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:ATRA-induced differentiation of HL-60 cells was studied using targeted mass-spectrometry including selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) approach. PRM experiment was performed in time-course manner, without peptide standards usage. PRM data was inspected in Skyline 3.1 software. In order to check peptide identity we developed spectrum library based on shotgun mass-spectrometry data, which has been obtained for HL-60 cells protein samples at 0, 3, 24, 48 and 96h after ATRA treatment.
Project description:To examine the properties of 14N and 15N labeled peptides using Parallel Reaction Monitoring (PRM) on a high resolution, high accuracy mass spectrometer we first set up this DDA experiment. The purpose of this experiment was to choose peptides that are good candidates for targeted quantification. The retention times, mass, and charge from 42 candidate peptides were acquired for targeted quantification.
Project description:TARGETED PROTEOMICS FOR THE DETECTION OF SARS-COV-2 PROTEINS: SARS-CoV-2, COVID-19, proteomics, targeted mass spectrometry, LC-MS, parallel reaction monitoring (PRM), limit of detection
Project description:TARGETED PROTEOMICS FOR THE DETECTION OF SARS-COV-2 PROTEINS: SARS-CoV-2, COVID-19, proteomics, targeted mass spectrometry, LC-MS, parallel reaction monitoring (PRM), limit of detection
Project description:Comprehensive LC-PRM-MS approach where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system to study the microheterogeneity of serum haptoglobin (Hp) extracted from 15 patients with cirrhosis and 15 with hepatocellular carcinoma (HCC).
Project description:Raw264.7 cells were cultured in serum-free DMEM containing DMSO or DMXAA for 4 h, and the conditioned media were collected. The precipitated proteins were directly digested with trypsin/Lys-C mix. Three biological replicates of the samples were individually prepared and analyzed by targeted LC-MS/MS using parallel reaction monitoring (PRM).
Project description:In this study, targeted parallel reaction monitoring assays for phosphopeptides were configured by mining data from large-scale experiments. The capabilities of the method were assessed by performing several benchmarking experiments. The accuracy of retention time prediction using a large-scale database was assessed by using BSA peptides or human phosphopeptides to predict the retention time of distinct peptides in a subsequent run. Data-driven phosphopeptide sequence and charge state was compared to heuristics-based selection using unscheduled PRM assays. Phosphoproteome analysis was performed in technical quadruplicate using parallel reaction monitoring, data-independent acquisition, and data-dependent acquisition. A label-free quantitative parallel reaction monitoring experiment was performed on phosphopeptides enriched from cells stimulated -/+ IGF-1 (n=6).
Project description:Development, implementation, and evaluation of a new data acquisition scheme called internal standard triggered-parallel reaction monitoring (IS-PRM) to increase the scale of targeted quantitative experiments while retaining high detection and quantification performance. All the details about the dataset, the associated sample preparation and liquid chromatography coupled to tandem mass spectrometry methods, and the data processing procedures are provided in the manuscript by Gallien et al., entitled "Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring", Molecular and Cellular Proteomics.
Project description:Development, implementation, and evaluation of a new data acquisition scheme called internal standard triggered-parallel reaction monitoring (IS-PRM) to increase the scale of targeted quantitative experiments while retaining high detection and quantification performance. All the details about the dataset, the associated sample preparation and liquid chromatography coupled to tandem mass spectrometry methods, and the data processing procedures are provided in the manuscript by Gallien et al., entitled "Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring", Molecular and Cellular Proteomics.