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

Area of Science:

• Biochemistry
• Surface Chemistry
• Analytical Chemistry

Background:

• On-chip phosphorylation analysis requires efficient peptide immobilization for accurate kinase activity measurement.
• Previous methods using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) resulted in low phosphorylation efficiency (20% at 2 h).

Purpose of the Study:

• To enhance the efficiency of on-chip peptide phosphorylation by optimizing surface chemistry.
• To investigate the role of poly(ethylene glycol) (PEG) linkers in peptide immobilization for improved kinase substrate analysis.

Main Methods:

• Immobilization of cysteine-terminated peptides on amine-modified gold surfaces using SSMCC.
• Comparison of direct immobilization versus immobilization using PEG-inserted peptides and PEG-containing cross-linkers.
• Utilizing fluorescence-labeled peptides to study immobilization density and linker effects.
• Assessing phosphorylation efficiency with specific kinases like cSrc and protein kinase A.

Main Results:

• Immobilization via a PEG-inserted peptide and SSMCC improved cSrc phosphorylation efficiency to 60% at 1 h.
• No phosphorylation was detected when peptides were immobilized with a PEG-containing cross-linker.
• Longer cross-linkers, including PEG, led to lower immobilization density.
• The positive effect of PEG on phosphorylation efficiency was observed for cSrc but not protein kinase A, suggesting kinase-dependent effects.

Conclusions:

• A flexible PEG linker, when combined with SSMCC and a PEGylated peptide, is a promising surface chemistry for improving on-chip phosphorylation analysis.
• Peptide mobility and accessibility are enhanced by PEG linkers, boosting phosphorylation efficiency.
• Immobilization density is critical and influenced by cross-linker length; optimization of the immobilization protocol is essential.