| Metadata |
| datasetIdentifier | PASS01587 |
| datasetType | SWATH |
| submitter | Harshi Weerakoon <harshitw83@yahoo.com> |
| submitter_organization | QIMR Berghofer Medical Research Institute |
| lab_head_full_name | Associate Professor Michelle M. Hill |
| lab_head_email | michelle.hill@qimrberghofer.edu.au |
| lab_head_organization | QIMR Berghofer Medical Research Institute |
| lab_head_country | Australia |
| datasetTag | HumanTcellLibrary |
| datasetTitle | A primary human T-cell spectral library to facilitate large scale quantitative T-cell proteomics |
| publicReleaseDate | 2020-10-19 00:00:00 |
| finalizedDate | 2020-10-19 03:12:40 |
| summary | Data independent analysis (DIA) exemplified by Sequential window acquisition of all theoretical mass spectra (SWATH-MS) provides robust quantitative proteomics data, but applications to human T-cell studies are limited by the lack of a primary human T-cell spectral library. To address this resource gap, we generated a high-quality spectral library containing data for 4,833 distinct proteins from human T-cells across genetically unrelated donors. This library covers ~24% proteins in UniProt/SwissProt human reviewed proteome. SWATH-MS analysis of 18 primary T-cell samples using the new human T-cell spectral library reliably identified and quantified 2,850 proteins at 1% false discovery rate (FDR), whereas the larger Pan-human spectral library identified and quantified 2,794 T-cell proteins, only 38% overlap with our data. Combining the two libraries resulted in quantification of 4,078 proteins in the SWATH-MS application of above primary human T-cells. Collectively, this large data archive will be a useful as a public resource for basic and clinical proteomic studies when analyzing human T-cells, adds to the public Pan-human spectral library and highlights the differences when comparing cell lines versus sorted primary human T immune cells. |
| contributors | Harshi Weerakoon
Jeremy Potriquet
Alok Shah
Sarah Reed
Buddhika Jayakody
Charu Kapil
Mukul, K. Midha
Robert L. Moritz
Ailin Lepletier
Jason Mulvenna
John J. Miles
Michelle M. Hill
|
| publication | Manuscript in preparation |
| growth | CD3/28 Dynabeads (Thermo Fisher scientific, USA) at 1:1 cell to bead ratio was used to obtain activated T-cells in which in vitro expansion was done for 7 days at 370C in a humidified, 5% CO2 incubator. |
| treatment | |
| extraction | A buffer composed of 100 mM Thriethylammonium bicarbonate (TEAB), 1% sodium dodecyl sulfate (SDS), and 5mM MgCl2 (Sigma, USA) supplemented with 1x Roche complete protease inhibitors was used in the cell lysis, while Dithiothreitol (DTT) and Indole-3-acetic acid (IAA) were used for reduction and alkylation respectively. The detergent removal was performed by the filter aided sample preparation method in which 10 ml, 10 KDa Amicon molecular weight cutoff filter tubes (Merk Millipore, USA) were used. |
| separation | Peptides were fractionated in Agilent 3100 OFFGEL fractionator (Agilent technologies, USA) using a 24 cm, pH 3-10 IPG strip (GE Health Care, USA) |
| digestion | To digest purified proteins sequencing grade modified trypsin (Sigma, USA) at protein to trypsin ratio of 50:1 was added and incubated overnight at 370C in a humidified incubator |
| acquisition | Prior to LC-MS/MS analysis, all the samples were spiked with the indexed retention time (iRT) peptides (1:100) (Biognosys, Switzerland). Peptide samples were chromatographically separated by a 10 uL injection on an Eksigent Nano Ultra 1D+ system (AB Sciex, USA) using a 15 cm long ChromXP C18-CL column (particle size 3 µm, 120 Å, 150mm x 0.075mm). A pre-concentration step (10 min) was performed employing a Chromxp trap (C18-CL, 5 µm, 120 Å, 0.3 x 10 mm) before commencement of the gradient. A flow rate of 500 nL/ min was used for all experiments. The mobile phase consisted of solvent A (0.1% formic acid) and solvent B (100 acetonitrile/0.1% formic acid) were used for the three consecutive linear gradients (90 min in total) for peptide elution: 5-10% solvent B (acetonitrile/0.1% formic acid) over 2 min, 10-40% solvent B over 58 min and 40-50% solvent B over 5 min. A final gradient from 50% to 95% solvent B in 10 min was used to clean the column. Eluates from the RP-HPLC column were directly introduced into the NanoSpray II ionisation source of a TripleTOF 5600 MS/MS System (AB Sciex, USA) operated in positive ion electrospray mode. The peptides and the DDA-MS acquisition protocol was created using Analyst 1.5.1 software (AB Sciex, USA) in which a 300 – 2000 (m/z) precursor mass range with 250 ms accumulation time and 100–2000 (m/z) product mass range with 100 ms accumulation time were selected in all the analysis. Ions observed in the TOF-MS scan exceeding a threshold of 50 counts and a charge state of +2 to +4 were set to trigger the acquisition of product ion spectra for a maximum of 10 of the most intense ions. |
| informatics | Trans-proteomic pipeline (TPP) was used to generate a spectral library from acquired DDA-MS data. A brief description of the protocol is given below.
DDA-MS data were first converted into mzML file format (msconvert, ProteoWizard) and each data set was searched using X!Tandem with k-score plugin and Comet against UniProtKB/Swiss-Prot human reviewed database with added decoy database and iRT sequence. PeptideProphet and iProphet were used to score and combine the results while MAYU was used to calculate FDR. Data were filtered through the iProphet probability cut-off corresponding to protein FDR at 1%. spectraST was used to generate individual spectral libraries normalised to iRT peptides. After building a consensus spectral library by combining individual libraries, Spectrast2tsv (msproteomicstools 0.8.0) was used to generate the final assay library for primary human T-cells. |
| instruments | AB SCIEX TripleTOF 5600 |
| species | Human |
| massModifications | Static: C+57.021464
Variable: M+15.994915 |
