krull2024.RdThey develop a new strategy for data-independent acquisition (DIA) that leverages the co-analysis of low-input samples alongside a corresponding enhancer (ME) of higher input. Using DIA-ME, they investigate the proteomic response of U-2 OS cells to interferon gamma (IFN-y) at the single-cell level.
krull2024A QFeatures object with 159 assays, each assay being a SingleCellExperiment object.
Assay 1-158: DIA-NN main output report table split for each acquisition run. First 15 run acquires 10 single cells (MEs) and, remaining 143 run acquires 1 single cell. It contains the results of the spectrum identification and quantification.
proteins: DIA-NN protein group matrix, containing normalised
quantities for 1553 protein groups in 143 single cells. Proteins
are filtered at (Q.Value <= 0.01), (Lib.Q.Value <= 0.01), and
(Lib.PG.Q.Value <= 0.01).
The colData(krull2024()) contains cell type annotations. The description
of the rowData fields for the different assays can be found in the
DIA-NN documentation.
The data were downloaded from PRIDE
repository
with accession ID PXD053464.
The data were acquired using the following setup. More information
can be found in the source article (see References).
Cell isolation: cells were detached with trypsin digestion, followed by dilution in 1.5 mL PBS, and isolated using BD FACSAria III instrument.
Sample preparation: Sorted single cells were collected in lysis buffer (50 mM TEAB, pH 8.5, and 0.025% DDM), denatured at 70 degrees Celsius for 30 minutes. Samples were acidified with 0.5% FA and transferred to auto sampler plates for mass spectrometry analysis.
Separation: Peptides were injected in a 2 microliter volume onto a (25 cm x 75 micrometer) ID column at a flow rate of 300 nL/min, separated using a gradient of ACN in water with 0.1% FA over 15 minutes, connected to a nano-ESI source.
Ionization: Ionization was performed using a 1,500 V capillary voltage with 3.0 L/min dry gas and a dry temperature of 180 degrees Celsius. MS data acquisition was conducted in diaPASEF mode using a timsTOF Pro mass spectrometer.
Mass spectrometry: MS1 scans covered a range of 200-1,700 m/z, while DIA window isolation targeted 475-1,000 m/z with eight DIA scans per cycle. Fragmentation was triggered by collision energy ranging from 45 eV to 27 eV depending on the ion mobility.
Data analysis: Data was processed using DIA-NN (v1.8.0) and Spectronaut 18 in a library-free approach, using deep learning for spectrum prediction, retention times, and ion mobility.
The data were collected from the PRIDE
repository
in the 03_SingleCell_Searches.zip file.
We loaded the DIA-NN main report table and generated a sample
annotation table based on the MS file names. We next combined the
sample annotation and the DIANN tables into a QFeatures object
following the scp data structure. We loaded the proteins group
matrix as a SingleCellExperiment object, and added the protein data
as a new assay and link the precursors to proteins using the
Protein.Group variable from the rowData.
Krull, K. K., Ali, S. A., & Krijgsveld, J. 2024. "Enhanced feature matching in single-cell proteomics characterizes IFN-γ response and co-existence of Cell States." Nature Communications, 15(1). Link to article
# \donttest{
krull2024()
#> see ?scpdata and browseVignettes('scpdata') for documentation
#> loading from cache
#> An instance of class QFeatures containing 159 set(s):
#> [1] D:\Data\KK\KK35\03_MEs\20230629_KK_10SC_control_01_A11_1_14185.d: SingleCellExperiment with 3521 rows and 1 columns
#> [2] D:\Data\KK\KK35\03_MEs\20230629_KK_10SC_control_02_B11_1_14186.d: SingleCellExperiment with 3349 rows and 1 columns
#> [3] D:\Data\KK\KK35\03_MEs\20230629_KK_10SC_control_03_C11_1_14188.d: SingleCellExperiment with 3277 rows and 1 columns
#> ...
#> [157] D:\Data\KK\KK35\20230630_KK_SC_IFNy_79_E10_1_14262.d: SingleCellExperiment with 1110 rows and 1 columns
#> [158] D:\Data\KK\KK35\20230630_KK_SC_IFNy_81_G10_1_14264.d: SingleCellExperiment with 1483 rows and 1 columns
#> [159] proteins: SingleCellExperiment with 1553 rows and 143 columns
# }