Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.Proteomics is the large-scale analysis of whole proteins and their role in biological systems. Abundance-based proteomics assigns protein functions in normal and pathological processes by quantification of global differences in protein expression levels (1). This classic approach identifies functional biomarkers by comparing samples from healthy individuals and patients. However, this abundance-based approach provides only indirect information about protein function (2). The abundance of a protein is not necessarily correlated with its activity because protein activities are predominantly regulated by a series of post-translational modifications (1, 2). Activity-based proteomics (activity-based protein profiling) is therefore considered an alternative approach to assigning protein functions in biological processes of interest (3). In this approach, specific activity-based probes using fluorescent, radioactive, and affinity tags are usually designed for detection of protein activity (2, 4–6). Activity-based proteomics identifies markers by comparative analyses of activity profiles between healthy and diseased cells and tissues (3, 7, 8). This approach is also used for profiling enzyme inhibitors, for developing therapeutic reagents, and for diagnosis (2, 5). Another functional proteomic approach using large-scale analysis is interactomics or interaction proteomics, which is a useful method for understanding the regulation of proteins in biological systems (9). To elucidate bioactive protein interactions with proteins or ligands, a number of technologies are currently used including the yeast two-hybrid system, affinity purification and mass spectrometry, the protein fragment complementation assay, the luminescence-based mammalian interactome, and protein arrays (9–13). Global differences in the dynamics of the interactome between healthy and diseased individuals provide new insights into causes of disease and can be used for biomarker identification and drug discovery (14–16). Thus, combinatory analyses of abundance, activity, and interaction have great potential in revealing regulation mechanisms and functions of proteins, although such an integrated proteomic approach has not been widely used.These proteomic methods have been coupled with various detection methods including one- or two-dimensional gel electrophoresis, one- or two-dimensional liquid chromatography and tandem mass spectrometry, surface plasmon resonance, and fluorometric assays for analyses of the proteome (9, 11). In combination with specific probes, colorimetric and fluorometric assays using multiwell plates have been extensively used for the determination of the abundance and activity of various proteins. Although often limited by the amount of sample, these methods nonetheless facilitate real-time measurement of changes in protein activity and high-throughput analyses of protein abundances and activities (17). Surface plasmon resonance, a method that does not necessitate labeling of proteins, has also been used for analysis of protein abundance, activity, and binding affinity (18–20). Using only very small amounts of sample, the microarray combined with fluorometric probes is a promising technology for the rapid analysis of a wide variety of biomolecular interactions, protein abundances, and activities. This approach has been used for serodiagnosis and identification of biomarkers by abundance-based protein profiling in human sera (21–25). It has also been used for kinetic studies of carbohydrate-protein (17) and peptide-protein interactions (26, 27). In addition, this technology has been used for the rapid determination of enzyme activities and for the identification of enzyme substrates and inhibitors (24, 28–33). However, combinatory profiling of protein activities and interactions based on array technology has yet to be reported.Using protein arrays, we propose as a model system an integrative proteomic approach for simultaneous profiling of the transamidating activity and interactions of transglutaminase 2 (TG2) with TG2-related proteins. TG2, known as tissue transglutaminase, is a member of the calcium-dependent transglutaminase family. Its activity and interactions are associated with a wide variety of diseases and cellular events (34). TG2 is implicated in the pathogenesis of a wide variety of diseases including inflammatory diseases such as celiac sprue, neurodegenerative disorders such as Huntington''s, Alzheimer''s, and Parkinson''s disease, as well as cancers, cardiovascular diseases, and diabetes (34–36). TG2 is also involved in various cellular events including cell growth, cell differentiation, cell adhesion, extracellular matrix crosslinking, and apoptosis (34, 37, 38). In the present study, the transamidating activity and binding affinity of TG2 for 16 proteins were simultaneously monitored using Cy5-conjugated TG2 and protein arrays (Fig. 1). Using this large-scale analysis, we constructed a quantitative activity-interaction (AI)¹ map to describe the quantitative interaction of TG2 with its related proteins. Thus, this integrative proteomic approach can be used to characterize functions and regulation mechanisms of a target protein in many biological processes of interest.Open in a separate windowFig. 1.Schematic diagram for the simultaneous analysis of transamidating activity and interaction of TG2 with TG2-related proteins. BAPA, 5-(biotinamido)pentylamine; Pr, protein; SA, streptavidin; TG2, transglutaminase 2.