Higher Temperature Porous Graphitic Carbon Separations Differentially Impact Distinct Glycopeptide Classes

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Glycoproteomics relies on chromatography for glycopeptide separation.
• Porous graphitic carbon (PGC) offers advantages over traditional phases like reversed-phase and HILIC.
• Higher column temperatures in PGC can enhance profiling depth and resolution.

Purpose of the Study:

• To investigate the impact of increased column temperature on PGC-based glycopeptide identification.
• To analyze qualitative and semiquantitative effects of elevated temperatures on glycopeptide analysis.
• To determine how different glycan structures respond to high column temperatures.

Main Methods:

• Mass spectrometry-based glycoproteomics.
• Analysis of enriched bovine and human glycopeptides using PGC chromatography.
• Evaluation of glycopeptide identification rates, signal intensity, resolution, and spectral count linearity at varying column temperatures.

Main Results:

• Increased column temperature positively impacts high mannose glycopeptides.
• Elevated temperatures negatively affect sialylated glycopeptides, reducing identification rates and quantitative accuracy.
• Temperature-dependent effects were observed on signal intensity, resolution, and spectral count linearity.

Conclusions:

• PGC chromatography at high temperatures offers benefits for certain glycopeptide analyses but poses challenges for others.
• Careful optimization of column temperature is crucial for accurate glycopeptide identification and quantification.
• Findings provide guidance for leveraging PGC separations in glycoproteomics while highlighting limitations for label-free quantification.