Mass spectrometry-based N- and C-terminomics for the identification of protein termini in model organisms and clinical specimens

Proteins are highly complex biopolymers that fulfill crucial functions in biological systems. They are synthesized at ribosomes, which translate mRNA information into an amino acid sequence. Upon synthesis, proteins present both an N‑ and C‑terminus, which flank the peptide backbone at both ends. Yet, proteins in vivo are only insufficiently described by this transfer of information. Commonly, proteins are highly modified, which can fundamentally change their structure and function. This includes the proteolytic processing, resulting in novel protein termini. The entirety of proteins within a cell is defined by the term proteome, while the entirety of protein termini is described as the terminome.

The presented thesis developed and utilized mass spectrometry-based protein analytics methods for proteomics and terminomics, to study proteins and their termini in a biological model system and within clinical context. In the first chapter, a novel C‑terminomics workflow is established, which utilized SCX chromatography coupled with peptide‑level charge‑reversal derivatization to map the cellular C‑terminome. The workflow enabled a thorough analysis of the cellular terminome and further tested the application of enzymatic 18O labelling for the detection of protein C‑termini in complex proteome samples.

In the second chapter, quantitative proteomics and terminomics methods were utilized to study clinical biopsy specimens from atopic dermatitis (AD) patients. Samples from four patients and five healthy controls were collected, both by invasive punch biopsies and minimally invasive tape‑stripping. The comparison of patient lesion, non-lesion and control specimens revealed multiple proteins with significant abundance differences, both novel and previously reported. Further, an N‑terminomics study was performed on tape-stripped specimens, enabling the first reported proteome-wide study of protein N‑termini within AD skin.

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Zitierform:
Kaleja, P., 2025. Mass spectrometry-based N- and C-terminomics for the identification of protein termini in model organisms and clinical specimens.
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