Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells

Reagents.

Tetrafluoroethylene (TFE), dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, formic acid, and BSA were obtained from Sigma-Aldrich (St. Louis, MO). Affymetrix/USB trypsin enzyme was purchased from Thermo Fisher Scientific (Rockford, IL). Synthetic crude heavy peptides labeled with ¹³C/¹⁵N on the C-terminal arginine or lysine and pure light peptides with >95% purity were purchased from Thermo Fisher Scientific (San Jose, CA).

Cell Culture.

The MCF7 breast cancer cell line was obtained from the American Type Culture Collection (Manassas, VA) and was grown as previously described.⁴⁴ 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 of 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 and grown until near confluence.

MCF7 Cell Lysates.

MCF7 cells were rinsed twice with ice-cold PBS and harvested in 1 mL of 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, 8 M 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).

RT-PCR Tube Pretreatment.

Reverse transcription polymerase chain reaction (RT-PCR) tubes (GeneMte) with 0.2 mL of volume were coated with Shewanella oneidensis lysate digests. The selection of cell lysate digests rather than cell lysates or standard proteins for coating the tube is because the absorption amount of proteins on the tube surface is hard to determine and the large amount absorption may have severe effects on sample digestion and SRM detection. The tubes were pretreated by the addition of 100 μL of S. oneidensis digests at a concentration of 0.2 μg/μL with incubation for overnight at room temperature. The pretreatment solution was removed, and the RT-PCR tube was rinsed three times with HPLC-grade water, and then air-dried in the fume hood. The dried RT-PCR tube was stored until further use. The coated nonmammalian peptides may fall off from the RT-PCR tube wall but should not affect SRM-based targeted quantification of mammalian peptides in terms of sensitivity and accuracy.

MCF7 Cell Equivalents for Optimization of Trypsin Amount and Denaturing Temperature.

A stock of 1 μg/μL of bulk MCF7 cell lysate was diluted to 10 ng/μL (equivalent to ~100 cells/μL) with ~10 ng/,μL BSA in 25 mM NH₄HCO₃. An amount of 1 μL of 10 ng/μL MCF7 cell lysates was added to the S. oneidensis-coated RT-PCR tube with the addition of 100 fmol of crude heavy peptide standards to optimize the single-tube TFE digestion method. To evaluate the impact of trypsin concentration on digestion efficiency, 10 ng of MCF7 cell lysates was digested at different trypsin concentrations (1, 2.5, 5, 10, 15, 20 ng/μL) with two processing replicates per each concentration. Furthermore, 10 ng MCF7 cell lysates were also used to evaluate digestion efficiency for three different denaturing temperatures (60 °C, 75 °C, 90 °C) with two processing replicates per each temperature.

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 of crude heavy peptide standards. To evaluate the processing reproducibility, three processing replicates were performed with each containing 5 ng of MCF7 cell lysates (equivalent to 50 cells) and 30 fmol of crude peptide standards.

Intact MCF7 Cells Isolated by Fluorescence-Activated Cell Sorting (FACS).

The cultured MCF7 cells were washed with PBS for one time and detached using 0.25% trypsin (Gibco, Grand Island, NY). To avoid cell clumping, after detaching they were dispersed into a single-cell suspension by passing three times through a 25 gauge needle. The cells were suspended in PBS, and then pelleted by centrifuging 5 min at 500g. This process was repeated for five times to remove the remaining PBS and trypsin. After that the cells were resuspended in PBS and passed through a 35 μm mesh cap (BD Biosciences, Canaan, CT) to remove large aggregates. 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 plate (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 1000g 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 in a −80 °C freezer until further analysis.

Optimized Single-Tube TFE Digestion Protocol.

