Isobaric Peptide Labeling on Digital Microfluidics for Quantitative Low Cell Number Proteomics.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Digital microfluidics (DMF) offers miniaturized, automated liquid handling for bioanalysis.
• Current DMF applications in proteomics are limited to qualitative analyses due to detergent incompatibility with organic solvents.
• Isobaric peptide labeling techniques, like TMT labeling, are crucial for multiplexed quantitative proteomics but face challenges on DMF platforms.

Purpose of the Study:

• To develop an on-chip isobaric peptide labeling strategy for digital microfluidics (DMF).
• To identify a detergent compatible with organic solvents for reproducible droplet movement and TMT labeling on DMF.
• To enable high-throughput, low-cell-number quantitative proteomics using DMF.

Main Methods:

• Screening of commercially available detergents for compatibility with organic solvents and droplet movement on DMF.
• Identification and testing of 3-dodecyloxypropyl-1-β-d-maltopyranoside (DDOPM) for TMT labeling efficiency.
• Evaluation of the on-DMF TMT labeling protocol using protein digests and Jurkat T cells treated with doxorubicin.

Main Results:

• DDOPM was identified as a detergent enabling facile droplet movement and micelle formation in organic solvents for TMT labeling.
• The on-DMF TMT labeling strategy showed comparable efficiency to conventional methods.
• The protocol successfully identified differentially abundant proteins in Jurkat T cells using as few as 75 cells per replicate.

Conclusions:

• A novel on-DMF isobaric labeling strategy was successfully developed using the DDOPM detergent.
• This advancement facilitates multiplexed quantitative proteomics on DMF devices.
• The developed method represents a significant step towards high-throughput, low-cell-number proteomic analyses.