petrosius2023_mES.RdProfiling mouse embryonic stem cells across ground-state (m2i) and differentiation-permissive (m15) culture conditions. The data were acquired using orbitrap-based data-independent acquisition (DIA). The objective was to demonstrate the capability of their approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes, and highlighting differences in the expression of key metabolic enzymes in distinct cell subclusters.
petrosius2023_mESA QFeatures object with 605 assays, each assay being a SingleCellExperiment object:
Assay 1-603: PSM data acquired with an orbitrap-based data-independent acquisition (DIA) protocol, hence those assays contain single column that contains the quantitative information.
peptides: peptide data containing quantitative data for 9884
peptides and 603 single-cells.
proteins: protein data containing quantitative data for 4270
proteins and 603 single-cells.
Sample annotation is stored in colData(petrosius2023_mES()).
The peptide and protein data can be downloaded from the
Dataverse
The raw data and the quantification data can also be found in
the MassIVE repository MSV000092429:
ftp://MSV000092429@massive.ucsd.edu/.
The data were acquired using the following setup. More information
can be found in the source article (see References).
Sample isolation: Cell sorting was done on a Sony MA900 cell sorter using a 130 μm sorting chip. Cells were sorted at single-cell resolution, into a 384-well Eppendorf LoBind PCR plate (Eppendorf AG) containing 1 μL of lysis buffer.
Sample preparation: Single-cell protein lysates were digested with 2 ng of Trypsin (Sigma cat. Nr. T6567) supplied in 1 μL of digestion buffer (100mM TEAB pH 8.5, 1:5000 (v/v) benzonase (Sigma cat. Nr. E1014)). The digestion was carried out overnight at 37 °C, and subsequently acidified by the addition of 1 μL 1% (v/v) trifluoroacetic acid (TFA). All liquid dispensing was done using an I-DOT One instrument (Dispendix).
Liquid chromatography: The Evosep one liquid chromatography system was used for DIA isolation window survey and HRMS1-DIA experiments.The standard 31 min or 58min pre-defined Whisper gradients were used, where peptide elution is carried out with 100 nl/min flow rate. A 15 cm × 75 μm ID column (PepSep) with 1.9 μm C18 beads (Dr. Maisch, Germany) and a 10 μm ID silica electrospray emitter (PepSep) was used. Both LC systems were coupled online to an orbitrap Eclipse TribridMass Spectrometer (ThermoFisher Scientific) via an EasySpray ion source connected to a FAIMSPro device.
Mass spectrometry: The mass spectrometer was operated in positive mode with the FAIMSPro interface compensation voltage set to −45 V. MS1 scans were carried out at 120,000 resolution with an automatic gain control (AGC) of 300% and maximum injection time set to auto. For the DIA isolation window survey a scan range of 500–900 was used and 400–1000 rest of the experiments. Higher energy collisional dissociation (HCD) was used for precursor fragmentation with a normalized collision energy (NCE) of 33% and MS2 scan AGC target was set to 1000%.
Raw data processing: The mESC raw data files were processed with Spectronaut 17 and protein abundance tables exported and analyzed further with python.
The data were provided by the Author and is accessible at the Dataverse The folder ('20240205_111248_mESC_SNEcombine_m15-m2i/') contains the following files of interest:
20240205_111251_PEPQuant (Normal).tsv: the PSM level data
20240205_111251_Peptide Quant (Normal).tsv: the peptide level data
20240205_111251_PGQuant (Normal).tsv: the protein level data
The metadata were downloaded from the Zenodo repository.
sample_facs.csv: the metadata
We formatted the quantification table so that columns match with the
metadata. Then, both tables are then combined in a single
QFeatures object using the scp::readSCP() function.
The peptide data were formated to a SingleCellExperiment object and the
sample metadata were matched to the column names and stored in the colData.
The object is then added to the QFeatures object and the rows of the PSM
data are linked to the rows of the peptide data based on the peptide sequence
information through an AssayLink object.
The protein data were formated to a SingleCellExperiment object and
the sample metadata were matched to the column names and stored in the
colData. The object is then added to the QFeatures object and the rows
of the peptide data are linked to the rows of the protein data based on the
protein sequence information through an AssayLink object.
Source article: Petrosius, V., Aragon-Fernandez, P., Üresin, N. et al. "Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition." Nat Commun 14, 5910 (2023). (link to article).
# \donttest{
petrosius2023_mES()
#> see ?scpdata and browseVignettes('scpdata') for documentation
#> loading from cache
#> An instance of class QFeatures containing 605 assays:
#> [1] 20230421_EV_PAF_FAIMS_U3000_uPAC_wishDIA_plate1_scMESC_m15_1: SingleCellExperiment with 633 rows and 1 columns
#> [2] 20230421_EV_PAF_FAIMS_U3000_uPAC_wishDIA_plate1_scMESC_m15_10: SingleCellExperiment with 2951 rows and 1 columns
#> [3] 20230421_EV_PAF_FAIMS_U3000_uPAC_wishDIA_plate1_scMESC_m15_100: SingleCellExperiment with 918 rows and 1 columns
#> ...
#> [603] 20230421_EV_PAF_FAIMS_U3000_uPAC_wishDIA_plate1_scMESC_m2i_99: SingleCellExperiment with 3264 rows and 1 columns
#> [604] peptides: SingleCellExperiment with 9884 rows and 603 columns
#> [605] proteins: SingleCellExperiment with 4270 rows and 603 columns
# }