By Dean A. Haycock

Special To BioWorld Today

It may have made the Human Genome Project possible and continues to be the basis of modern molecular genetics, but deciphering DNA sequences can be annoying.

Gel electrophoresis is tedious and slow. It requires enzymatic degradation of DNA and then loading the samples into wells pressed into slabs of gelatin. Driven by an electric current running through the gel, the DNA fragments carrying the genetic code slowly migrate to their places and await fixing and staining before identification.

Isn't there some technology that can accept an injected sample of DNA and return a sequence while the scientist waits? No. But recent progress in analytic chemistry raises hope that more efficient, researcher-friendly tools for determining the structure of large biomolecules such as DNA may be possible.

The most recent hint that macromolecules may someday be analyzed more efficiently and rapidly appears in the February 12 , 1998, Nature article "Impact energy measurement in time-of-flight mass spectrometry with cryogenic microcalorimeters."

Results obtained from an experimental mass spectrometer described in this paper suggest that similar technology may someday make it possible to study macromolecules with molecular masses well above the cut-off of 20,000 — 100,000 daltons imposed by the limitations of today's equipment.

In conventional mass spectrometry, compounds are identified by sorting gaseous ions derived from the compounds in electric and magnetic fields. The term mass spectrometry actually represents a group of techniques that use these principles to distinguish among atoms, ions and molecules on the basis of mass.

Operating under high vacuum, mass spectrometers have four main parts: a system through which the sample is introduced; an energy source which fragments the sample; an analyzer which separates the fragments according to mass; and a detector. Different types of mass spectrometers can precisely measure the mass of ions, detect the presence of isotopes and measure the relative amounts of ions in a mixture.

Biochemists have found MALDI-TOF (or matrix-assisted laser-desorption-ionization time-of-flight) spectrometry useful for studying proteins and other biomolecules. These systems can reveal the structure of a biomolecule by analyzing the spectrum of ions produced when the molecule is fragmented. The fragments are identified by mass and relative abundance. In effect, the molecule is "taken apart" in reality and then conceptually "put back together again," thus revealing its structure.

Biologists using mass spectrometry, however, have a problem. It works well for relatively small molecules but biologists inevitably want to study large molecules such as proteins, carbohydrates and nucleic acids. Due to intrinsic inefficiencies and noise in the equipment, it is easier to distinguish differences in molecules of low mass than in molecules of high mass.

For example, adding one to 100 produces a one percent increase. Adding one to 100,000 creates a 0.001 percent increase, which is easy to miss in noise. This and other limitations cause difficulties at large mass.

Gene C. Hilton, a physicist at the National Institute of Standards and Technology, in Boulder, Colo., and his colleagues are developing a new type of mass spectrometry that appears to be suitable for use with large molecules. The device is an improvement of previously described experimental mass spectrometries that use cryogenic detectors to measure minuscule pulses of heat deposited by a sample after it is hit by a particle.

A conventional mass spectrometry detector kicks out an electron or an ion when a particle strikes. This signal is then amplified. With larger and larger molecules, the sensitivity of this system decreases. Because cryogenic detectors measure the heat released from a sample, the sensitivity of cryogenic detectors, for the most part, is independent of the mass of the compound.

Matthias Frank and his colleagues at the Lawrence Livermore National Laboratory, in Berkeley, Calif., have shown that such detectors can gather information from a single macromolecule as large as 750,000 daltons. The cryogenic detectors used in these experiments, however, do not record the total impact energy produced during analysis.

Hilton and his co-authors described a significant improvement in this type of mass spectrometry.

"What is unique about our detectors is that they measure all of the energy that is deposited when the particles strike the detector. The other cryogenic detectors actually throw away a good portion of it," Hilton told BioWorld Today.

Whereas conventional detectors can report that a particle has hit, cryogenic detectors can report both the hit and how much energy it contained. The authors were able to provide the first calibrated measurement of the energy detected in their system. They and other authors showed that cryogenic detectors will not become less efficient when studying larger and larger molecules.

Practical Application Far From Reality

Using a new type of cryogenic microcalorimeter fabricated on a thin silicon membrane, the authors were able to detect all of the energy deposited by a particle impact. This allowed them to obtain an energy resolution an order of magnitude better than any previously described.

"Mass spec — however you make it work [with] our detectors or otherwise — could be used to obtain a 100 to 1,000 times speed-up over gel electrophoresis. We are a long way from that," Hilton said.

For one thing, it will be necessary to make the detector bigger than it now is. "Our detector was 200 by 200 micrometers in area, whereas the detectors people normally use for mass spec are an inch [square]. It is going to be harder to make them bigger — not impossible — but it is not easy either," Hilton said.

Also the cryogenic technology requires development.

"In order to make a system that any biologist could have in his lab, we need to go a long way," Hilton said.

While Hilton stressed the work that needs to be done before this experimental cryogenic detector is available commercially, he noted that a company based in Boudry, Switzerland, GenSpec SA, already has been established to develop mass spectrometers with cryogenic detectors.

Frank believes that cryogenic detectors could eventually lead not only to improved detection of large molecules but also to improved understanding of what happens at the "front end" of the process.

"I believe this will help develop better mass spectrometry," Frank said. *