Abstract

Advanced Molecular Tools for Proteome Analyses

Masood Kamali-Moghaddam, Associated Professor, Uppsala University

The possibility to detect and analyze proteins in their biological environments with increased specificity and sensitivity will provide opportunities to use also very rare molecules as reliable biomarkers of diseases. We have recently developed a proximity ligation assay, where very high specificity and sensitivity of target molecule detection results from the requirement of multiple recognition events, combined with extremely high efficiency of signal detection due to amplification of DNA molecules that form in the detection reactions. A multiplex form of the assay allows parallel analyses of panels of proteins in minute amounts of samples, while other forms of the assay facilitates detection of high-order biological complexes. In proximity ligation assays, affinity probes, such as antibodies, are attached to oligonucleotides to form proximity probes. Once a target molecule is recognized by a set of proximity probes the DNA strands are connected to each other via enzymatic ligation to form a DNA template that can be amplified and quantified as the measure of the target molecule’s concentration. In the in situ PLA the PCR amplification step is replaced by rolling-circle-amplification. Here, we illustrate the application of PLA for screening and validation of protein biomarkers in different diseases such as cancer and autoimmune disorders, and the use of PLA for detection of high-order protein complexes ¬– such as microvesicles – as biomarkers for prostate cancer.