The pivotal role of reactivity in the design of novel biotinylation reagents for the chemical-proteomics-based identification of vascular accessible biomarkers.
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. Thus, one prerequisite is the identification of a robust, disease-specific, vascular accessible biomarker localized on the surface of diseased cells, in the surrounding extracellular matrix or on newly formed blood vessels. One avenue towards the identification of such biomarkers consists in the enrichment of the vascular accessible surface proteome fraction prior to analysis. This can be achieved by covalent modification of the target proteins with membrane-impermeable ester derivatives of biotin, followed by streptavidin-based affinity capturing. The properties of the respective reagents are determined by the linker between the biotin moiety and the reactive group for protein coupling. In the frame of this study, novel, reactivity-improved peptide-based biotinylation reagents as well as reagents based on highly hydrophilic heparin linkers were synthesized and validated. The comprehensive evaluation of different biotinylation reagent classes with aliphatic, PEGylated, peptide-based and heparin-based linkers on single model protein BSA, HeLa cells as well as perfused kidney tissue revealed that the linker-dependent chemical reactivity is the crucial factor for the design of novel biotinylation reagents for vascular targeting approaches. 


Significance 

To obtain a reliable identification and stable quantification of vascular accessible protein targets by means of mass spectrometry, covalent modification with a membrane-impermeable ester derivative of biotin, followed by streptavidin-based affinity capturing, is frequently applied for in vivo or ex vivo biomarker identification studies. Nevertheless, no comprehensive evaluation of different biotinylation reagent classes has been performed so far. Within this study, we systematically evaluated novel peptide- and heparin-based biotinylation reagents as well as established compounds based on aliphatic and PEGylated linkers. We identified the linker-dependant chemical reactivity of biotinylation reagents to be the critical factor for the design of novel reagents with high efficiency. The novel, site-specifically activated peptide-based reagents were found to be efficient compounds for application in mass spectrometry-based discovery of novel vascular-accessible biomarkers.
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Sabrina A
SA
Hanke
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Alexander
A
Kerner
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Wiebke M
WM
Nadler
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Andreas
A
Trumpp
B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany.
Yixin
Y
Zhang
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address: christophroesli@gmail.com.
Christoph P
CP
Rösli
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D019921:Biotinylation / Q000379:methods*; D001808:Blood Vessels / Q000378:metabolism*; D015195:Drug Design; D006801:Humans; D007202:Indicators and Reagents / Q000138:chemical synthesis; D058990:Molecular Targeted Therapy; D040901:Proteomics / Q000379:methods*
D015415:Biomarkers; D007202:Indicators and Reagents
Biotin; In vivo biotinylation; Mass spectrometry; Proteomics; Technology; Vascular biomarker