Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection.
Analysis of interactive networks between proteins and other molecular constituents is of paramount importance to delineate complex cellular processes. In order to facilitate this process, new technologies that allow rapid, high-throughput parallel screening, as well as identification of constituents, are necessary. A particularly powerful combination in this regard could be the use of multiprotein microarrays coupled with mass spectrometry (MS). In the initial step of the method development we applied MS to single-protein microarrays. We demonstrated that even a simplified version of the method allows rapid parallel label-free assay of specific protein interactions with multiple metabolites derived from complex artificial and natural mixtures. The microarrays fabricated by the electrospray deposition technique and cross-linked in glutaraldehyde vapor were brought into contact with droplets of solution containing either a natural extract of baker's yeast cells or an artificial cocktail of metabolites. After washing, the microarrays were placed into 75% methanol to denature proteins and release specifically bound metabolites. The eluates were then analyzed by electrospray ionization mass spectrometry (ESI-MS) to simultaneously detect all the metabolites bound. Such a procedure applied to ten different proteins demonstrated that 50-400 ng of cross-linked protein is enough to obtain ion intensities from metabolites that are well distinguishable above noise. The compatibility of microplates and different microarray designs with MS detection is discussed.
Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia. vmorozov@beckmaninstitute.org
Victor N
VN
Morozov
Tamara Ya
TY
Morozova
Kenneth L
KL
Johnson
Stephen
S
Naylor
D002457:Cell Extracts / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015003:Yeasts
D002457:Cell Extracts; D011506:Proteins