Analysis of Protein Interaction and Function with a 3-Dimensional MALDI-MS Protein Array

Abstract

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.

Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

“…Gavin et al [37] demonstrated desorption of intact proteins captured on hydrogel-immobilized antibodies followed by MS analysis. The method for array manufacturing there differs substantially from the technology for hydrogel-arrays used in this work.…”

“…Recently Gavin et al [37] demonstrated desorption of intact proteins captured on hydrogel-immobilized antibodies followed by MS analysis. The procedure allows the analysis of a single sample at a time and problems with mass resolution in the higher mass range seem inevitable.…”

Surface plasmon resonance imaging in arrays coupled with mass spectrometry (SUPRA–MS): proof of concept of on-chip characterization of a potential breast cancer marker in human plasma

“…In this field, the hyphenation of immunoassays with mass spectrometry seems very promising in that it enables qualitative or quantitative measurements with the possibility of analyzing part of the sequence of a captured protein [4][5][6][7][8][9][10][11]. Since 2000, biomolecular interaction analysis-mass spectrometry (BIA-MS) has been used to investigate two analytical approaches: the off-chip approach, resulting in elution of bound analytes before mass spectrometric analysis [12][13][14][15] and the on-chip approach, based on direct analysis of the arrays with MALDI-TOF-MS [16][17][18][19].…”

Global protein interactome exploration through mining genome-scale data in Arabidopsis thaliana

“…Some of them, like AFM, FRET and BREF, can only be applied on the small scale protein-protein interaction detection, while others, such as Yeast Two Hybrid, Affinity Purification and Protein Arrays, are suitable for high throughput purpose. High throughput technologies, like yeast two-hybrid system (Y2H) and affinity purification, have been applied for genome-wide detection of protein protein intearction in several species, including Homo Sapiens [1-4], Drosophila Melanogaster [5,6], Saccharomyces Cerevisiae, [7-9]and Caenorhabditis elegans [10]. …”