PNNL - Carrier-assisted LC-SRM

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Calibration 21:30:49101326681713
Processing reproducibility of 21:30:4910122466312
Intact MCF7 21:30:491824481501224
Carrier-assisted single-tube processing approach for targeted proteomics analysis of low numbers of mammalian cells

  • Organism: Homo sapiens
  • Instrument: TSQ Vantage
  • SpikeIn: Yes
  • Keywords: Carrier-assisted, cLC-SRM, sensitivity, single-cell proteomics, targeted proteomics
  • Lab head: Tujin Shi
Cell heterogeneity is inherent in multicellular organisms and their cellular processes. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of high-quality antibodies. Herein we report a simple, easily implemented, mass spectrometry-based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible quantification of most EGFR pathway proteins expressed at levels ≥30,000 and ≥3,000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells, and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.
Experiment Description
LC-SRM Analysis: The selection of surrogate peptides for EGFR pathway proteins and the SRM assays were described previously. High-purity light peptides (>95%) were used to calibrate crude heavy peptide concentrations. Samples were analyzed using a nanoACQUITY UPLC® (Waters Corporation, Milford, MA) coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). The UPLC s nanoACQUITY UPLC BEH 1.7 µm C18 column (75 µm i.d. × 20 cm), was connected to a chemically etched 20 μm i.d. fused silica electrospray emitter via a stainless metal union. Solvents used were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in 90% acetonitrile (mobile phase B). ~4 μL out of the total ~15 µL peptide sample was directly loaded onto the BEH C18 column from the RT-PCR tube without using a trapping column. Sample loading and separation were performed at a flow rate of 350 nL/min and 300 nL/min, respectively. The binary LC gradient was used: 5-20% B in 26 min, 20-25% B in 10 min, 25-40% B in 8 min, 40-95% B in 1 min and at 95% B for 7 min for a total of 52 min and the analytical column was re-equilibrated at 99.5% A for 8 min. The TSQ Vantage mass spectrometer was operated with ion spray voltages of 2,400 ± 100 V, a capillary offset voltage of 35 V, a skimmer offset voltage of -5 V and a capillary inlet temperature of 220° C. The tube lens voltages were obtained from automatic tuning and calibration without further optimization. The retention time scheduled SRM mode was applied for SRM data collection with the scan window of ≥6 mins. The cycle time was set to 1 s, and the dwell time for each transition was automatically adjusted depending on the number of transitions scanned at different retention time windows. A minimal dwell time 10 ms was used for each SRM transition. All the EGFR pathway proteins were simultaneously monitored in a single LC-SRM analysis. Data Analysis: Skyline software was used for all SRM data analysis. The raw data were initially imported into Skyline software for visualization of chromatograms of target peptides to determine the detectability of target peptides. For each peptide the best transition without matrix interference was used for precise quantification. Two criteria were used to determine the peak detection and integration: (1) same retention time and (2) approximately the same relative SRM peak intensity ratios across multiple transitions between endogenous (light) peptide and heavy peptide internal standards. Standard derivation (SD) and coefficient of variation (CV) were obtained from multiple technical or biological replicates. All the data were manually inspected to ensure correct peak detection and accurate integration. The signal-to-noise ratio (S/N) was calculated by the peak apex intensity over the highest background noise within a retention time region of ±15s for the target peptide. The limit of detection (LOD) and the limit of quantification (LOQ) were defined as the lowest concentration points of each target protein at which the S/N ratio of surrogate peptides was at least 3 and 7, respectively. For conservatively determining the LOQ values, in addition to the requirement of S/N ≥7, one additional criteria was applied: surrogate peptide response over different cell numbers must be within the linear dynamic range. All calibration curves were plotted using Microsoft Excel 2007. The RAW data from TSQ Vantage were loaded into Skyline software to display graphs of extracted ion chromatograms (XICs) of multiple transitions of target proteins monitored.
Sample Description
Cell Culture: The MCF7 breast cancer cell line was obtained from the American Type Culture Collection (Manassas, VA) and was grown as previously described. Briefly, MCF7 cells were maintained in 15 cm dishes in ATCC-formulated Eagle’s Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 0.01 mg/mL human recombinant insulin and a final concentration 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were grown at 37 °C with 95% O2 and 5% CO2. Cells were seeded into 15 cm culture plates, grown until near confluence. MCF7 Cell Lysates: MCF7 cells were rinsed twice with ice cold PBS and harvested in 1 mL ice cold. PBS containing 1% phosphatase inhibitor cocktail (Pierce, Rockford, IL) and 10 mM NaF (Sigma Aldrich, St. Louis, MO). Cells were centrifuged at 1500 rpm for 10 min at 4°C and excess PBS was carefully aspirated from cell pellet. Cell pellets were resuspended in ice-cold cell lysis buffer (250 mM HEPES, 8M Urea, 150 mM NaCl, 1% Triton X-100, pH 6.0) at a ratio of ~3:1 lysis buffer to cell pellet. Cell lysates were centrifuged at 14,000 rpm at 4 °C for 10 min and soluble protein fraction was retained. Protein concentrations were determined by the BCA assay (Pierce, Rockford, IL). MCF7 Cell Equivalents for Evaluation of Platform Performance: A stock of 1 µg/µL of bulk MCF7 cell lysates was diluted to different protein concentrations (0.5, 1, 2, 5, 10, 20 ng/µL equivalent to 5, 10, 20, 50, 100, 200 cells/µL) with ~10 ng/µL BSA in 25 mM NH4HCO3. The sample dilution was performed in the S. oneidensis-coated RT-PCR tubes. From each point, 1 µL was transferred to the new S. oneidensis-coated RT-PCR tube with the addition of 30 fmol crude heavy peptide standards. To evaluate the processing reproducibility, three processing replicates were performed with each containing 5 ng MCF7 cell lysates (equivalent to 50 cells) and 30 fmol crude peptide standards. Intact MCF7 Cells Isolated by Fluorescence-Activated Cell Sorting (FACS) A BD Influx flow cytometer (BD Biosciences, San Jose, CA) was used to deposit cells into the precoated RT-PCR tubes. Alignment into a Hard-Shell 96-Well PCR Plates (Bio Rad, Hercules, CA) was done using fluorescent beads (Spherotech, Lake Forest, IL) after which the coated RT-PCR tubes are placed into the plates for cell collection. For unstained MCF7 cells, forward and side scatter detectors were used for cell identification. Once sorting gates were established, cells were sorted into the RT-PCR tubes using the 1-Drop Single sort mode. After isolation of the desired number of cells into the RT-PCR tube, the isolated cells were immediately centrifuged at 1000 g for 10 min at 4 °C to keep the cells at the bottom of the tube to avoid potential cell loss. The RT-PCR tubes with the isolated cells were stored into -80 °C freezer until further analysis.
Created on 9/17/18, 9:31 PM