Features of Peptide Fragmentation Spectra in Single-Cell Proteomics
Project description (from MassIVE):
SCP: This dataset contains 3 data types: trace samples consisting of 2ng and 0.2ng aliquots of HeLa protein digest standard, and single HeLa cells. Pierce™ HeLa protein digest standard and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA). Mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) were respectively prepared from LC-MS grade water and acetonitrile purchased from Honeywell (Charlotte, NC). The digest standard was reconstituted to a final concentration of 200 ng/µL with 100 µL of mobile phase A to form a stock solution. For the experiments, the stock samples were further diluted to 0.2 and 2 ng/µL using the same mobile phase. HeLa cells were cultured from cells purchased from American Type Culture Culture Collection (Manassas, VA). Single HeLa cells were prepared using the nanoPOTS workflow and analyzed by manual LC injection as described previously (29797682) except that cells were isolated into nanowells using the CellenONE platform (Lyon, France) instead of by fluorescence activated cell sorting. Columns: 30-µm-i.d. fused silica capillary columns from Polymicro (Phoenix, AZ) were packed with different materials: Jupiter C18 3.0 µm, 300 Å particles and Kinetex C18 core shell particles of 1.3 µm, 100 Å µm were purchased from Phenomenex (Torrance, CA); BEH C18, 1.7 µm, 130 Å was from Waters (Milford, MA). Column lengths were adjusted to keep the pressure and the linear velocity constant for all columns. The lengths were 50, 9 and 16 cm for Jupiter, Kinetex and BEH columns respectively. Solid-phase-extraction (SPE) columns were prepared by packing Jupiter C18 particles into 100-µm-i.d. × 5-cm-long fused silica capillaries. The file names contain the sample size and lc packing material. doi:10.25345/C5NV69
Bulk: This data was originally uploaded to pride project PXD011163. More details can be found there. Cells were lysed, reduced, and alkylated in lysis buffer (1% SDC, 10 mM TCEP, 40 mM CAA, and 100 mM TRIS, pH 8.0) supplemented with complete EDTA-free protease inhibitor mixture and phosSTOP phosphatase inhibitor mixture. Cells were heated for 5 min at 95 C, sonicated with a Bioruptor Plus, and diluted 1:10 with 50 mM ammonium bicarbonate, pH 8.0. Proteins were digested overnight at 37 C with trypsin and Lys-C (enzyme:substrate ratio of 1:50 and 1:75). SDC was precipitated by acidification to 5% of formic acid. Samples were desalted using Sep-Pak C18 cartridges and directly subjected to phosphopeptide enrichment. Samples for proteome analysis were instead dried down and stored at -80 C until nLC-MS analysis. Phosphopeptides enrichment was performed using Fe(III)-NTA in an automated fashion using the AssayMAP Bravo Platform. Reversed phase nLC-MS/MS analysis was performed with an Agilent 1290 Infinity UHPLC system coupled to an Orbitrap Q Exactive Plus mass spectrometer, or Orbitrap Fusion mass spectrometer for the phosphoproteome analysis. The UHPLC was equipped with a double frit trapping column (Reprosil C18, 3 um, 2 cm x 100 um) and a single frit analytical column (Poroshell EC-C18, 2.7 um, 50 cm x 75 um). Trapping was performed in solvent A (0.1% FA in water) at 5 uL/min, while for the elution the flow rate was passively split to 300 nL/min. The linear gradient was as follows: 13-40% solvent B (0.1% FA in 80% ACN) in 220 min, or 8-32% in 95 min for phosphopeptide analysis. Total analysis time was 235 min for the proteome samples and 110 min for the phosphoproteome samples. The mass spectrometers were operated in data-dependent mode. The Orbitrap Q Exactive Plus full-scan MS spectra from m/z 375-1600 were acquired at a resolution of 35000 (FWHM) after accumulation to a target value of 3e6. Up to 10 most intense precursor ions were selected for fragmentation, with the isolation window set to 1.5 m/z. HCD fragmentation was performed at normalized collision energy of 25% after the accumulation to a target value of 5e4. MS/MS was acquired at a resolution of 17500 (FWHM). The Orbitrap Fusion full-scan MS spectra from m/z 375-1500 were acquired at a resolution of 120000 (FWHM) after accumulation to a target value of 4e5. The most intense peptide ions fitting within a 3 s cycle were selected for HCD fragmentation, with the isolation window set to 1.6 m/z, and a normalized collision energy of 30%, after the accumulation to a target value of 5e4. MS/MS was acquired at a resolution of 30000 (FWHM). doi:10.25345/C5BN6F
The mzML files are downloaded from the MassIVE datasets MSV000087524 (doi:10.25345/C5NV69)(SCP), MSV000087689 (doi:10.25345/C5BN6F) (bulk) and redistruted here are:
D19_15um30cm_SC1.mzML
OR11_20160122_PG_HeLa_CVB3_CT_A.mzML
The identification files were created with the search engine Sage, and uploaded on zenodo: DOI:10.5281/zenodo.19370231 following the experiment's guidelines.
D19_15um30cm_SC1.sage.tsv
OR11_20160122_PG_HeLa_CVB3_CT_A.sage.tsv
Dataset license: CC0 1.0 Universal (CC0 1.0)
Boekweg, Hannah, Daisha Van Der Watt, Thy Truong, S. Madisyn Johnston, Amanda J. Guise, Edward D. Plowey, Ryan T. Kelly, and Samuel H. Payne. 2022. “Features of Peptide Fragmentation Spectra in Single-Cell Proteomics.” Journal of Proteome Research 21 (1): 182–88.