Peptide microarrays for the characterization of antigenic regions of human chromogranin A

Abstract

Microarraying peptides is a powerful proteomics technique for studying molecular recognition events. Since peptides have small molecular mass, they are not easily accessible when adsorbed onto solid supports. Moreover, peptides can lack a well-defined three-dimensional structure, and therefore a correct orientation is essential to promote the interaction with their target. In this work, we investigated the suitability as a peptide array substrate of a glass slide coated with a copolymer of N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and [3-(methacryloyl-oxy)propyl]trimethoxysilyl. This polymeric surface was used as substrate for peptides in the characterization of linear antigenic sites of human chromogranin A, a useful tissue and serum marker for neuroendocrine tumors and a precursor of many biologically active peptides. The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which linear epitopes are freely exposed despite peptide random orientation. The results reported in this article are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.

Human body fluid proteome analysis

“…This study points to a new source of producing thousands of purified proteins for protein array fabrication. Peptide microarrays displaying biologically active small peptides in a high-density format provide an attractive technique to probe complex samples such as serum [141][142][143]. A technical difficulty in fabrication of peptide arrays is that peptides, usually with small molecular mass, are not easily accessible when adsorbed onto solid supports.…”

“…Peptides also lack a well-defined 3-D structure, and therefore a correct orientation is essential to promote the interaction between peptides and their targets. Incorporation of elongated spacer molecules or modification of the solid substrate can significantly improve the accessibility of a target molecule [142]. In addition, protein arrays can also be prepared with the cellular proteins expressed in a cell line or the proteins expressed in a cell-free in vitro transcription/translation system [144,145].…”

Spatially addressable protein array: ssDNA‐directed assembly for antibody microarray

“…Over the past two decades, the emergence of microarray technology and high-throughput analysis of genomic and proteomic information enabled the mapping of the functional roles of many proteins and these have become important disciplines for characterizing complex cellular functions [1][2][3][4][5][6][7]. Among the various microarray techniques developed, protein chip technology has been growing rapidly with novel strategies for the screening of protein-protein, proteinligand, or antigen-antibody interactions [8][9][10].…”

Surface Behavior and Molecular Recognition in DNA Microarrays from N,N‐Dimethylacrylamide Terpolymers with Activated Esters as Linking Groups

“…[22] Over the last few years, a new terpolymer made of DMA (with 97% of moles in the monomer feed), NAS (2%) and 3-(trimethoxysilyl)propyl methacrylate (MAPS, 1%) has been synthesized by free radical copolymerization and used as a functional coating for the fabrication of DNA, protein and peptide microarrays. [23][24][25] The terpolymer binds efficiently to glass both with covalent bonds due to MAPS's silane functionalities, and with physico-chemical multiple interactions with the substrate, such as hydrogen bonding and van der Waals forces because of the DMA. The last monomer, NAS, enables covalent binding of aminomodified oligonucleotides.…”

“…[23] In this study, the effect of terpolymer molecular weight was studied and was correlated to the physico-chemical and functional surface behavior of the resulting microarray. Four different molecular weights of such a terpolymer, indicated as copoly(DMA-NAS-MAPS), were synthesized by changing the monomer feed concentration (the terpolymer studied previously [23][24][25] was obtained by the lowest examined monomer feed concentration). Glass slides were coated by dipping in diluted aqueous solutions.…”