woo2022_macrophage: Woo et al. 2022 (Cell Syst.): LPS-treated macrophages

In UCLouvain-CBIO/scpdata: Single-Cell Proteomics Data Package

Woo et al. 2022 (Cell Syst.): LPS-treated macrophages

Description

Single-cell data from macrophages subjected to 3 LPS treatments. The data were acquired using the TIFF (transfer identification based on FAIMS filtering) acquisition method. The data contain 155 single cells: 54 control cells (no treatment), 52 cells treated with LPS during 24h and 49 cells treated with LPS during 49h.

Usage

woo2022_macrophage

Format

A QFeatures object with 5 assays, each assay being a SingleCellExperiment object:

• ⁠peptides_[intensity or LFQ]⁠: 2 assays containing peptide quantities or normalized quantities using the maxLFQ method as computed by MaxQuant.

• ⁠proteins_[intensity or iBAQ or LFQ]⁠: 3 assays containing protein quantities or normalized proteins using the iBAQ or maxLFQ methods as computed by MaxQuant.

Sample annotation is stored in colData(woo_macrophage()).

Acquisition protocol

The data were acquired using the following setup. More information can be found in the source article (see References).

• Sample isolation: cultured RAW 264.7 cells treated or not with 100 ng/ul LPS. The cells were sorted using the Influx II cell sorter and deposited on a nanoPOTS chip.

• Sample preparation: cells are lysed using using a DDM+DTT lysis and reduction buffer. The proteins are alkylated with IAA and digested with LysC and trypsin. Samples are then acidified with FA, vacuum dried and stored in freezer until data acquisition.

• Liquid chromatography: peptides are loaded using an in-house autosampler (Williams et al. 2020). The samples are concentrated through a SPE column (4cm x 100µm i.d. packed with 5µm C18) with microflow LC pump. The peptides are then eluted from an LC column (25cm x 50 µm i.d. packed with 1.7µm C18) from a 60 min gradient (100nL/min).

• Mass spectrometry: MS/MS was performed on an Orbitrap Fusion Lumos Tribrid MS with FAIMSpro coupled to a 2.4 kV ESI. FAIMS setup: 4-CV method (-45, -55, -65, -75 V). MS1 setup: resolution = 120.000, range = 350-1500 m/z,AGC target of 1E6, accumulation of 254ms. MS2 setup: 30% HCD, resolution AGC 2E4, accumulation of 254ms.

• Raw data processing: preprocessing using Maxquant v1.6.2.10 that use Andromeda search engine (with UniProtKB 2016-21-29). MBR was enabled.

Data collection

All data were collected from the MASSIVE repository (accession ID: MSV000085937).

The peptide and protein data were extracted from the peptides_RAW_LPS_scProteomics.txt or proteinGroups_RAW_LPS_scProteomics.txt files, respectively, in the RAW_LPS_SingleCellProteomics folders.

The tables were split so that intensities, maxLFQ, and iBAQ data are contained in separate tables. Tables are then converted to SingleCellExperiment objects. Sample annotations were inferred from the sample names. All data is combined in a QFeatures object. AssayLinks were stored between peptide assays and their corresponding proteins assays based on the leading razor protein (hence only unique peptides are linked to proteins).

The script to reproduce the QFeatures object is available at system.file("scripts", "make-data_woo2022_macrophage.R", package = "scpdata")

Source

The peptide and protein data can be downloaded from the MASSIVE repository MSV000085937

References

Source article: Woo, Jongmin, Geremy C. Clair, Sarah M. Williams, Song Feng, Chia-Feng Tsai, Ronald J. Moore, William B. Chrisler, et al. 2022. “Three-Dimensional Feature Matching Improves Coverage for Single-Cell Proteomics Based on Ion Mobility Filtering.” Cell Systems 13 (5): 426–34.e4. (link to article).

Examples

woo2022_macrophage()

UCLouvain-CBIO/scpdata documentation built on April 21, 2024, 12:33 a.m.