Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Elevating column temperature enhances peptide chromatographic separation.
• High temperatures can cause artificial peptide modifications, compromising analysis quality.

Purpose of the Study:

• To present a novel high-temperature LC-MS method that minimizes peptide modification and degradation.
• To improve peptide separation and identification in LC-MS analyses.

Main Methods:

• Utilized a short inline trap column at near-ambient temperature upstream of a heated separation column.
• Optimized trap column retentivity and dimensions to reduce peptide residence time in the heated column.
• Applied the method to peptide mapping of trastuzumab and exploratory LC-MS proteomic analyses.

Main Results:

• Increased peak capacity 1.4-fold in a 110-min peptide mapping of trastuzumab.
• Achieved 10% more peptide identifications in proteomic analyses compared to 30 °C.
• Reduced temperature-related artifacts (N-terminal pyroGlu, oxidized Met) by 66% and 63% respectively, compared to direct injection at 60 °C.

Conclusions:

• The novel method retains benefits of high column temperature while significantly reducing peptide modifications.
• This approach enhances the reliability of peptide analysis and quality control for biopharmaceuticals.
• Offers a valuable tool for improving LC-MS methodology in research and industry.