cong2020AC.RdSingle-cell proteomics using the nanoPOTS sample processing device in combination with ultranarrow-bore (20um i.d.) packed-column LC separations and the Orbitrap Eclipse Tribrid MS. The dataset contains label-free quantitative information at PSM, peptide and protein level. The samples are single Hela cells. Bulk samples (100 and 20 cells) were also included in the experiment to increase the idendtification rate thanks to between-run matching (cf MaxQuant).
cong2020ACA QFeatures object with 9 assays, each assay being a SingleCellExperiment object:
100/20 HeLa cells: 2 assays containing PSM data for a bulk
of 100 or 20 HeLa cells, respectively.
Blank: assay containing the PSM data for a blank sample
Single cell X: 4 assays containing PSM data for a single cell.
The X indicates the replicate number.
peptides: quantitative data for 12590 peptides in 7 samples
(all runs combined).
proteins: quantitative data for 1801 proteins in 7 samples
(all runs combined).
Sample annotation is stored in colData(cong2020AC()).
All files can be downloaded from the PRIDE repository PXD016921. The source link is: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/02/PXD016921
The data were acquired using the following setup. More information
can be found in the source article (see References).
Cell isolation: The HeLa cells were diluted and aspired using a microcapillary with a pulled tip.
Sample preparation performed using the nanoPOTs device. Protein extraction using RapiGest (+ DTT) + alkylation (IAA) + Lys-C digestion + cleave RapiGest (formic acid)
Separation: UltiMate 3000 RSLCnano pump with a home-packed nanoLC column (60cm x 20um i.d.; approx. 20 nL/min)
Ionization: ESI (2,000V; Nanospray Flex)
Mass spectrometry: Thermo Fisher Orbitrap Fusion Eclipse. MS1 settings: accumulation time = 246ms; resolution = 120,000; AGC = 1E6. MS/MS settings depend on quantity. All: AGC = 1E5. 20-100 cels: accumulation time = 246ms; resolution = 120,000. Single cells: accumulation time = 500ms; resolution = 240,000.
Data analysis: MaxQuant (v1.6.3.3) + Excel
The PSM, peptide and protein data were collected from the PRIDE
repository (accession ID: PXD016921). We downloaded the
evidence.txt file containing the PSM identification and
quantification results. The sample annotation was inferred from
the samples names. The data were then converted to a QFeatures
object using the scp::readSCP() function.
The peptide data were processed similarly from the peptides.txt
file. The quantitative column names were adpated to match the PSM
data. The peptide data were added to QFeatures object and link
between the features were stored.
The protein data were similarly processed from the
proteinGroups.txt file. The quantitative column names were
adapted to match the PSM data. The peptide data were added to
QFeatures object and link between the features were stored.
Cong, Yongzheng, Yiran Liang, Khatereh Motamedchaboki, Romain Huguet, Thy Truong, Rui Zhao, Yufeng Shen, Daniel Lopez-Ferrer, Ying Zhu, and Ryan T. Kelly. 2020. “Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry.” Analytical Chemistry, January. (link to article).
# \donttest{
cong2020AC()
#> see ?scpdata and browseVignettes('scpdata') for documentation
#> loading from cache
#> An instance of class QFeatures containing 9 assays:
#> [1] 100 HeLa cells: SingleCellExperiment with 11487 rows and 1 columns
#> [2] 20 HeLa cells: SingleCellExperiment with 9313 rows and 1 columns
#> [3] Blank: SingleCellExperiment with 136 rows and 1 columns
#> ...
#> [7] Single cell 4: SingleCellExperiment with 6771 rows and 1 columns
#> [8] peptides: SingleCellExperiment with 12590 rows and 7 columns
#> [9] proteins: SingleCellExperiment with 1801 rows and 7 columns
# }