LC-MS accurate quantification of a tryptic peptide co-eluted with an isobaric interference by using in-source...

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Chromatography

Background:

• Isobaric and isomeric interferences are significant challenges in liquid chromatography-mass spectrometry (LC-MS).
• Accurate quantification of target analytes is often hindered by co-eluting isobaric compounds.
• Existing methods may require extensive chromatographic optimization to resolve such interferences.

Purpose of the Study:

• To develop a method for identifying isobaric contamination and quantifying analytes accurately despite co-eluting interferences.
• To introduce a strategy combining in-source purification and fragmentation for enhanced LC-MS analysis.
• To demonstrate the utility of this approach using peptide mixtures.

Main Methods:

• Integration of in-source purification with chromatographic separation.
• Total fragmentation of precursor ions of isobaric interferences to achieve LC-pseudo-MS² capability.
• Utilizing the ratio of peak areas of tryptic peptides and their isotopically labeled internal standards for quantification and interference detection.

Main Results:

• The developed method successfully identified isobaric interferences and enabled accurate quantification.
• Accurate quantification of tryptic peptides was achieved without the need for chromatographic separation of interferences.
• Calculated concentrations for spiked samples showed high accuracy with narrow confidence intervals (e.g., 8.0 ± 0.5 μM and 15.7 ± 0.5 μM).

Conclusions:

• The combined in-source purification and fragmentation strategy effectively overcomes isobaric interference issues in LC-MS.
• This approach provides accurate quantification by eliminating the reliance on chromatographic resolution of interferences.
• The method offers a robust solution for complex sample matrices encountered in LC-MS analysis.