For MCF7 cell lysates, starting with 1 μg/μL stock, different concentrations were generated by serial dilution with 10 ng/μL BSA in 25 mM NH₄HCO₃. A pipette at the volume range of 0.2–2 μL was used to accurately deliver the desired low volume smaller than 2 μL with pipetting up and down for three times. First, 1 μL of MCF7 cell lysates at the desired concentration was added to the coated RT-PCR tube together with 4 μL of 25 mM NH₄HCO₃ and 0.3 μL of 100 fmol/μL heavy peptide standards with centrifugation at 5000 rpm for 3 min. Amounts of 9 μL of 100% TFE and 0.6 μL of 50 mM DTT were then added with a final 60% TFE and 2 mM DTT. Samples were centrifuged at 5000 rpm for 5 min, and then gently mixed at 850 rpm for 3 min. The sample were then incubated at 90 °C for 1 h for protein denaturation using the RT-PCR thermocycler (Thermo Fisher Scientific) with the heated lid option followed by cooling to room temperature with centrifugation at 5000 rpm for 3 min. A total of 0.5 μL of 60 mM IAA was added to the RT-PCR tube for a final concentration of ~2 mM. The sample was then incubated in the dark at room temperature for 30 min with gentle shaking at 850 rpm. Prior to the addition of trypsin, the sample was concentrated to a volume of ~4 μL to reduce TFE volume for digestion using a SpeedVac concentrator. An amount of 9 μL of 25 mM NH₄HCO₃ was then added together with 2.25 μL of 100 ng/μL USB trypsin for a final concentration of ~15 ng/μL. The sample was gently mixed at 850 rpm for 3 min, and then incubated for overnight (~16 h) at 37 °C for digestion. After trypsin digestion 0.5 μL of 5% FA was added to stop the enzymatic reaction. The final sample was centrifuged at 14 000g for 1 h.

For FACS-isolated intact MCF7 cells, the only change was the first step in which 4 μL of 25 mM NH₄HCO₃, 1 μL of 10 ng/μL BSA in 25 mM NH₄HCO₃, and 0.3 μL of 100 fmol/μL crude heavy peptide standards (total 30 fmol) were added to the sample tube. Amounts of 9 μL of 100% TFE and 0.6 μL of 50 mM DTT were then added with the final 60% TFE and 2 mM DTT. The sample was centrifuged at 5000 rpm for 5 min, and then gently mixed at 850 rpm for 3 min. The sample was sonicated for 1 min, and then placed on ice for 1 min for a total of five cycles.

LC–SRM Analysis.

The selection of surrogate peptides for epidermal growth factor receptor (EGFR) pathway proteins and the SRM assays were described previously.^42,45 SRM assays for EGFR pathway proteins and the transitions selected are summarized in Tables S1 and S2. High-purity light peptides (>95%) were used to calibrate crude heavy peptide concentrations. The determined copy numbers per cell for EGFR pathway proteins across different human cell lines are listed in Table S3.

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). An amount of ~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 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 2400 ± 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 min. 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.⁴⁶ 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 ±15 s 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.

Optimization of Protein Digestion.

Starting with the standard TFE digestion protocol and using a ratio of trypsin to protein (w/w) of 1:50, 10 ng of MCF7 cell lysates was processed and digested in the coated RT-PCR tube with minimal sample dilution (total volume of ~15 μL) (Figure 1B). Following LC–SRM analysis, no SRM signal was detected, even for the most abundant PEBP1 protein. We attributed this to the very low enzyme and protein concentrations (~13 pg/μL for trypsin and ~667 pg/μL protein), which would result in a relatively low digestion efficiency as compared to standard solution digestion protocols (≥10 ng/μL for trypsin and ≥1 μg/μL protein).^49,50 Because of the limited protein concentrations that can be obtained from small numbers of mammalian cells, we felt it necessary to compensate by significantly increasing the trypsin concentration.

A series of trypsin concentrations at 1–20 ng/μL were evaluated using 10 ng of MCF7 cell lysates. Four surrogate peptides derived from three high-abundance proteins were measured by LC–SRM (Table S4A). As trypsin concentrations increased from 1 to 10 ng/μL, the abundance of all endogenous peptides (i.e., the L/H peak area ratio) increased significantly by at least 6-fold. Increasing trypsin concentration beyond 15 ng/μL produced variable results and no further significant increase in L/H ratio for any of the tested peptides (Figure 2A). Therefore, trypsin concentration at 15 ng/μL was selected for the single-tube TFE digestion method. The decreased abundance for peptide LVVVGAGGVGK at 20 ng/μL suggested that ion suppression was occurring, which was supported by global proteomics analysis (Figure S2). Global profiling of 10 ng of MCF7 cell lysate digests at different trypsin concentrations showed that a majority of the highly abundant peptides originated from trypsin autodigestion (Figure S2A). Importantly, elevated trypsin levels also appeared to act as an effective carrier to reduce absorption loss at both the protein and peptide levels because of the total amount of trypsin and its corresponding peptides. When compared to 10 ng of BSA carrier, the total amount of trypsin was ~225 ng (i.e., ~15 ng/μL with a total sample volume of ~15 μL).

Optimizing Denaturing Temperature.

