williams2020_tmt: Williams et al. 2020 (Anal. Chem.): 3 AML cell line

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

Williams et al. 2020 (Anal. Chem.): 3 AML cell line

Description

Single-cell label data from three acute myeloid leukemia cell line culture (MOLM-14, K562, CMK). The data were acquired using a TMT-based quantification protocole and the nanoPOTS technology. The objective was to demonstrate successful use of the new nanoPOTS autosampler presented in the source article. The samples contain either carrier (10 ng), reference (0.2ng), empty or single-cell samples..

Usage

williams2020_tmt

Format

A QFeatures object with 4 assays, each assay being a SingleCellExperiment object:

• ⁠peptides_[intensity or corrected]⁠: 2 assays containing peptide reporter ion intensities or corrected reporter ion intensities as computed by MaxQuant.

• ⁠proteins_[intensity or corrected]⁠: 2 assays containing protein reporter ion intensities or corrected reporter ion intensities as computed by MaxQuant.

Sample annotation is stored in colData(williams2020_tmt()).

Acquisition protocol

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

• Sample isolation: cultured MOLM-14, K562 or CMK cells were isolated using flow-cytometry based cell sorting and deposit on nanoPOTS microwells

• Sample preparation: cells are lysed using using a DDM lysis buffer. Proteins are digested with trypsin followed by TMT labelling and quanching with HA. The samples are then acidified with FA, pooled in a single samples (adding carrier and reference peptide mixtures), and dried until LC-MS/MS analysis.

• Liquid chromatography: peptides are loaded using the new autosampler described in the paper. Samples are loaded using a a homemade miniature syringe pump. The samples are then desalted and concentrated through a SPE column (4cm x 100µm i.d. packed with 5µm C18) with microflow LC pump. The peptides are then eluted from a long LC column (60cm x 50 µm i.d. packed with 3µm C18) coupled to a nanoflox LC pump at 150nL/mL (elution time is not expliceted).

• Mass spectrometry: MS/MS was performed on an Orbitrap Fusion Lumos Tribrid MS coupled to a 2kV ESI. MS1 setup: Orbitrap analyzer at resolution 120.000, AGC target of 1E6, accumulation of 246ms. MS2 setup: Orbitrap with HCD at resolution 120.000, AGC target of 1E6, accumulation of 246ms.

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

Data collection

All data were collected from the MASSIVE repository (accession ID: MSV000085230).

The peptide and protein data were extracted from the Peptides_AML_SingleCell.txt or ProteinGroups_AML_SingleCell.txt files, respectively, in the AML_SingleCell folders.

The tables were duplicated so that intensisities and corrected intensities are contained in separate tables. Tables are then converted to SingleCellExperiment objects. Sample annotations were inferred from the sample names, from table S2 and from the Experimental Section of the paper. 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_williams2020_tmt.R", package = "scpdata")

Source

The PSM and protein data can be downloaded from the MASSIVE repository MSV000085230.

References

Source article: Williams, Sarah M., Andrey V. Liyu, Chia-Feng Tsai, Ronald J. Moore, Daniel J. Orton, William B. Chrisler, Matthew J. Gaffrey, et al. 2020. “Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell Proteomics.” Analytical Chemistry 92 (15): 10588–96. (link to article).

Examples

williams2020_tmt()

UCLouvain-CBIO/scpdata documentation built on Oct. 29, 2024, 4:22 p.m.