news
Protein arrays and MS: a marriage of convenience Fluorescence-based protein arand hydrophobic SAMs break (a) ray technology often assumes down over time in complex that protein–protein interactions solutions because nonspecific are strictly monogamous. But analyte adsorption occurs on what if the immobilized protein the hydrophobic background. is promiscuous in its choice of However, the combination of interaction partners? Ideally, hydrophobic SAMs and the researchers could identify unroughened porous gold created (b) anticipated interactions from a superhydrophobic surface. To a complex mixture of analytes prevent nonspecific adsorption, and determine whether there is a pocket of trapped air sepamore than one bound species rated liquid droplets from the per protein spot. With these underlying superhydrophobic issues in mind, collaborators surface. The superhydrophoRobert J. Cotter and Heng Zhu bic background confined the at the Johns Hopkins Univer(a) Binding of analyte to immobilized protein on a flat gold surMALDI matrix solution to indisity School of Medicine merged face does not occur at a sufficient density to be detected by vidual protein spots and avoided protein arrays with MS to probe MALDI TOFMS. (b) Deposition of porous gold increases the sur- cross-contamination. complex mixtures of peptides face area of the protein spot (SEM image at left), which allows To test the technique, Cotter with antibody arrays (Anal. increased protein immobilization, analyte binding, and S/N for and colleagues coupled anti-V5, Chem. 2008, 80, 1448–1458). the MALDI TOF mass spectrum. Scale bars are 10 μm. anti-HA, and anti-c-myc anti“Our approach was to marry bodies to different hydrophilic two techniques—protein arrays and ionization method’s efficacy for surface- SAM spots and manually spotted plasma MS—that have been widely used in captured analytes. But MALDI requires spiked with the cognate peptides. Then, proteomics,” says Cotter. By using MS the addition of a matrix in an organic matrix solution was added to the protein to identify molecules bound to spots on solvent, which easily spreads across most spots, and the researchers performed a protein array, researchers could probe surfaces. To avoid cross-contamination, MALDI TOFMS and MS/MS sequenccomplex mixtures without fluorescence the matrix solution must be somehow ing. The expected m/z was obtained for labeling. However, MS-based detection localized to individual protein spots on peptides bound to each antibody-immois not as sensitive as fluorescence. Cotthe array. In addition, the researchers bilized spot. In addition, MS detected a ter says, “A major technical problem is needed to block nonspecific binding of degradation product of the V5 peptide getting enough protein immobilized on analytes to the gold substrate. that would not have been identified by the surface of the array to achieve a satTo accomplish these objectives, fluorescence-based methods. MALDI isfactory S/N ratio in the MS analysis.” Cotter and colleagues patterned selfTOFMS imaging produced a 3D repre To increase the density of immoassembled monolayers (SAMs) onto sentation of the signals of the bound bilized protein at each spot in an arthe porous gold surface. The SAMs analytes, which were assigned different ray, Kenyon Evans-Nguyen, a postdocwere composed of long alkyl chains colors, at each protein spot. toral fellow in Cotter’s lab, developed a with a thiol group at one terminus and The researchers are now focusing method to electrochemically deposit po- either a carboxyl or a methyl group their efforts on practical applications of rous gold on a flat gold slide. Just as a at the other end. The thiol group atthe technology. Cotter says, “On the dihigh-rise apartment building accommo- tached to the gold surface, whereas the agnostic front, I’m interested in making dates more tenants than a single-story carboxyl group was used to immobilize arrays of MHC [major histocompatibilstructure with the same footprint, the proteins of interest at spots on the array. ity complex] compounds to pull down nanostructured porous gold immobiHydrophilic, carboxy-terminated SAM displays of antigens that could be used lized substantially more protein than a spots were printed on the porous gold to diagnose diseases.” Because hundreds flat gold surface of the same spot size. substrate with an ink-jet printer, and of antigens can bind to each MHC molConsequently, more analyte bound to the printed surface was immersed in a ecule, an MS-based detection system each protein spot; this amplified the dodecanethiol solution to form hydrowould be convenient for the identificaMS signal. phobic, methyl-terminated SAMs in the tion and quantification of multiple antiMALDI TOFMS was used to anaunprinted regions. gens bound to a single protein spot. a lyze the protein arrays because of the Ordinarily, patterns of hydrophilic —Laura Cassiday Ma r c h 1 , 2 0 0 8 / A n a l y t i c a l C h e m i s t r y
1357
