New tools for precision proteomics

All proteins aren't peas in a pod, but some sure look that way.

That's where the latest generation of mass spectrometers comes in. They can pick out differences between certain proteins that the previous generation of instruments cannot.

The arrival this year of two new mass spectrometers puts the Hutchinson Center "current with the state-of-the-art," said Phil Gafken, staff scientist and director of the Proteomics shared resource. "The new instruments are about 10-fold more sensitive than the old instruments."

That doesn't mean the Center's two older mass spectrometers are obsolete. It does mean that investigators can now — when the situation demands — discern differences between proteins that otherwise would be undetectable.

What a hammer and saw are to carpenters, mass spectrometers are to proteomics researchers, who seek to characterize proteins in complex mixtures, including protein patterns in blood, urine and tissue that may serve as biomarkers to detect and/or predict disease at an early stage.

The challenge is enormous. Each of the 20,000 or so genes in the human genome may produce five to 10 protein products and each of those is subject to potential modifications. Do the math and it means there may be "a million different proteins floating around," Gafken said.

Mass spectrometers sift proteins by mass and electrical charge, collect data about the proteins and then analyze the data by computer. The Center purchased the two new instruments primarily to help investigators better characterize modified versions of the same protein — a task easier said than done given how subtle the differences can be. Yet those differences can yield important biological information. "If there are two species (variations) possible for the same protein, you need the ability to determine which one of those species is present or if both species are present," Gafken said.

Histones are an example of proteins that exist in modified forms. Two key types of histone modifications look very similar via mass spectrometry, but are very different biologically. "Histones can affect gene regulation and a number of Center investigators are studying them, but it's important to be able to identify which modifications are present," Gafken said. "The older mass spectrometers could not resolve the differences between the two modifications, but the new ones can."

Analyzing intact proteins

One of the new mass spectrometers, the LTQ-FT, employs a new strategy for protein analysis. Traditional protein analysis uses a bottom-up approach that involves breaking proteins into smaller pieces called peptides, analyzing the peptides and reconstructing the peptides back into proteins. "That's perfectly fine for certain types of analysis," Gafken said. "But the problem is you have the potential to lose information about proteins."

Thanks to improved technology, the LTQ-FT can take a top-down approach, analyzing intact proteins to give investigators a more global — and thereby precise — look. Although the top-down scheme is not yet a fully developed approach, the Center's LTQ-FT will be prepared to support top-down studies in the near future.

While both are used to characterize proteins, the new mass spectrometers get their work done at different paces. The 4800 MALDI Tof/Tof runs narrow inquiries of small samples and can complete thousands every day, Gafken said. Meanwhile, the LTQ-FT runs larger inquiries of more complex samples, completing only 20 or so a day. "Each has its role," Gafken said.

So far, one of the most significant uses of the new instruments relates to Dr. Jim Olson's exploration of tumor painting. Olson, a pediatric cancer specialist and an investigator in the Clinical Research Division, is collaborating with colleagues at the University of Washington to tag tumor cells in mice with a dye that shines brightly when exposed to certain wavelengths of light.

The ultimate goal of Olson's research is to provide surgeons with a tool to help them precisely remove tumors from the brain. However, before the technique can be used on humans, more must be known about the peptide that transports the dye to the tumor and which dyes work best — answers being produced by the 4800 MALDI Tof/Tof.

"The 4800 is doing a fantastic job on Dr. Olson's project," Gafken said.

The LTQ-FT was funded in part by a grant from the M.J. Murdock Charitable Trust. Funding for the 4800, in its entirety, came from an anonymous private donor.

Gafken, Eng offer mass spectrometry-based proteomics training Aug. 14-18

Shared Resources presents "Mass Spectrometry-based Proteomics" with Phil Gafken and Jimmy Eng, Aug. 14-18 in B1-072/074 and J2-115. The course, which meets from 2-5 p.m. each day, is recommended for anyone who is currently utilizing proteomics or plans to use proteomics in the near future. The course is intended to educate novice users of the technology to the level where they have a solid understanding of it and they could apply proteomics to their research.

The first three days of the course will include details about mass spectrometers, protein identification and quantification by mass spectrometry, characterizing post-translational modifications, data acquisition, analysis of complex mixtures and preparing samples for mass spectrometry.

Day four will include database search algorithms and the analysis of proteomics data. The final day of the course will take place in the computer-training center and participants will receive hands-on demonstrations on how to use software tools available at the Center for analyzing proteomics data and results.

Space is limited. Anyone interested in the course should register with Karen Miller by e-mail at kamiller@fhcrc.org.