Colorful quality control of chromatographic sample preparation

Area of Science:

• Proteomics
• Analytical Chemistry
• Chromatography

Background:

• Multidimensional chromatography is crucial for proteomic biomarker discovery, generating numerous fractions requiring comparison.
• Processing variations in sequential chromatographic steps can introduce random deviations, potentially falsifying proteomic results.
• Robust quality control is essential to ensure the reliability of proteomic data derived from complex chromatographic separations.

Purpose of the Study:

• To evaluate the suitability of colored phycobilins as internal standards for quality control in multidimensional chromatographic separations.
• To assess the impact of phycobilins on sample processing and subsequent mass spectrometric analysis.
• To determine if phycobilins can effectively detect deviations in chromatographic elution protocols.

Main Methods:

• Utilized colored phycobilins as internal standards during multidimensional chromatographic runs.
• Performed size exclusion and anion exchange chromatography with phycobilins alone, in combination, and with plasma proteins.
• Analyzed elution reproducibility and peak location stability of phycobilins.
• Assessed the effect of phycobilins on mass spectrometric analysis, including protein coverage and peptide/protein numbers.

Main Results:

• Phycobilin elution exhibited high reproducibility, with stable main peak locations under proper chromatographic performance.
• Phycobilins did not interfere with sample processing when run alone, combined, or with plasma proteins.
• Characteristic peak shifts in phycobilins effectively indicated deviations caused by common elution protocol failures.
• Mass spectrometry analysis remained unaffected, with no alteration in protein coverage, peptide numbers, or protein numbers.

Conclusions:

• Colored phycobilins are effective internal standards for quality control in multidimensional chromatography.
• Phycobilins provide a reliable method for evaluating the robustness and reproducibility of chromatographic applications.
• The use of phycobilins enhances the accuracy and trustworthiness of proteomic biomarker discovery.