petrosius2023_AstralAML.RdSingle cell proteomics data from FACS sorted cells from the OCI-AML8227 model. The dataset contains leukemic stem cells (LSC; CD34+, CD38-), progenitor cells (CD34+, CD38+), CD38+ blasts (CD34-, CD38+) and CD38- blasts (CD34-, CD38-). It contains quantitative information at PSM, peptide and protein levels. Data was acquired using an Orbitrap Astral mass spectrometer. Direct DIA analysis was performed with Spectronaut version 17.
petrosius2023_AstralAMLA QFeatures object with 217 assays, each assay being a SingleCellExperiment object:
Assays 1-215: PSM data from the Spectronaut PEPQuant file with LFQ quantities from the FG.MS1Quantity column.
peptides: Peptide data resulting from the PSM to peptide
aggregation the 215 PSM assays. Resulting peptide assays were
joined into a single assay.
proteins: Protein data from the Spectronaut PGQuant file with
LFQ quantities from the PG.Quantity column.
The colData(petrosius2023_AstralAML()) contains cell type annotation, batch
annotation and FACS data. The description of the rowData fields
can be found in the Spectronaut user manual.
The PSM data, protein data and sample annotations can be downloaded from the dataset 'Astral AML single-cell data from Petrosius et al. 2023 preprint' in the Dataverse.
The data were acquired using the following setup. More information can be found in the source article (see References).
Cell isolation: Cell sorting was done on a FACS Aria III or Aria II instrument, controlled by the DIVA software package and operated with a 100 μm nozzle. Cells were sorted at single-cell resolution, into a 384-well Eppendorf LoBind PCR plate containing 1 μL of lysis buffer.
Sample preparation Single-cell protein lysates were digested overnight at 37°C with 2 ng of Trypsin supplied in 1 μL of digestion buffer. Digestion was stopped by the addition of 1 μL 1% (v/v) trifluoroacetic acid (TFA). All liquid dispensing was done using an I-DOT One instrument.
Liquid chromatography: Chromatographic separation of peptides was conducted on a vanquish Neo UHPLC system connected to a 50 cm uPAC Neo Low-load and an EASY-spray. Autosampler and injection valves were configured to perform direct injections from a 384 well plate using a 25 uL injection loop on 11.8 min gradients.
Mass spectrometry: Acquisition was conducted with an Orbitrap Astral mass spectrometer operated in positive mode with the FAIMSPro interface compensation voltage set to −45 V. MS1 scans were acquired with the Orbitrap at a resolution of 120,000 and a scan range of 400 to 900 m/z with normalized automatic gain control (AGC) target of 300 % and maximum injection time of 246 ms. Data independent acquisition of MS2 spectra was performed in the Astral using loop control set to 0.7 seconds per cycle with varying isolation window widths and injection times. Fragmentation of precursor ions was performed using higher energy collisional dissociation (HCD) using a normalized collision energy (NCE) of 25 %. AGC target was set to 800 %.
Raw data processing: Raw files were processed using Spectronaut version 17. Direct DIA analysis was performed in pipeline mode. Pulsar searches were performed without fixed modifications. N-terminal acetylation and methionine oxidation were set as variable modifications. Quantification level was set to MS1 and the quantity type set to area under the curve.
The data were provided by the authors and is accessible at the Dataverse The dataset ('Astral AML single-cell data from Petrosius et al. 2023 preprint') contains the following files of interest:
20240201_130747_PEPQuant (Normal).tsv: the PSM level data
20240201_130747_PGQuant (Normal).tsv: the protein level data
index_map.csv: FACS data.
msRuns_overview.csv: Sample annotations.
We added the FACS data to the sample annotations in a single table.
Both annotations and PSM features tables are then combined in a
single QFeatures object using the scp::readSCP() function.
The peptide data were obtained by aggregation of the PSM data to the peptide level. All of the resulting peptides assays were joined into a single assays. Individual peptides assays were discarded.
The protein data were formatted from the 20240201_130747_PGQuant (Normal).tsv
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.
Note that the QFeatures object has not been further processed and has therefore not been normalized, log-transformed or batch-corrected.
Valdemaras Petrosius, Pedro Aragon-Fernandez, Tabiwang N. Arrey, Nil Üresin, Benjamin Furtwängler, Hamish Stewart, Eduard Denisov, Johannes Petzoldt, Amelia C. Peterson, Christian Hock, Eugen Damoc, Alexander Makarov, Vlad Zabrouskov, Bo T. Porse and Erwin M. Schoof. 2023. "Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics." biorxiv. https://doi.org/10.1101/2023.06.06.543943 DOI:10.1101/2023.06.06.543943
# \donttest{
petrosius2023_AstralAML()
#> see ?scpdata and browseVignettes('scpdata') for documentation
#> loading from cache
#> An instance of class QFeatures containing 217 assays:
#> [1] CD38_neg_1: SingleCellExperiment with 4282 rows and 1 columns
#> [2] CD38_neg_10: SingleCellExperiment with 3393 rows and 1 columns
#> [3] CD38_neg_11: SingleCellExperiment with 4530 rows and 1 columns
#> ...
#> [215] prog_9: SingleCellExperiment with 4560 rows and 1 columns
#> [216] peptides: SingleCellExperiment with 11549 rows and 215 columns
#> [217] proteins: SingleCellExperiment with 2904 rows and 215 columns
# }