Search results for "data-independent acquisition"
showing 10 items of 11 documents
Label-Free Proteomics of Quantity-Limited Samples Using Ion Mobility-Assisted Data-Independent Acquisition Mass Spectrometry
2021
Over the past two decades, unbiased data-independent acquisition (DIA) approaches have gained increasing popularity in the bottom-up proteomics field. Here, we describe an ion mobility separation enhanced DIA workflow for large-scale label-free quantitative proteomics studies where starting material is limited. We set a special focus on the single pot solid-phase-enhanced sample preparation (SP3) protocol, which is well suited for the processing of quantity-limited samples.
Label-free profiling of skeletal muscle using high-definition mass spectrometry
2014
We report automated and time efficient (2 h per sample) profiling of muscle using ultra-performance liquid chromatography (LC) coupled directly with high-definition mass spectrometry (HDMSE). Soluble proteins extracted from rat gastrocnemius (n=10) were digested with trypsin and analysed in duplicate using a 90 min RPLC gradient. Protein identification and label-free quantitation were performed from HDMSE spectra analysed using TransOmics Informatics for Proteomics software. In total 1,514 proteins were identified. Of these, 811 had at least 3 unique peptides and were subsequently used to assess the dynamic range and precision of LC-HDMSE label-free profiling. Proteins analysed by LC-HDMSE …
In-depth evaluation of software tools for data-independent acquisition based label-free quantification.
2015
Label-free quantification (LFQ) based on data-independent acquisition workflows currently experiences increasing popularity. Several software tools have been recently published or are commercially available. The present study focuses on the evaluation of three different software packages (Progenesis, synapter, and ISOQuant) supporting ion mobility enhanced data-independent acquisition data. In order to benchmark the LFQ performance of the different tools, we generated two hybrid proteome samples of defined quantitative composition containing tryptically digested proteomes of three different species (mouse, yeast, Escherichia coli). This model dataset simulates complex biological samples con…
Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization o…
2021
Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in…
Suspect, non-target and target screening of emerging pollutants using data independent acquisition: Assessment of a Mediterranean River basin
2019
A single workflow based on three approaches (target, suspected and non-target screening) using liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) in data independent acquisition mode (DIA) was developed to assess the presence of emerging pollutants (EPs) in water and sediments from a Mediterranean River Basin. Identification of potential contaminants was based on mass accuracy, isotopic ratio pattern, theoretical fragmentation, and retention time using Waters UNIFI software. In the suspect screening against a library containing 2200 components, 68 contaminants were tentatively identified, 6 of which were confirmed and quantified with analytical standar…
Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry *
2015
Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS t…
Biomedical applications of ion mobility-enhanced data-independent acquisition-based label-free quantitative proteomics.
2014
Mass spectrometry-based proteomics greatly benefited from recent improvements in instrument performance and the development of bioinformatics solutions facilitating the high-throughput quantification of proteins in complex biological samples. In addition to quantification approaches using stable isotope labeling, label-free quantification has emerged as the method of choice for many laboratories. Over the last years, data-independent acquisition approaches have gained increasing popularity. The integration of ion mobility separation into commercial instruments enabled researchers to achieve deep proteome coverage from limiting sample amounts. Additionally, ion mobility provides a new dimens…
Data-independent acquisition strategies for quantitative proteomics
2013
In shotgun proteomics, data-dependent precursor acquisition (DDA) is widely used to profile protein components in complex samples. Although very popular, there are some inherent limitations to the DDA approach, such as irreproducible precursor ion selection, under-sampling and long instrument cycle times. Unbiased ‘data-independent acquisition’ (DIA) strategies try to overcome those limitations. In MSE, which is supported by Waters Q-TOF instrument platforms, such as the Synapt G2-S, a wide band pass filter is used for precursor selection. During acquisition, alternating MS scans are collected at low and high collision energy (CE), providing precursor and fragment ion information, respectiv…
In‐depth protein profiling of the postsynaptic density from mouse hippocampus using data‐independent acquisition proteomics
2014
Located at neuronal terminals, the postsynaptic density (PSD) is a highly complex network of cytoskeletal scaffolding and signaling proteins responsible for the transduction and modulation of glutamatergic signaling between neurons. Using ion-mobility enhanced data-independent label-free LC-MS/MS, we established a reference proteome of crude synaptosomes, synaptic junctions, and PSD derived from mouse hippocampus including TOP3-based absolute quantification values for identified proteins. The final dataset across all fractions comprised 49 491 peptides corresponding to 4558 protein groups. Of these, 2102 protein groups were identified in highly purified PSD in at least two biological replic…
LipidMS: An R Package for Lipid Annotation in Untargeted Liquid Chromatography-Data Independent Acquisition-Mass Spectrometry Lipidomics.
2018
High resolution LC-MS untargeted lipidomics using data independent acquisition (DIA) has the potential to increase lipidome coverage, as it enables the continuous and unbiased acquisition of all eluting ions. However, the loss of the link between the precursor and the product ions combined with the high dimensionality of DIA data sets hinder accurate feature annotation. Here, we present LipidMS, an R package aimed to confidently identify lipid species in untargeted LC-DIA-MS. To this end, LipidMS combines a coelution score, which links precursor and fragment ions with fragmentation and intensity rules. Depending on the MS evidence reached by the identification function survey, LipidMS provi…