Behavior of Alpha-2-Macroglobulin unique peptides in biological samples

Yıldız, Pelin
Proteomics is the comprehensive study of proteins and proteoforms. Proteomics research enables the identification of new protein biomarkers for diagnostic applications and investigates novel targets for drug development. In bottom-up (shotgun) proteomics, proteins are digested using proteases, and corresponding peptides are analyzed by mass spectrometry (MS). The peptide-centric approach focuses on MS-based identification and/or quantification of only unique peptide(s) of the protein. However, proteins often contain multiple unique peptides. Therefore, the selection of the unique peptide representing the protein is crucial for both qualitative and quantitative proteomics. Here, we investigated the relationship between protein concentration and unique peptide responses under conventional proteolytic digestion conditions. Alpha-2-macroglobulin (A2MG), a clinically important protein associated with liver, lung, neurological diseases, and prostate cancer, was selected as a reference protein. Two common proteases, trypsin, and a trypsin/Lys-C mixture, were used for proteolytic digestion. Protein-peptide correlation, digestion efficiency, matrix-effect, and concentration-effect were evaluated for protein standard, human serum, and bovine serum. Twelve unique A2MG peptides were monitored using triple quadrupole mass spectrometry (QQQ-MS) operated in multiple reaction monitoring mode (MRM). The protein-unique peptide correlations were assessed at eight protein concentration levels. While a linear correlation was observed for all unique peptides at high concentration levels (0.0536 – 0.1071 μg/μl), peptide correlation at low concentration levels (0.0071 – 0.0357 μg/μl) were variable for both enzymes. The same investigation was performed in human serum and bovine serum at three A2MG protein levels. The results showed that the change in protein level was not reflected in peptide levels. We further investigated the relationship between protein-peptide correlation and certain peptide parameters such as pI value, peptide length, locations in the protein structure, and the presence of reactive amino acids. Outcomes of the research suggested that location of the peptides in the protein structure is the main factor which affects the linear peptide correlation since peptides located inner regions of the structure did not show linear correlation with other target peptides. Also, the peptides with lowest pI values show opposite correlation among all twelve A2MG unique peptides. It was observed that the twelve A2MG unique peptides behave different at different protein concentrations, as well as, various biological matrices. This is the first study investigating dynamic protein-peptide correlations in biological samples. The behavior of peptides at different concentrations and biological environments is critical for protein-based biomarker studies.


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Citation Formats
P. Yıldız, “Behavior of Alpha-2-Macroglobulin unique peptides in biological samples,” M.S. - Master of Science, Middle East Technical University, 2021.