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Rapid Commun Mass Spectrom. 2013 May 30;27(10):1149-54. doi: 10.1002/rcm.6555.

Covalent protein immobilization with a parylene-H film for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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1
Department of Materials Sciences and Engineering, Yonsei University, Seoul, Korea.

Abstract

RATIONALE:

For the sensitive analysis of receptor-ligand interactions by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), receptor proteins should be immobilized on a target plate with a high surface density. In this work, a parylene-H film with formyl groups was developed for the efficient covalent immobilization of receptor proteins for MALDI-TOF MS.

METHODS:

The parylene-H film was thermally deposited on a target plate and receptor proteins were covalently immobilized. The surface properties of the parylene-H film were analyzed by atomic force microscopy (AFM) and cyclic voltammetry (CV). The immobilization efficiency of the parylene-H film was analyzed by fluorescence imaging with streptavidin and fluorescence-labeled biotin. MALDI-TOF MS was performed using the parylene-H-coated target plate with streptavidin and different concentrations of biotinylated peptide as the receptor and ligand, respectively.

RESULTS:

The parylene-H film on a target plate had a flat surface (Rq : ±2.755 nm) without any pinholes and could be regarded to be electrically conductive under an electric potential of 30 kV. The fluorescence image proved that the parylene-H film improved the protein immobilization efficiency as well as ligand detection sensitivity. The mass spectra quantitatively revealed peaks from the ligand molecules without any interference peaks from the immobilized receptor proteins.

CONCLUSIONS:

A parylene-H film with formyl groups was thermally deposited on a target plate and the receptor protein was covalently immobilized on the target plate. The interactions of ligand molecules with the immobilized receptor proteins were quantitatively analyzed by MALDI-TOF MS.

PMID:
23592120
DOI:
10.1002/rcm.6555
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