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Research ProfileS
By now, many proteomics researchers are familiar with the criticisms of SELDI technology: it has a reputation for generating results that are not highly reproducible, and some scientists say that pattern-based clinical diagnoses are not reliable (Anal. Chem. 2003, 75, 472A–476A). About a year ago, Günther Bonn and colleagues at Leopold-Franzens University (Austria) developed material-enhanced laser desorption/ionization (MELDI) as an alternative. In this issue of JPR (pp 44–53), the researchers demonstrate a new workflow that allows scientists to identify candidate biomarkers that are selected in MELDI screens. They also test fullerenes as new carrier materials for MELDI. The entire process is highly reproducible and rapid. According to Bonn, MELDI is like LC without the column. “We have particles that are similar to the ones used in conventional chromatography, and we bring these together directly with biofluids, such as serum, plasma, or urine samples,” he explains. After an incubation and a few washing steps, the proteins and peptides that are bound to the particles can be analyzed directly by MS. Profiles similar to those obtained with SELDI are generated. The entire process is automated for highthroughput screening. But a profile is only the starting point for biomarker discovery, says Bonn. The researchers wanted to know which proteins were responsible for the differences in the various MELDI profiles that they observed with human serum samples. The problem was that the sample complexity was still too high for protein identification by MS/MS after a typical MELDI MS analysis. But when the researchers included a µLC step after the elution, the MS signals were more intense, and MALDI MS/MS analyses could be conducted. Proteins and peptides with m/z < 4000 can be identified with this protocol. Larger proteins can be digested, then identified by ESI-MS/MS or MALDI MS/MS.
The µLC step improves the analysis by “covering a larger mass range,” says Bonn. His group used a novel monolithic stationary phase, poly(p-methylstyrene-co-1,2-bis-p-(vinylphenyl)ethane (MSt/BVPE), that they developed. Compared with other commercially available monolithic materials, MSt/BVPE speeds the separation process. Because small molecules can be separated with this material, it also is ideal for metabolomics studies.
GÜNTHER BONN
A new top-down proteomics workflow
Carbon soccer balls? Derivatives of [60]fullerene were used in a new MELDI workflow that allows researchers to identify candidate biomarkers.
On the MELDI side of the workflow, the researchers have been testing different carrier materials, such as cellulose, silica, and various polymers. For example, in another paper in this issue of JPR (pp 382–386), they report the use of derivatized poly(glycidyl methacrylate/divinyl benzene) particles. In the current work, however, they evaluated derivatives of [60]fullerenes, which are spheres composed of 60 carbon atoms. Why fullerenes? “If you have fullerenes, then the absorption of MALDI laser energy is quite high, [and this process] increases the sensitivity,” says Bonn. In addition, the 3D fullerene particles have a higher surface area than the 2D layers used for SELDI; this
20 Journal of Proteome Research • Vol. 6, No. 1, 2007
property increases the sensitivity and selectivity of the method, he adds. Another advantage of using fullerenes is that researchers can easily regulate the binding properties of these molecules. For example, [60]fullerenoacetic acid is hydrophilic, dioctadecyl methano[60]fullerene is hydrophobic, and Cu(II)-IDA-[60]fullerene is specific for histidine residues. Different protein and peptide patterns are observed when various fullerene derivatives are used; the patterns depend on the binding properties of the derivatives. The MELDI profiles obtained with the derivatized fullerenes were highly reproducible, but the researchers did not stop at this step. Instead, they continued their study and identified interesting proteins and peptides in serum with the new workflow. As a proof of principle, they identified peptides from samples that were screened with the fullerene derivatives. Kininogen-1 precursor, clusterin precursor (also known as apolipoprotein J), and complement C3 precursor were present in the serum samples. Clusterin precursor is a g lycoprotein found in the inner-ear and cerebrospinal fluids, and it is involved in neurodegenerative disorders. Bonn says he was surprised that everything seemed to come together with the use of fullerenes and a new workflow. “The hardest part was to figure out the elution process so that everything was reproducible,” he says. The elution conditions depend on the particles that are used, so researchers must reoptimize every time they change to a different type of particle. In the end, however, “It was very interesting to observe high recovery and high reproducibility,” says Bonn. The researchers are continuing to improve the MELDI method and are trying to better understand how similar carrier materials can sometimes provide data in different mass ranges. They also are attempting to combine particles with different properties in the same MELDI analysis to select a broader group of candidate biomarkers. —Katie Cottingham
© 2007 American Chemical Society
