Abstract
The development of analytical methods for the quantification of peptides in biological matrices has become increasingly important with the continuously growing attention for these large bio-molecules in all kinds of biomedical research. For example, endogenous peptides often serve as templates for the development of novel (peptidic) therapeutic compounds, while their
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roles in physiological processes are extensively explored for applications in disease (pre) diagnosis. In recent years, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has developed into one of the most valuable analytical techniques that could overcome most problems generally associated with the bioanalysis of peptides, as it allows highly sensitive and specific analyses. The suitability of LC-MS/MS for the quantification of peptides in biological matrices has been overviewed in the introduction of this thesis, which thereafter focuses on the development and validation of various LC-MS/MS methods for different types of peptides, including peptide drugs, endogenous bioactive peptides and peptide fragments from endogenous blood proteins. The developed methods have been used for the absolute quantification of the specific peptides in clinical serum samples to evaluate their potential as serum markers for different types of cancer. For example, the antimicrobial human neutrophil peptides-1, -2 and -3 (HNP-1, 2 and -3) were quantified in serum samples from colorectal cancer patients. The developed LC-MS/MS method allowed for the first time simultaneous quantification of individual concentrations of these three structurally almost similar peptides and revealed significantly up-regulated serum concentrations of HNP-1 and -2 in colorectal cancer patients compared to serum from colonoscopy-assessed controls. Furthermore, two different LC-MS/MS methods were developed for the absolute quantification of proteolytic peptide fragments from common blood proteins, which have previously been postulated as exhibiting diagnostic information for the detection of different types of cancer. The suitability and importance of the developed LC-MS/MS methods was shown by the measurement of clinical serum samples from breast cancer patients and matched controls. These analyses showed significantly up-regulated serum concentrations in breast cancer for des-Arg9-bradykinin, a metabolite from bradykinin, and for several peptides derived from the blood protein inter alpha-trypsin inhibitor heavy chain 4 (ITIH4). Moreover, serum concentrations of the up-regulated peptides were significantly decreased after surgical removal of the breast cancer tumor, indicating the potential of these peptides in the follow-up of breast cancer. The developed LC-MS/MS methods were further exploited to more specifically investigate the influence of pre-analytical factors on the serum concentrations of the different peptide fragments, required for future studies to finally elucidate the role of these peptides in different types of cancer and their potential as cancer biomarkers.
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