ChIP-ing Away at a Proteomics Bottleneck

Courtesy of Andrew Gooley, Proteome Systems Among proteomics techniques, protein microarrays may be getting a lot of buzz these days, but two-dimensional gels still do most of the work. That's because protein arrays present a number of technical challenges that have limited their implementation.1 Proteins vary wildly in stability, solubility, viscosity, and ease of synthesis, for instance. And, the in vitro translated proteins generally used to construct protein biochips often lack the wide d

Sep 8, 2003
Jeffrey Perkel
Courtesy of Andrew Gooley, Proteome Systems

Among proteomics techniques, protein microarrays may be getting a lot of buzz these days, but two-dimensional gels still do most of the work. That's because protein arrays present a number of technical challenges that have limited their implementation.1 Proteins vary wildly in stability, solubility, viscosity, and ease of synthesis, for instance. And, the in vitro translated proteins generally used to construct protein biochips often lack the wide diversity of co- and post-translational modifications that can affect protein activity in vivo.

But protein arrays aren't dead in the water. Recently, Andrew Gooley, chief scientific officer at Proteome Systems, New South Wales, Australia, and colleagues asserted that the product of two-dimensional gel electrophoresis is basically a protein array.2 The difference between such an array and traditional microarrays, the authors explained in their manuscript, is that each protein's position is defined not by the user, but by its isoelectric point and molecular weight.

To prove their point, the researchers blotted 2-D gels to PVDF membranes, adhered the membranes to MALDI target plates, and probed them using mass spectrometry. To process the proteins for MS analysis, they skipped the traditional excision, digestion, extraction, cleanup, and target-spotting steps3 and instead employed a prototype chemical inkjet printer (ChIP) system jointly developed by Proteome Systems, Shimadzu Biotech of Kyoto, Japan, and Microfab Technologies of Plano, Texas. That device, set for early-access availability in September, could mark the wave of the future for proteomics, says 2-D gel aficionado Vito DelVecchio of the University of Scranton, Pa. "It will make things a lot easier," he says. "It's making things a lot more high-throughput."

And according to Don Thompson, North American sales manager for Shimadzu Biotech, applications are not limited to proteomics. "It's a kind of enabling technology," he says. The company has its own ideas about potential uses (which Thompson declined to enumerate), but, "We want to get it in front of people who are out-of-the-box thinkers, who can come up with some killer applications."

A RAINBOW OF CHEMISTRIES Thompson describes the ChIP system as a combination imaging system and nanochemistry dispenser. An embedded scanner images the stained blot attached to the MALDI target for target picking, after which the target moves on a stage to specified positions under the piezoelectric printing heads, which then dispense picoliter to nanoliter quantities of the desired reagents. The coordinates of each spot are fed to the mass spectrometer for subsequent peptide mass fingerprinting or peptide sequence analysis.

Researchers can use the ChIP system to squeeze more information from precious samples. The spotting zones range from 200 to 300 µm in diameter, and Thompson says the system can easily lay down a 4 x 4 reagent grid on a 2-mm protein spot. "You can print a whole rainbow of chemistries on the one sample," explains Gooley. Printed spots can contain trypsin or other proteases, an endoglycosidase to monitor glycosylation, antibodies for protein identification, and so on. A side-mounted camera helps researchers to optimize printing conditions for each head, and since the printing heads are noncontact, scientists need not worry about source contamination.

The system has a number of other advantages. The lack of liquid-handling steps means the process should be more or less 100% efficient, says DelVecchio, who has never used the system. And in contrast to gels, which last only a few days, the membranes are stable enough for long-term storage. Researchers can therefore reprobe blots archived months earlier as new antibodies become available, explains Gooley, iteratively characterizing important spots. Finally, the technique's sensitivity--it can identify femtomole quantities of protein--is on par with in-gel methods, though the sequence coverage per reaction is currently inferior. But Thompson notes researchers can boost coverage by using multiple proteinases.

Shimadzu Biotech and Proteome Systems are positioning the ChIP system for proteomics applications, but the system's flexibility supports other uses too, says Gooley. One possibility is tissue microchemistry, in which the system directly deposits antibodies onto a tissue sample for immunofluorescence or immunohistochemical analysis.

In any event, don't expect traditional proteomics robots to become obsolete, says Gooley. Instead, he says, ChIP offers protein biochemists greater flexibility when dealing with precious samples or reagents. Nor will it make the field more accessible to neophytes, as the learning curve remains too steep, predicts Scranton's DelVecchio, who still does 2-D gel-based proteomics using liquid-handling robots. Nevertheless, he says, "I think I would buy one."

--Jeffrey M. Perkel

References
1. L. Lane, "Protein microarrays at the cusp," The Scientist, 17[14]:42-4, July 14, 2003.

2. A.J. Sloane et al., "High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies," Mol Cell Proteomics, 1:490-9, 2002.

3. J.M. Perkel, "Technologies vie for dominance," The Scientist, 17[5]:20-2, March 10, 2003.

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