single, large room that represents the highest concentration of MS expertise and equipment within the NIH intramural community.
The room currently houses 15 mass spectrometers and has enough space for additional instruments in the future. Housing all of the mass spectrometers in a single room simplifies the building engineering requirements; more importantly, however, is that having them all in the same room encourages people with expertise in MS to interact and share their knowledge.
There are several types of mass spectrometers. The new facility’s collection includes ion-trap, Orbitrap, Fourier transform ion cyclotron resonance (FTICR), triple quadrupole, and time-of-flight (TOF) mass spectrometers.
While all of these instruments can determine molecular mass, each type of mass spectrometer has a unique capability. Some (i.e., ion-traps, Orbitraps, FTICR) are designed for high-throughput protein sequencing. These instruments are used for global analysis to determine differences in protein abundances between different biological samples. They are also optimized for identifying post-translational modifications across entire proteomes or within a single purified protein.
The triple quadrupole mass spectrometers are designed for molecular quantitation and are used primarily for quantifying the absolute abundance of metabolites (and proteins) in human serum, urine, or tissue.
The TOF mass spectrometers are used for intact protein molecular weight determination or characterization of proteins isolated from complex mixtures. In addition, one of the TOF instruments in the lab is used for MS imaging, which involves the interrogation of an entire tissue section to determine the location of various ions within the sample.
Recent Findings Using MS
Xia Xu, Ph.D. (LPAT), recently measured the absolute quantity of 15 estrogen metabolites in the NCI-60 cell panel using triple quadrupole mass spectrometry.1 Quite surprisingly, he found that many of the cancer cell lines not commonly associated with steroid hormones (e.g., melanoma and leukemia) contained significant amounts of endogenous estrogens and estrogen metabolites. These results suggest that human tumors outside of those associated with the female reproductive system may be beneficially treated using therapy aimed at estrogen biosynthesis and estrogen-related signaling pathways.
In an example using an ion-trap mass spectrometer, Ming Zhou, Ph.D. (LPAT), collaborated with Yossef Raviv, Ph.D. (CCR), utilizing a photoaffinity-labeling approach to identify new proteins that facilitate HIV-1 transfer.2 They identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1 transfer between antigen presenting cells (APC) and CD4+ target cells. This result was validated as antibodies directed against ATP synthase inhibited APC-mediated transfer of multiple strains HIV-1 to CD4+ target cells. This identification of ATP synthase’s role in HIV-1 transfer provides new targets for inhibiting HIV-1 proliferation in vivo.
ACL Works Closely with Protein Groups
The mission of the ACL is to develop and/or characterize antibodies or other affinity binding reagents and provide a source of both the reagents and information that would be available to the public. The first antibodies were raised against recombinant full-length proteins produced in a collaboration with Argonne National Laboratory. Two outside companies were contracted to produce the antibodies, and the ACL selected them from submitted candidate clones. The aim was to have three antibodies for each target.
Since its establishment in 2005, the ACL has raised antibodies against proteins produced by the Protein Expression Lab (PEL) for intramural investigators, proteins produced by the Structural Genomics Consortium in Toronto, the University of Copenhagen, and both proteins and peptides requested by extramural investigators. All of the antibodies and cell lines are available at cost from the University of Iowa Developmental Studies Hybridoma Bank.
With the move to the ATRF, the ACL may now interact more easily with both the PCL and PEL groups, and continue collaborations to broaden its capabilities.
Raising Antibodies
The ACL is expanding in terms of the ability to raise both antibodies to peptides and peptides with modifications, such as phosphorylation, that are known to be important in cancer.
A collaboration with PEL has demonstrated the ability to raise an antibody to insoluble proteins through immunization with inclusion bodies. This has provided three antibodies to the PSPHL protein, representing the only antibodies that have ever been raised against this protein. The ACL has also begun raising antibodies to other proteins from PEL that were raised for the Nanotechnology Characterization Lab (NCL) and one of their collaborations. The capabilities of the ACL allow for the selection of antibodies that can be used for a highly specific end purpose defined by the NCL collaboration.
According to Gordon Whiteley, Ph.D., director of ACL, “To date, we have approximately 230 antibodies available that have been screened from over 2,500 candidate antibodies. These antibodies have been used extensively, according to our provider in Iowa. Furthermore, many are being incorporated in diagnostic tests by various commercial vendors.”
Protein Chemistry
PCL uses MALDI-MS in combination with traditional protein chemistry methods to identify proteins and their modifications. In a study recently published with Ira Pastan, M.D.,3 Oleg Chertov, Ph.D., identified a specific modification on a protein that confers resistance in pediatric patients with acute lymphoblastic leukemia from treatment with immunotoxins. PCL also has a variety of technologies that support investigators in characterizing how proteins interact with their binding partners including new drug candidates.
Andrew Stephen, Ph.D., acting director of the PCL commented, “I think the most exciting thing about moving to the new building is having all this different expertise [in the Advanced Technology Program] now in one location. Being co-located, I expect we’ll have more chance to discuss science and share our expertise, leading us to a more integrated approach to solving problems.” In addition, he said, the co-location will enable more creative technology development because staff from different labs will be involved in the same project.
Attracting Partners
Having a program with experts in a multitude of technologies with collaborations with NCI principal investigators and new partnership space all in one place is likely to be an inviting prospect to companies interested in developing new technologies and partnering with NCI scientists.
Having beta space adjacent to these laboratories will be invaluable in partnering with companies who wish to develop technologies or beta test early versions of new technologies.
Timothy Veenstra, Ph.D., director of LPAT, noted that, as the laboratory interactions strengthen, “we anticipate expanding in new areas to provide NCI scientists access to cutting-edge technologies. There are already plans to develop a metabolomics discovery capability to find cancer-related metabolites in studies utilizing either human samples or mouse models. To be successful in this new area is going to require the collective expertise available within the ATRF and the sharing of knowledge between individual scientists. Fortunately, the ATRF has provided the necessary geometry and now it is simply up to individuals to take advantage of the personal knowledge within their surroundings.”
References
1. Xu X and Veenstra TD. (2012). Genome Med. 4:31-35.
2. Yavlovich A et al. (2012). Ectopic ATP synthase facilitates transfer of HIV-1 from antigen presenting cells to CD4+ target cells. Blood. In press,
3. Wei H et al. (2012). Proc Natl Acad Sci U S A. 109(18):6898-903.
Andrew Stephen,Ph.D., is acting director of the Protein Chemistry Laboratory; Timothy Veenstra, Ph.D., is director of the Laboratory of Proteomics and Analytical Technologies; and Gordon Whitely, Ph.D., is director of the Antibody Characterization Laboratory.