Direct-S: a directed mass spectrometry method for biomarker verification in native serum

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Serum is a key bio-specimen for biomarker monitoring.
• Direct analysis of low-abundance biomarkers in serum presents significant challenges.
• Existing methods often require depletion or pre-fractionation of high-abundance proteins.

Purpose of the Study:

• To establish a method for direct quantification of protein biomarkers in native serum.
• To overcome limitations in analyzing low-abundance biomarkers.
• To develop a sensitive, efficient, and accurate assay for serum biomarker verification.

Main Methods:

• Development of a directed mass spectrometry (inclusion list driven MS) method named Direct-S.
• Utilized an (18)O-labeling technique for internal standards of targeted peptides.
• Employed tandem mass spectrometry (MS/MS) with targeted peptide fragmentation.
• Used (16)O/(18)O ion pairs and internal standard characteristics for peptide identification.

Main Results:

• Direct-S enabled direct quantification of protein biomarkers (A1AT, LG3BP, CTSD) in native serum.
• Demonstrated good linearity (r > 0.9845) from 20 fmol to 0.5 nmol.
• Achieved reliable quantification across five orders of magnitude, down to 71 pg μL(-1).
• Successfully quantified candidate biomarkers in serum samples from colorectal cancer patients and healthy individuals.

Conclusions:

• Direct-S is a low-cost, convenient, and accurate method for serum biomarker verification.
• The method eliminates the need for high-abundance protein depletion or pre-fractionation.
• Direct-S offers improved sensitivity and efficiency for analyzing low-abundance biomarkers in complex matrices like serum.