TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/19620/
Y1  - 2015///
ID  - heidok19620
TI  - Development of Novel Reagents for the Selective Enrichment of Vascular Accessible Proteins and the Identification of Disease-Specific Biomarkers
AV  - public
A1  - Hanke, Sabrina Annette
KW  - Proteomics
KW  -  Bioconjugation
KW  -  Mass Spectrometry
N2  - A promising approach for the development of novel therapeutics with fewer side effects in healthy tissues is the targeted delivery of bioactive molecules directly to the site of disease. The prerequisite is the identification of a robust, disease-specific biomarker, targetable either with monoclonal antibodies interfering with the target?s biological function or with antibody-drug conjugates delivering a therapeutic payload such as a cytotoxic drug. Hence, the target molecules of interest have to be vascular-accessible and are therefore located on the surface of diseased cells, on newly formed blood vessels or in the perivascular extracellular matrix. Proteomic approaches for the identification of novel biomarkers have to deal with (i) the high dynamic range of the proteome over at least seven orders of magnitude, (ii) the low abundance of the highly diverse plasma membrane proteome fraction, as well as with (iii) the hydrophobic character of membrane proteins. The proteins of interest are therefore often under-represented in mass spectrometric datasets of full proteome samples and cannot be stably quantified. One avenue towards the enrichment of the vascular-accessible surface proteome fraction prior to mass spectrometric analysis is the covalent modification of the target proteins with a membrane-impermeable ester-derivative of biotin, followed by streptavidin-based affinity capturing. The biotinylation of potential biomarkers is fast and efficient and can be performed by in vitro labelling of cells, via in vivo perfusion of mice or ex vivo using surgically resected tissue material.

The work in this thesis focused on the synthesis and multi-step validation of two novel, multiply-charged peptide-based as well as of two novel heparin-based biotinylation reagents. Furthermore, two alkyne-tagged reagents for bioorthogonal click-chemistry based enrichment were designed. The reagents? reactivity was assessed by coupling to BSA in different ratios and analysis with linear MALDI mass spectrometry. Subsequent validation was performed in vitro on HeLa cells and in vivo via perfusion of healthy NSG mice. Biotinylation efficacy was examined using FACS analysis, ELISA and Western Blot of cell and tissue samples. Cell surface or perivascular biotinylation was visualized by confocal laser scanning microscopy. Mass spectrometric analyses of the accessible proteome fractions were performed in comparison to the commercial reagents Sulfo-NHS-LC-biotin and NHS-PEG12-biotin and PBS- or non-treated negative controls on biotinylated HeLa cells and kidney or liver tissue using a MALDI TOF/TOF? 5800 system (AB SCIEX). Mass spectrometric data were analysed in terms of identified protein and proteotypic peptide numbers as well as of protein localization. Relative quantification based on MS1 signal intensities was performed using the in-house developed software MSQBAT. SRM-analysis of some medium- and low-abundant cell surface and extracellular matrix proteins was performed in comparison to full proteome samples on a QTRAP® 6500 system (AB SCIEX).

The properties of any biotinylation reagent are determined by the linker between the biotin residue for affinity purification and the reactive group for protein coupling. Increase in size and polarity influences the reagents? selectivity and reactivity. Site-specific activation of the novel peptide-based biotinylation reagents could significantly improve the reactivity in comparison to non-specifically activated NHS-?-Ala-(L-Asp)3-biotin published by Strassberger et al. in 2010. Alkyne-tagged reagents revealed comparable reactivity to the biotinylation reagents resulting from the similarity of the peptide-based linkers. Due to their enormous size and the increased steric hindrance, heparin-based reagents are less reactive than the smaller peptide-based and commercial reagents. The reactivity difference is also mirrored in the mass spectrometric datasets. A total of 1574 proteins could be identified within the in vitro analysis. The proof-of-principle study could demonstrate the stable identification of a 38-49% fraction of plasma membrane or extracellular matrix annotated proteins with the peptide-based reagents. 1965 proteins were found in the kidney dataset with a comparable fraction of 40-45% surface annotated proteins. Within the liver dataset, 1531 proteins could be identified with a slightly increased intracellular protein fraction (27-34% surface proteome fraction). Reasons for the typical background proteome identification in all samples were further assessed by different sample preparation strategies within this thesis. In short, it can be stated, that the success of biomarker studies with biotinylation reagents are dependent of the vascularisation of the target tissue to enable high biotinylation rates as well of elaborated sample preparation protocols: For example, delipidation is crucial for the work with tissue samples. A slight reactivity decrease of the novel peptide-based reagents compared to commercial Sulfo-NHS-LC-biotin was detected due to the sterically more hindered peptide linker. Nevertheless, the enrichment of the targeted surface proteome is very stable: 40% of the quantified proteins are more than 2-fold up- or down-regulated in comparable fractions between the peptide-based and the commercial reagent (kidney dataset). The number of identified proteotypic peptides per plasma membrane or extracellular matrix annotated protein is significantly increased compared to the negative controls and to the intracellular proteome fraction, which is the basis for enabling a stable protein quantification. 

The mass spectrometric studies revealed that the novel peptide-based reagents provide a reliable technology platform for the enrichment of vascular accessible proteins: Plasma membrane or extracellular matrix annotated biomarker candidates can be stably identified and quantified based on high numbers of proteotypic peptides. Furthermore, stable SRM-based quantification of medium- and low-abundant targets is enabled.
ER  -