MECHANISM OF ALKALI-INDUCED DECOMPOSITION OF 2-HYDROXY- AND 2-ALKOXYALKYLCOBALOXIMES.

MECHANISM OF ALKALI-INDUCED DECOMPOSITION OF 2-HYDROXY- AND 2-ALKOXYALKYLCOBALOXIMES

In Situ Detection of Protein Complexes and Modifications by Chemical Ligation Proximity Assay

Protein function is often regulated by protein–protein interactions and post-translational modifications. Detection of these important biological phenomena in fixed biological samples could serve as an invaluable tool in biomedical research, drug development, as well as clinical cancer diagnostics and prognostics. We report here a novel methodology which utilizes unique antibody bioconjugates capable of forming proximity induced chemical ligation to enable in situ detection of proximal targets in fixed biological samples. Using this new methodology, we demonstrate in situ visualization of various protein heterodimers/complexes and post-translational modifications such as phosphorylation and ubiquitination. This new method offers high specificity, sensitivity, flexibility, and ease of use. In addition, the assay preserves critical contextual and heterogeneity information on biomarkers in clinically relevant samples

Enzymatically Amplified Mass Tags for Tissue Mass Spectrometry Imaging

Tissue mass spectrometry imaging (MSI) is a rapidly developing technology which promises to provide biomarker molecular information within tissue context, which is an unmet medical need in the era of personalized medicine. However, challenges associated with tissue specimens as well as the MSI technical limitations have hindered the practical applications of this technology. We report here a mass tag based MSI method that combines the strength of signal amplification by immuno-enzymatic reactions with the superior detection characteristics of mass spectrometry to enable matrix-free MSI of protein biomarkers in formalin fixed paraffin embedded (FFPE) tissues. The method involves binding of the target protein with a primary antibody with high affinity and specificity, followed by binding with a secondary antibody–enzyme conjugate. Enzyme substrates suitable for mass spectrometry detection are locally deposited at the site of the target through enzymatically catalyzed transformation. The precipitates thus serve as mass tags to be detected in mass spectrometry to represent the target biomolecule in tissue. Two enzymes and various substrates have been identified and successfully used to demonstrate the feasibility of this novel MSI method to image protein targets in FFPE tissue samples

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates

Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays