PNNL - Carrier-assisted targeted proteomics

Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicatesCalibration Curves
Detectability of EGFR pathway proteins (100 intact HMEC) by 15:59:17121326781213
SRM signal comparsion between 10 and 100 intact HMEC by 15:59:174482464
CalibCurv_HMEC-RAS (1-1000 cell equivalents) 15:59:171126101
cPRISM-SRM reproducubility in 10 and 20 cell 15:59:172241262
CalibCurv_HMEC-SHC1 (1-1000 cell equivalents) 15:59:17112681
CalibCurv_HMEC-KNRAS (1-1000 cell equivalents) 15:59:171126101
Entire cPRISM-SRM reproducibility (100 intact HMEC) 15:59:1755103045
cLC-SRM for evaluation of peptide recovery (1-20 cell equivalents) 15:59:17661238126
CalibCurv_HMEC-EGFR (1-1000 cell equivalents) 15:59:17112691
CalibCurv_HMEC-ADAM17 (1-1000 cell equivalents) 15:59:17112481
CalibCurv_HMEC-NRAS (1-1000 cell equivalents) 15:59:17112481
CalibCurv_HMEC-HRAS (1-1000 cell equivalents) 15:59:17112881
Facile carrier-assisted targeted mass spectrometric approach for proteomic analysis of low numbers of mammalian cells

  • Organism: Homo sapiens
  • Instrument: TSQ Vantage
  • SpikeIn: No
There is an unmet technical challenge for mass spectrometry (MS)-based proteomics analysis of single mammalian cells. Quantitative proteomic analysis of single cells has been exclusively achieved by antibody-based immunoassays but is limited by the availability of high-quality antibodies. Herein we report a facile targeted MS-based proteomics method, termed cPRISM-SRM (carrier-assisted high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected reaction monitoring), for reliable analysis of low numbers of mammalian cells. The method capitalizes on using ‘carrier protein’ to assist processing of low numbers of cells with minimal loss, high-resolution PRISM separation for target peptide enrichment, and sensitive SRM for protein quantification. We have demonstrated that cPRISM-SRM has sufficient sensitivity to quantify proteins expressed at ≥200,000 copies per cell at the single-cell level and ≥3,000 copies per cell in 100 mammalian cells. We envision that with further improvement cPRISM-SRM has the potential to move toward targeted MS-based single-cell proteomics.
Experiment Description
A small number of cells are isolated either by serial dilution or cell sorting and collected into a container with large amounts (~50 µg) of carrier proteins to prevent undesired sample loss. Commonly used digestion protocols are adopted for sample processing to generate tryptic peptides without any further modification. After digestion and sample cleanup heavy isotope-labeled internal standards are added to the peptide mixtures. Highly sensitive PRISM-SRM is then used for precise quantification of surrogate peptides from proteins of interest with reducing the significantly increased dynamic concentration range caused by the addition of carrier proteins. The freely-available open-source Skyline software is used for SRM data analysis.
Sample Description
HMEC cell equivalents. Typically, one mammalian cell can generate ~100 pg of the total peptides. A stock of 1 µg/µL of bulk HMEC cell digest was spiked into ~25 µg of BSA digest at peptide amounts of 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 ng, equivalent to 0, 1, 5, 10, 20, 50, 100, 200, 500, and 1000 HMEC cells, respectively (three replicates for 10 and 20 HMEC equivalents). Crude heavy peptide standards of EGFR pathway proteins were added to each sample with the final concentration of 2 fmol/µL. The final volume of each sample was 50 µL with BSA peptide concentration at ~0.5 µg/µL. Intact HMEC cells. A stock of ~104 cells/mL (= ~102 cells/10µL) was generated with a 1:100 dilution of 10 µL of ~1 million cells/mL (= ~104 cells/10µL). 1 and 10 µL of the stock HMEC cells (i.e., ~10 and 100 intact HMEC cells) were added to low-binding Eppendorf tubes preloaded with ~50 µg of carrier BSA, respectively, with the final volume of 50 µL for each sample. Thus, for each intact HMEC cells the same amount of carrier BSA was used to prevent sample loss. Standard urea digestion protocol was used for sample processing without further optimization. ~50% of peptide recovery was achieved for each sample (i.e., ~25 µg of total peptides).
Created on 4/17/18, 3:59 PM