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R E S E A R C H Fluorescence and MALDI on a single chip
P R O F I L E
Although peptides are easy to analyze with MALDI-TOFMS, large protein targets are a bit more difficult. When Thomas Laurell and colleagues at Lund proteins were the intended antigens, University (Sweden) have developed a the researchers performed an on-chip pore chip protein array (PCPA) that can digestion step prior to the MS analysis. be used to efficiently screen complex However, this approach had an unanprotein mixtures and obtain the identiticipated side effect: The antibody was ties of interacting proteins from a single also digested. Fragments of the antibody porous silicon surface. With a few more were present in digested samples; these tweaks, the PCPA, which is described in were visible as interfering peaks in the this issue of JPR (pp 988–994), could be mass spectrum. But Laurell doesn’t used in the clinic to assay human serum think this is a huge problem. “If you for antigens that have been implicated know which antibody is in the spot, you in various disease states. also should know which peptides to In PCPA experiments, an antibody expect,” he says. array is spotted onto a porous silicon Laurell says that the version of the surface. Human serum that has been PCPA detailed in this issue of JPR is a pre-labeled in bulk with a fluorescent first step and that later versions may be tag flows over the antibody array. Only more sensitive and even have surfaces those spots that emit a fluorescent sigdedicated to specific nal, which indicates protein classes. binding between the There are a few antibody and an antiother techniques gen, are then chosen that are more sensifor MALDI-TOFMS. tive, he says, but this Laurell and his assay is unique in colleagues have done that both the fluoextensive work with Large-molecule probing rescence screen and porous silicon surthe MALDI analysis faces. Just last year, are done on the the researchers pubsame chip, which lished a paper in AnaSmall-molecule reduces labor and lytical Chemistry probing MS analysis time. (2003, 75, 6968–6974) In the future, describing a fluoresAntibody on surface Incubation with Selective binding of Washed surface with Laurell thinks the cence-based antifluorescently labeled fluorescently labeled labeled antigen ready for PCPA will be a verbody–antigen binding sample target molecule fluorescence or MS readout satile and efficient assay using such a way of analyzing husurface. But a fluoresDouble your pleasure. A schematic of the PCPA experiments, which feature dual fluoresman serum samples. cence assay doesn’t cence and MALDI readouts. Depending on the tell you everything specific application you would like to at hand, the array can be designed with Some experimental steps for the know, cautions Laurell. Researchers a proteome of antibodies or only a few assay vary, depending on whether a pepshould make sure that the bound molespecific antibodies. This flexibility will tide or a protein is the targeted analyte. cule is really the antigen they are trying allow assay multiplexing for clinical use, For example, when a peptide was tarto target, he says. “You could make the says Laurell. Once antibodies are progeted, the researchers had to place a assumption that it is [the correct antiduced that recognize disease biomarkspacer between the surface of the chip gen], but you can never be sure because ers, he says, researchers can spot a panand the peptide’s antibody. Laurell postunonspecific binding could occur and el of those antibodies onto the PCPA lates that because antibodies that recogthere are homologues and other moleand quickly screen through many panize peptides are small, there may not be cules that may have the same epitope tient serum samples. enough space for the antibody to bind to that matches the binding site,” explains —Katie Cottingham both the surface and the peptide. Laurell. “This is something that is
© 2004 American Chemical Society
always of concern for the users and the virologists who are reading these chips.” Because the researchers also worked on chip-based MALDI techniques, Laurell says it was a “natural step” to attempt to combine the screening power of the fluorescence assay with the precision of MALDI-TOFMS for the identification of bound antigens. Although combining the two techniques sounds simple, Laurell admits that this was his team’s biggest challenge. “We ran our hands into the wall quite a few times trying to get [both optimized],” he says. “Either we had a good MALDI signal or we had good fluorescence, but not both at the same time.” After some tinkering, the group finally achieved a compromise in which both fluorescence and MALDI work well in the assay.
Journal of Proteome Research • Vol. 3, No. 5, 2004
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