Optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis

Analytical Chemistry
Kazuki InamoriYoshiki Katayama

Abstract

We investigated the optimal surface chemistry of peptide immobilization for on-chip phosphorylation analysis. In our previous study, we used a heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) to immobilize cysteine-terminated peptides on an amine-modified gold surface. The study revealed that the phosphorylation efficiency and rate were low (only 20% at 2 h) comparing with the reaction in solution. In this study, to improve the phosphorylation efficiency, the kinase substrates were immobilized via poly(ethylene glycol) (PEG), a flexible, hydrophilic polymer. An improvement in cSrc phosphorylation was achieved (60% at 1 h) from using a PEG-inserted peptide and SSMCC. However, no phosphorylation could be detected when the peptide was immobilized with a PEG-containing cross-linker. Fluorescence-labeled peptide studies revealed that the use of longer cross-linkers resulted in lower immobilization density. We considered that the flexible PEG linker was preferable to secure high phosphorylation efficiency for the immobilized peptide, probably due to the improvement of cSrc accessibility and peptide mobility, but the immobilization protocol is critical for keeping high density o...Continue Reading

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Citations

Jun 21, 2013·Analytical and Bioanalytical Chemistry·Roza TrzcinskaBarbara Trzebicka
May 24, 2011·Molecular Biotechnology·Alexandra ThieleMike Schutkowski
Jan 15, 2011·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Takashi ShimomuraYoshiki Katayama
Sep 3, 2010·Analytical Biochemistry·Guangchang ZhouStephen J Kron
Dec 18, 2009·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Oct 16, 2014·Biochimica Et Biophysica Acta·Eiji KinoshitaTohru Koike
Apr 13, 2013·Chemical Society Reviews·Christopher J GraySabine L Flitsch
Sep 24, 2010·Bioconjugate Chemistry·Guangchang ZhouStephen J Kron
Oct 1, 2010·ACS Applied Materials & Interfaces·Stella H NorthScott G Walton
Apr 5, 2014·Langmuir : the ACS Journal of Surfaces and Colloids·Roza TrzcinskaBarbara Trzebicka
May 30, 2014·Journal of Proteome Research·Haruna ImamuraYasushi Ishihama
Apr 26, 2014·Journal of the American Chemical Society·Kelsey I MacConaghyMark P Stoykovich

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