Quantitative peptide bioanalysis using column-switching nano liquid chromatography/mass spectrometry

Area of Science:

• Bioanalysis
• Analytical Chemistry
• Biochemistry

Background:

• Endogenous peptides circulate at low pmol l-1 concentrations in bodily fluids, demanding highly sensitive bioanalytical methods.
• Analyzing these minute analyte concentrations in complex biological matrices presents significant challenges.

Purpose of the Study:

• To develop a miniaturized liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method for sensitive peptide quantification.
• To couple the LC/ESI-MS system with an on-line preconcentration (PC) system to enhance sensitivity for low-concentration analytes.

Main Methods:

• A miniaturized LC/ESI-MS method was developed using custom nanoLC columns (75 microns i.d.) and a micro-ESI interface.
• An on-line preconcentration system utilizing strong anion-exchange material was integrated to handle large sample volumes.
• The method was applied to analyze endothelin peptides (ETs) in cell supernatants.

Main Results:

• Simultaneous analysis of ET isoforms was achieved with detection limits as low as 30 pmol l-1 (1.5 fmol on column) in cell supernatants.
• The method demonstrated linearity from 50 to 2000 pmol l-1, with correlation coefficients of 0.99 for two ET isoforms.
• Investigation included assessment of matrix effects and recovery for method validation.

Conclusions:

• The developed nanoLC/ESI-MS method coupled with on-line preconcentration is highly effective for bioanalysis of low-abundance endogenous peptides.
• This sensitive bioanalytical approach enables the quantification of potent vasoconstrictors like endothelin peptides in complex biological samples.