Ian Burbulis

Multiplex protein detection with DNA readout via mass spectrometry.

Multiplex protein quantification has been constrained by issues of assay specificity, sensitivity and throughput. This research presents a novel approach that overcomes these limitations using antibody-oligonucleotide conjugates for immuno-polymerase chain reaction (immuno-PCR) or proximity ligation, coupled with competitive PCR and MALDI-TOF mass spectrometry. Employing these combinations of technologies, we demonstrate multiplex detection and quantification of up to eight proteins, spanning wide dynamic ranges from femtomolar concentrations, using only microliter sample volumes.

Reagents for investigating MAPK signalling in model yeast species

Here we present a set of resources (bacterial expression plasmids and antibodies) for the interrogation of proteins involved in yeast MAPK signalling. We constructed bacterial protein expression plasmids for 25 proteins involved in MAPK signalling in budding yeast. From these constructs we expressed and purified proteins and generated rabbit polyclonal antibodies against 13 proteins in the pheromone MAPK pathway. We verified the specificity of the antibodies and employed them to follow pathway proteins in cells stimulated with pheromone. We show that these reagents can be used to detect pheromone‐induced post‐translational modifications and changes in the oligomeric state of pathway proteins. In addition to recognizing their target proteins in Saccharomyces cerevisiae, these antibodies allow the detection of predicted orthologues in the distant evolutionary relatives Kluyveromyces lactis and Schizosaccharomyces pombe. These antibodies are new tools for investigating MAPK signalling in model yeast species and may be useful for studying MAPK signalling in higher eukaryotes. Copyright

Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

ABSTRACT Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.