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GNPS - Hela Single Cell QC kelly lab

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ryan Kelly

PROVIDER: MSV000087524 | GNPS | Wed May 26 09:31:00 BST 2021

REPOSITORIES: GNPS

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Project description:In the light of the ongoing single-cell revolution, scientific disciplines are combining forces with the ultimate goal to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, LC separation, MS/MS data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and FAIMS. As compared to the currently commercially available pillar array columns, this dedicated limited sample column has a reduced cross section (factor of 2) and micro pillar dimensions that have been further downscaled (also by a factor of 2; 2.5 µm instead of 5 µm diameter), resulting in improved chromatography at reduced void times. A dilution series ranging from 5 to 0.05 ng/µL was prepared using trace amounts of PEG in the sample solvent to reduce adsorptive effects in the autosampler vial, sample stability up to 24h after dilution was demonstrated. Comparative processing of the MS/MS data with different database search algorithms (with and without second search feature activated and with and without rescoring based on fragmentation pattern prediction) pointed out the benefits of using Sequest HT together with INFERYS when analyzing samples at a concentration below 1 ng/µL. On average (data from quadruplicate runs), we were able to successfully identify 2855 unique protein groups from just 1 ng of HeLa lysate and this using a 60 min non-linear LC solvent gradient at a flow rate of 250 nL/min, hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10 (2436 proteins identified from 10 ng of HeLa lysate in 2019). By successfully identifying 1486 and 2210 proteins (average values, quadruplicates) from as little as 250 and 500 pg of HeLa lysate respectively, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

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GNPS - Hela Single Cell QC kelly lab

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