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Ion mobility fractionation enhanced mass spectrometry acquisition method for single-cell proteomics

ABSTRACT: Development and evaluation of the TIFF (Transferring Identification based on FAIMS Fractionation) method by benchmarking with standard methods. Single HeLa cells, LPS-treated RAW cells, and non-depleted human lung dissociated single cells were analyzed as applications of the TIFF method for single cell proteomics.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Ying Zhu

PROVIDER: MSV000085937 | MassIVE | Mon Aug 10 12:54:00 BST 2020

REPOSITORIES: MassIVE

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Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering.

Woo Jongmin J Clair Geremy C GC Williams Sarah M SM Feng Song S Tsai Chia-Feng CF Moore Ronald J RJ Chrisler William B WB Smith Richard D RD Kelly Ryan T RT Paša-Tolić Ljiljana L Ansong Charles C Zhu Ying Y

Cell systems 20220316 5

Single-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label- ...[more]

PMID: 35298923

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Ion mobility fractionation coupled to PASEF for enhanced proteome coverage

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2022-08-12 | PXD033129 | Pride

Single-cell RNA profiling of living cells with LC-seq

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Fractionation RNAseq in conjunction with Illumina TRUseq method

Project description:Fractionation RNAseq in conjunction with Illumina TRUseq method

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Deep mutational scanning and mobility-based selection for 6 RNA targets

Project description:RNA mutations are known to change mobility in native gels. What is not known is if mobility can serve as an effective tool to separate structurally similar (structural homologs) from structurally destabilized variants in a deep-mutation library of an RNA. Here we defined the proportion of a mutant in the native band from native polyacrylamide gel electrophoresis (PAGE) as a native-mobility-fitness score. The fitness scores of single and double mutants allowed a unsupervised, RNA-specific analysis to detect key secondary and tertiary base pairs through covariational signals. Subsequent amplification of these signals and their use as restraints for folding led to not only high-accuracy secondary structures with the F1-score > 0.9, but also quality tertiary-structure models between 3.6 Å and 7.7 Å RMSD from their native structures for the best in top 5 models for 6 RNAs tested including two CASP 15 difficult targets. This MobiSeq method should provide a simple and effective method for inferring 2D and 3D structures and improving mechanistic understanding of all structured RNAs.

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Single cell RNA-seq and machine learning reveal novel subpopulations and regulatory sequences in low-grade inflammatory monocytes

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2021-01-29 | GSE160450 | GEO

Enhanced methylome sequencing by recovery of unsequenceable fragments

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Enhanced Lipidome Coverage in Shotgun Analyses by using Gas-Phase Fractionation.

Project description:A high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging. Graphical Abstract ᅟ.

| S-EPMC5061616 | biostudies-literature

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Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS-QTOF Instrument.

Project description:Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm2), followed by parallel accumulation-serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min gradient. This result corresponded to ∼19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single-range TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by the average and median coefficient of variation values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.

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