Abstract
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.
Highlights
Proteomics is the large-scale analysis of whole proteins and their role in biological systems
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
Characterization of Cy5-conjugated TG2—To better characterize the regulation and functions of proteins in biological systems, we developed an integrative proteomic profiling method to simultaneously monitor the activities and interactions of target proteins with their related proteins in a highthroughput assay
Summary
Chemicals and Reagents—3-Aminopropyltrimethoxysilane, dithiothreitol, thrombin, Cy3-conjugated streptavidin, human serum albumin (HSA), fibrinogen and fibronectin (from human plasma), ␣-synuclein (human recombinant), retinoblastoma (human recombinant), E-cadherin (human recombinant), and G-actin were obtained from Sigma. 5-(biotinamido)pentylamine (BAPA) was obtained from Pierce Science (Rockford, IL). TG2-related proteins were labeled with Cy5 NHS ester and absorbance of Cy5-conjugates was determined using the Nanodrop® ND-1000 UV-Vis spectrophotometer. Protein concentrations bound to the array surface were calculated from fluorescence intensities obtained from washed sets using standard curves generated from dried sets of array spots. Sixteen proteins with the indicated concentrations were immobilized on the surface of well-type amine arrays for 1 h at 37 °C and blocked with 2 M BSA containing 0.1% (v/v) Tween 20 in phosphate buffered saline (PBS) (8.1 mM Na2HPO4, 1.2 mM KH2PO4, 2.7 mM KCl, and 138 mM NaCl, pH 7.4) for 30 min at 37 °C. Following washing with 0.1% (v/v) Tween-20 in PBS and with milli-Q-purified water, the arrays were dried under compressed air and scanned with a fluorescence scanner using 543 nm and 633 nm lasers (PerkinElmer Life and Analytical Sciences).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
