Toward Real-Time Proteomics: Blood to Biomarker Quantitation in under One Hour

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Traditional bottom-up proteomics is time-consuming due to multistep tryptic digestion.
• The slow turnaround of current proteomics methods limits clinical utility for dynamic disease monitoring.

Purpose of the Study:

• To evaluate a novel hyperthermoacidic archaeal (HTA) protease for rapid sample preparation in proteomics.
• To assess the combination of HTA protease with dual-trapping single analytical column (DTSC) liquid chromatography-mass spectrometry (LC-MS) for high-throughput quantitative proteomics.

Main Methods:

• Utilized the HTA protease 'Krakatoa' for single-step, rapid protein digestion (5-30 min) at pH 3 and >80 °C.
• Employed high-throughput DTSC LC-MS for analysis of processed biofluid samples.
• Applied targeted mass spectrometry (MS) for sensitive peptide and protein quantification.

Main Results:

• Quantified over 160 proteins from 1 μL of whole blood in under 1 hour.
• Successfully measured labile Angiotensin I and II peptides and over 150 proteins in serum and plasma from 53 individuals.
• Identified at least 46 proteins not detectable by traditional trypsin digestion.

Conclusions:

• The HTA protease combined with DTSC LC-MS enables real-time proteomics, drastically reducing analysis time.
• This rapid proteomics approach offers immediate potential for advancing clinical diagnostics and research applications.
• The methodology facilitates timely quantification of disease biomarkers for improved patient care.