Project description:TOP1cc-seq assay was performed for the quantitative detection of the transient DNA-protein covalent interactions upon acute stimulations.
Project description:The classification of haematolymphoid neoplasms has important clinical implications, yet current methods to determine cell lineage on formalin-fixed paraffin-embedded (FFPE) tissues are unsatisfactory, especially for neoplasms of NK- and T-cell lineages. Herein, we performed copy number analysis using the MIP platform on 86 cases, including T-, NK- and B-cell lymphomas, malignant cell lines and normal lymphocyte subsets. We demonstrate that the loss of T- and B-cell receptor gene loci correlates with cell lineage. The overall sensitivity of the assay in our cohort was 91% and 78%, while the specificity was 84% and 93%, for the corresponding antigen receptors in T- and B-cell samples, respectively. Fluorescence in situ hybridization showed no detectable translocation or deletion of the TCRA locus in samples showing loss of TCRA. Compared to PCR-based gene arrangement assay for lineage assignment, the MIP assay showed good reliability on FFPE samples, including cases more than 10 years old. In conclusion, the detection of the loss of the antigen receptor gene loci using the MIP array is a novel genetic marker of lymphoid cell lineage and a potentially valuable tool in the diagnosis and classification of lymphoid neoplasms especially of T- and NK-cell origin.
Project description:Botulinum neurotoxins (BoNTs) are a group of neurotoxins produced by Clostridium bacteria. Their effect on neuro-muscular connections through cleaving proteins of the SNARE complex results in blocking acetylcholine signal transduction. To prevent external intoxication by BoNT, analysis of potential sources of BoNT needs to be performed. Currently available methods target the presence of BoNTs molecules but only a fraction is screening real toxic activity. The FDA-approved mouse bioassay, based on exposing live mice to the potentially contaminated food, is the most common. Unfortunately, this assay is expensive, time consuming and ethically questionable. That is why there is a need for assays that can measure the activity of BoNTs enzymatically. We present an assay combining known EndoPep-MS assay with detection protein affinity chips manufactured by ion soft-landing technology. The toxic activity is indirectly determined by monitoring their endopeptidase activity as a change in molecular mass of cleaved substrate peptides exposed to BoNTs. Our modification utilizes a protein array modified by affinity molecules towards either the BoNTs or the substrate peptides. Both variants allow to perform the in-situ reaction and detection of substrate peptides by MALDI-ToF MS on the protein chip. This method demonstrated successful detection of active BoNT/A1 in both buffer and complex matrices, achieving a detection limit of 0.5 ng/mL.
Project description:There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected pa-tient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as RT-PCR are the gold standard, during the current pandemic the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers, and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detec-tion methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syn-cytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagno-sis of active infection and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of pro-teins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein we report a tar-geted mass spectrometry assay for the detection of SARS- CoV-2 spike protein and nucleoprotein in a relevant biologi-cal matrix. Recombinant full-length spike protein and nucleoprotein were digested and prototypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high resolution Orbitrap instrument. A spectral library, which con-tained 7 proteotypic peptides (4 from spike protein and 3 from nucleoprotein) and the top 3 to 4 transitionsMS2 spectra, was generated and evaluated. From the original spectral library, we selected 2 best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in-vitro de-rived mucus. The PRM assay provided a limit of detection (LOD) of ~200 attomoles and a limit of quantitation (LOQ) of ~ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2E5 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the pres-ence of SARS-CoV-2 in archived or recently collected biological fluids, in-vitro derived research materials, and wastewater samples