Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array.
Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.
Gavin
Igor M
IM
Biochip Technology Center, Argonne National Laboratory, IL 60439, USA. igavin@anl.gov
Kukhtin
Alexander
A
Glesne
David
D
Schabacker
Daniel
D
Chandler
Darrell P
DP
D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods