Project description:Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high throughput experiments. Most importantly, it enables results dependent acquisition (RDA) where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide- and glycan-moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks future development of RDA technology to transcend data acquisition.
2023-03-13 | PXD040716 | Pride
Project description:Early diagnosis of brain abscess using real-time electron microscopy and metagenomics.
| PRJEB40138 | ENA
Project description:Real time detection of AUFI pathogens using Nanopore sequencing: A feasibility and efficacy study
Project description:Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using Nextstrain.
| PRJEB36528 | ENA
Project description:EMG produced TPA metagenomics assembly of PRJEB36528 data set (Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using Nextstrain.).
Project description:In this project we want to compare ovarian cell cancer treated or non treated with cis-platinum. As acquisition strategy we have used a Real Time Search MS3 method in an Orbitrap Eclipse. The acquisition cycle began with an MS1 scanwhere the most intense ions were selected for fragmentation in the ion trap using CID. MS2 spectra were searched in real time with data acquisition using the sp-human database. MS2 spectra with an Xcorr greater than or equal to 1 and less than 10 ppm precursor mas error, triggered the submission of an MS3 spectrum to the instrument. MS3 spectrum, were collected using the multinotch MS3-based TMT method, in a way were ten MS2 fragment ions were captured in the MS3 precursor population using isolation waveforms with multiple frequency notches
Project description:P. aeruginosa isolates from keratitis and healthy conjunctival sacs were obtained. The transcriptome profile of P. aeruginosa was characterized by a high throughput RNA-seq strategy using the Illumina HiSeq 2500 platform. The DEGs were analyzed with DESeq and validated through quantitative real-time polymerase chain reaction (PCR) and with experimental mice.
Project description:In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and we present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. Here we show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.
2022-03-14 | PXD029320 | Pride
Project description:Rapid metagenomic identification of two major swine pathogens with real-time nanopore sequencing