Development of an efficient on-chip digestion system for protein analysis using MALDI-TOF MS

Area of Science:

• Biochemistry
• Analytical Chemistry
• Microfluidics

Background:

• Protein digestion is a critical step in proteomic analysis.
• Conventional methods can be time-consuming and require large sample volumes.
• Microfluidic devices offer potential for miniaturized and efficient biochemical assays.

Purpose of the Study:

• To develop and evaluate a solid-phase trypsin microreactor for electrokinetically-driven protein digestion.
• To couple the microreactor with MALDI-TOF MS for enhanced protein identification.
• To demonstrate the feasibility of using the microreactor for intact cell fingerprint analysis.

Main Methods:

• Fabrication of a poly(methyl methacrylate) (PMMA) microfluidic channel using hot embossing.
• Immobilization of trypsin onto UV-modified PMMA microposts within the microchannel.
• Electrokinetically-driven flow for protein digestion and subsequent off-line MALDI-TOF MS analysis.
• Evaluation using standard proteins (cytochrome c, BSA, phosphorylase b, beta-casein) and intact Escherichia coli cells.

Main Results:

• Achieved efficient digestion of cytochrome c with 97% sequence coverage in approximately 20 seconds.
• Obtained sequence coverages of 46% for BSA, 63% for phosphorylase b, and 79% for beta-casein.
• Demonstrated successful fingerprint analysis of intact Escherichia coli cells using the microreactor.

Conclusions:

• The developed solid-phase trypsin microreactor provides rapid and efficient protein digestion.
• The system is suitable for high-throughput proteomic analysis and identification.
• The microreactor technology shows promise for analyzing complex biological samples, including intact cells.