Dataset Information

MS quantification of circulating CFP-10, p24 and VP40

ABSTRACT: Liquid chromatography coupled mass spectrometry (LC-MS) operating in targeted mass spectrometry (MS) including multiple reaction monitoring (MRM) mode is an accurate analytical method. Integrating with nanoelectrospray-tandem MS (NanoES-MS/MS) and immunoprecipitation (IP), it becomes a promising method in detecting serum biomarkers which are at extremely low concentrations, such as mycobacterium tuberculosis (Mtb) secreted 10 kDa culture filtrate antigen (CFP-10). We employed an optimum denaturing condition for trypsin digestion and IP of targeted peptides. The condition shows several advantages over commonly used one, 1) it prevents further dilution of sample, thus enables as-needed sample volume, 2) It ensures liberation of trace amount of antigen peptide under a cost-effective enzyme to substrate mass ratio, 3) It avoids time-consuming and unnecessary reduction of disulfide bonds, as some pathogen antigens don't contain disulfide bond. More importantly, optimum MRM transitions of the targeted peptide and standardized data processing and interpretation algorithm provide confirmative information of it in serum samples. By changing the denaturing buffer, addition of a clean-up step, selection of optimum MRM transitions and better interpreting MS signal, this workflow showed an improved accuracy in clinical samples.

ORGANISM(S): Ebola Virus Mycobacterium Tuberculosis Sudan Ebolavirus Human Immunodeficiency Virus 1

SUBMITTER: Qingbo Shu

PROVIDER: PXD021149 | panorama | Tue Nov 09 00:00:00 GMT 2021

REPOSITORIES: PanoramaPublic

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Publications

Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants.

Shu Qingbo Q Kenny Tara T Fan Jia J Lyon Christopher J CJ Cazares Lisa H LH Hu Tony Y TY

PLoS pathogens 20211108 11

Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled m ...[more]

PMID: 34748613

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Project description:Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validation and clinical translation of protein biomarkers discovered by global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of specific endogenous (Endo) peptides with those of stable isotope labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrants sensitive and precise measurements required for targeted MS assays. Compared to MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensate for different ion suppression among transitions of a target peptide. However, PRM still suffers different ion suppressions between Endo and SIL peptides due to poor spray stability as the Endo and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed as wideband PRM (WBPRM), designed for high-throughput targeted MS approach. WBPRM employs a wide isolation window for simultaneous fragmentations of both Endo and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of target peptides. Compared to PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and accuracy of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a short experiment time. Its adaptability to a MS method provided by the manufacture makes it a facile method to implement for MS based assays, particularly in complex biological samples, where the needs for higher accuracy, sensitivity and efficiency are paramount.

Project description:Purpose: We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens. Experimental design: Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias. Results: In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate â¤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue. Conclusions and clinical relevance: These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics. Affymetrix GeneChip Mouse Genome 430 2.0 microarrays were used to profile whole tissues from 5 different tissue types of 25 tumor-bearing and 25 control mice of the Her2/Neu breast cancer mouse model. The 5 tissues tested were from breast, liver, spleen, blood cell, and thymus. The tumor-bearing mice were bitransgenic for MMTV-rtTA/TetO-NeuNT, and the control mice were transgenic for TetO-NeuNT only. The control mice were age- and cage-matched to the tumor-bearing mice. All samples were lysed and total RNA isolated and amplified prior to hybridization.

Dataset Information

MS quantification of circulating CFP-10, p24 and VP40

Publications

Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants.

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