Plastic microchip liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry using...

Area of Science:

• Analytical Chemistry
• Separation Science
• Mass Spectrometry

Background:

• Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a powerful tool for protein analysis.
• Improving the separation efficiency and sensitivity of MALDI-TOF-MS for complex peptide mixtures remains a challenge.

Purpose of the Study:

• To develop and characterize an integrated microchip liquid chromatography (LC) system for off-line coupling with MALDI-TOF-MS.
• To evaluate the performance of the microchip LC-MALDI-TOF-MS interface for peptide mixture analysis.

Main Methods:

• Fabrication of a microfluidic device with an array of monolithic LC columns using hot embossing on cyclic olefin copolymer (COC).
• Preparation of methacrylate monolithic columns via UV-initiated polymerization within microfluidic channels.
• Utilizing a pulsed electric field deposition system for non-contact transfer of column effluents to MALDI targets.

Main Results:

• Achieved approximately 59% amino acid sequence coverage for digested bovine serum albumin (BSA) using microchip LC-MALDI-MS, a significant improvement over MALDI-MS alone (23%).
• Demonstrated high sensitivity, detecting as low as 10 fmol/µL of spiked peptide in 1 µg of digested BSA (S/N = 10).
• Obtained more than double the amino acid sequence coverage for a mixture of three digested proteins (BSA, myoglobin, cytochrome c) compared to MALDI-MS alone.

Conclusions:

• The developed microchip LC-MALDI-TOF-MS system offers enhanced performance for analyzing complex peptide mixtures.
• This integrated approach significantly improves protein identification and characterization capabilities.
• The system shows promise for sensitive and efficient analysis of low-abundance proteins in complex biological samples.