From twenty peptides to a single protein: peptide centric quantification via different digestion methods

Date

2025-9-01

Author

Kul, Ceren

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Targeted proteomics provides robust, precise, and reproducible protein quantification that supports biomarker validation and clinical applications. Advancements in mass spectrometry (MS) enable high-throughput quantitative analysis of thousands of proteins via their unique peptides. In the peptide-centric approach, previous studies have reported that multiple unique peptides of a protein exhibit different correlations. Digestion methods affect protein denaturation and enzymatic efficiency, leading to variations in the peptide products. To date, the effects of peptide selection and digestion methods on peptide-based protein quantification have not been systematically evaluated. This study investigated all unique peptides representing a single protein under nine digestion methods commonly used in clinical proteomics. Alpha-2-Macroglobulin (A2MG), a clinically important biomarker, was selected as a reference protein. A multiple-reaction-monitoring (MRM) method was developed with twenty stable-isotope-labeled (SIL) peptides. The analytical performance of the MRM method was evaluated for linearity, sensitivity, and matrix effects. The twenty peptides were analyzed the protein standard and human serum, and the MRM method was applied clinically to serum samples from healthy individuals and polycystic ovary syndrome (PCOS) phenotypes (A–D). The study showed that variations in peptide responses were influenced by sequence length, hydrophobicity, structural location, reactive amino acids, and the reagents and efficiency of specific digestion methods. Clinical study showed that biomarkers should be evaluated at the peptide level for both diagnostic and prognostic applications, as peptide signals are influenced by method-related differences as well as by underlying biological variation. Further studies should focus on method standardization and on assessing the utility of peptide-centric markers.

Subject Keywords

Biomarker, Multiple reaction monitoring (MRM), Alpha-2- Macroglobulin (A2MG), Unique peptide, Digestion method

URI

https://hdl.handle.net/11511/116081

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Graduate School of Natural and Applied Sciences, Thesis