Hutch News

Ions on a foot race

May 17, 2001
Dr. Phil Gafken

Dr. Phil Gafken checks the paths of the ion-trap mass spectrometer, one of two instruments in the Center's Biotech Center used for protein identification.

photo by Clay Eals

Hutch scientists wanting to take advantage of sensitive protein-analysis techniques need look no further than the Weintraub building first floor.

With two mass spectrometers operated by staff scientist Dr. Phil Gafken, researchers easily can characterize the proteins they study in their labs.

"The typical use for mass-spec is protein identification," he said. "Often, a researcher will isolate a protein whose identity is unknown. With mass spec, you need only a small amount of protein to determine its identity."

Gafken, who joined the Hutch's Biotechnology Center last fall to oversee mass-spectrometry operations, runs two types of instruments and helps scientists design and interpret experiments.

Although each instrument has different advantages depending on the type of research problem, both operate on the same principle: determining mass and charge properties - also known as the mass-to-charge ratio - of molecules converted to their gaseous state.

Like DNA, proteins are large molecules made of smaller building blocks. But unlike DNA, which has only four building blocks, proteins contain assortments of 20 different kinds of amino acids. The order of each amino acid helps determine how the protein will fold into its characteristic three-dimensional shape, which enables it to carry out its biological function, which could be anything from breaking down glucose to providing the structural support for chromosomes.

Electrically charged molecules

Mass spectrometry relies on making molecules ionized, or electrically charged, which allows sensitive detectors to determine their mass-to-charge ratio. After the charge of the molecules is determined, its mass can be identified based on its chemical composition.

The two Hutch instruments, both of which are suited for protein identification, are known as MALDI-TOF and electrospray ionization ion-trap mass spectrometers.

MALDI-TOF - which stands for matrix-assisted laser-desorption ionization time-of-flight - is like a track-and-field event for molecules, Gafken said.

Samples are mixed with a solvent containing an organic compound, or matrix, and spotted onto a metal plate, called a target, and a laser is aimed at each spot on the target.The samples transform into a gas, and the constituent molecules race through a tube and hit a detector, which measures their "time of flight."

"Think of it as a foot race," he said. "The firing of the laser is the starter's pistol. The ions travel from a starting point, the spot on the target, through a flight tube and to a finish line, the detector."

MALDI-TOF is capable of analyzing small or large proteins, Gafken said.

"In theory, anything that ionizes you should be able to analyze with MALDI- TOF. However, the practical mass range for this particular instrument is 1000 daltons to 100,000 daltons," he said, referring to the unit used to measure protein mass.

Electrospray ionization ion-trap mass spectrometry is best for small to medium-sized proteins, Gafken said.

The ion-trap mass spectrometer in the Biotechnology Center is directly coupled with an instrument called a high-performance liquid-chromatography system (HPLC). Protein mixtures are injected onto the HPLC, which separates them based on their physical properties. The separated components immediately enter the mass spectrometer as a fine spray, which causes the liquid HPLC droplets to rapidly evaporate and leave charged particles. These particles enter the ion-trap, where they are dispersed and ejected onto a detector based on their mass-to-charge ratios.

"Most researchers are familiar with having an absorbance detector attached to their HPLC," Gafken said. "In my case, I have a ion-trap mass spectrometer attached to my HPLC that is doing all the detection."

Besides protein sequencing, mass spectrometry can detect subtle chemical modifications on proteins, which also affects their mass.

Dr. Christine Smith, Gottschling lab postdoc, uses mass spectrometry to detect changes on histones, a family of proteins that interact with DNA.

"Mass-spec has been great for figuring out which modifications are present on the histones and for determining their exact location," she said.

Experimental design, interpretation

Besides running the instruments, Gafken works with researchers on experimental design and interpretation.

"About a quarter of my time is spent consulting with people about their samples and how they might best use the technology," he said. "My most important function is to talk with them before the experiment is done to make sure they get the kind of data they're after. After the experiment is done, I explain the results."

As a Center shared resource, the mass Gafken's facility operates on a pay-per-use basis.

Janell Baldwin, director of Shared Resources, said that as mass spectrometry has become a more widely used tool in support of Center research, the facility needs expansion.

"We've developed a preliminary proposal with the help of faculty advisers and have submitted a budget request to provide support for further development of this resource," she said. "It's definitely one of the Center's long-term goals."

Expansion of the facility will include lab space, equipment and added staff.

Gafken encourages Hutch scientists interested in using mass spectrometry for their research to contact him at 206-667-2872 or e-mail him at pgafken@fhcrc.org. The facility's web site is http://sharedresources.fhcrc.org/.

 

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