Quantitation of surface-bound proteins on biochips using MALDI-TOF MS

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Accurate quantitation of surface-bound proteins is crucial for various applications, including diagnostics and drug discovery.
• Existing methods for measuring proteins on biochips often have limitations in accuracy and specificity.
• Development of novel, reliable techniques for absolute protein quantitation on solid supports is needed.

Purpose of the Study:

• To develop and validate a novel method for the absolute quantitation of proteins specifically bound to ligand-presenting biochips.
• To utilize matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for this purpose.
• To establish a reliable alternative to current protein quantitation tools for surface-bound analytes.

Main Methods:

• Proteins bound to a biochip were digested with trypsin.
• Peptide fragments were analyzed using MALDI-TOF mass spectrometry.
• An isotope-labeled internal standard (IS) with the same sequence as a reference peptide (RP) but different molecular weight was used for quantitation.
• Absolute protein amounts were determined by comparing mass intensities of the RP and IS.

Main Results:

• The method successfully quantitated glutathione S-transferase (GST) and a GST-fusion protein captured on glutathione-presenting biochips.
• A strong correlation was observed between glutathione densities on the biochips and the absolute quantity of captured proteins.
• The use of an IS in the same molecular milieu as the analyte ensured accurate quantitation.

Conclusions:

• The developed MALDI-TOF MS-based method provides accurate absolute quantitation of surface-bound proteins.
• This technique offers a valuable alternative to existing methods for analyzing proteins immobilized on biochips.
• The findings have implications for high-throughput screening, proteomics, and biochip assay development.