Another important factor to affect digestion efficiency is protein denaturation. For standard TFE digestion protocols, proteins are incubated at 60 °C.^47,48 However, 60 °C did not appear to be adequate for denaturing the low amounts of proteins in our samples (Figure 2B). In an attempt to further improve trypsin digestion efficiency, three denaturing temperatures (60 °C, 75 °C, 90 °C) were evaluated at a trypsin concentration of 15 ng/μL. Protein denaturation was performed in a PCR thermocycler with a heated lid option. Comparative analysis of the L/H area ratios for the three temperatures showed that increasing denaturing temperature improved the digestion efficiency (Figure 2B). When compared to the standard 60 °C, 90 °C improved L/H area ratios by at least 7-fold. Thus, 90 °C was selected as the optimal denaturing temperature.

Sensitivity and Reproducibility of cLC–SRM.

The performance of cLC–SRM on small amounts of cell lysates was evaluated by targeted quantification of 10 EGFR pathway proteins spanning a wide dynamic concentration range (Table S4B). We used pooled MCF7 cell lysates to eliminate variations due to stochastic sampling of small numbers of intact cells. Different amounts of MCF7 cell lysates with 0.5–20 ng of total proteins (equivalent to 5–200 cells) were processed with the optimized single-tube TFE digestion method followed by LC–SRM analysis. Multiple SRM technical replicates were performed for each data point (i.e., three replicates for 0–1 ng and two replicates for 2–20 ng) (Table S5). The obtained LODs and LOQs were used to evaluate the detection sensitivity. As shown in Figure 3A, XICs clearly shows transitions for ATADDELSFK derived from GRB2 present at ~220 000 copies per MCF7 cell. cLC–SRM was able to reproducibly quantify endogenous ATADDELSFK down to five MCF7 cell equivalents with an S/N ratio of 14 and ~1800 zmol of quantification sensitivity (Table 1 and Table S6). Furthermore, moderate-abundance PTPN11 at ~54 000 copies per cell was detected in 10 cell equivalents with an S/N ratio of 6 and ~900 zmol of detection sensitivity (Table 1 and Table S6). Sensitivity was further confirmed by confident detection of multiple endogenous peptides from other low-abundance EGFR pathway proteins in 20 MCF7 cell equivalents (e.g., FYGAEIVSALDYLHSEK derived from AKT1 at ~4000 copies per cell with S/N ratio of 5 and ~120 zmol of detection sensitivity, and SFEDLTDHPVTR derived from ADAM17 at ~10 000 copies per cell with S/N ratio of 3 and ~300 zmol of detection sensitivity) (Table 1 and Table S6). The resultant calibration curves displayed excellent linearity over a wide dynamic range of MCF7 cell equivalents (Figure 3, parts B and C, and Figures S3-S11) with LOQs of ~5–10 cells for high-abundance proteins, ~10–20 cells for moderate-abundance proteins, and ~50–200 cells for low-abundance proteins (Table 1). All these results suggest that cLC–SRM can provide ~100–4000 zmol of detection sensitivity and ~300–7000 zmol of quantification sensitivity.

The median SRM technical CV for all target peptides across all the data points was ~9% (Table S5), consistent with the technical reproducibility of well-characterized LC–SRM with CV below 10%.^42,45,51-54 The overall reproducibility of cLC–SRM was evaluated by targeted analysis of three processing replicates for 12 surrogate peptides derived from low- to high-abundance EGFR pathway proteins in 50 MCF7 cell equivalents (Table S7). The processing CVs ranged from 1.4% to 28.7% with the median value of 11.8%, suggesting high processing reproducibility of cLC–SRM. As expected, low- and moderate-abundance proteins have higher CVs than high-abundance proteins, which can be largely attributed to variations in SRM detection sensitivity.

Quantification of EGFR Pathway Proteins in FACS-Isolated MCF7 Cells.

With the demonstration of technique robustness using cell lysates, we next applied cLC–SRM to measure EGFR pathway proteins in 10 and 100 intact MCF7 cells isolated by FACS. Using our previously established SRM assays⁴³ (Tables S1 and S2), 32 core EGFR pathway proteins and feedback regulators with a wide dynamic concentration range (Table S3) were measured using 10 and 100 intact MCF7 cells with two biological replicates per each to evaluate practical applicability. Three technical replicates per biological replicate were performed to evaluate the overall performance of cLC–SRM for quantitative analysis of intact cells (Table S8). cLC–SRM enabled detection of high- and moderate-abundance proteins in 10 intact MCF7 cells with the median CV of 13.5% (Figures 4 and S12, and Tables 2 and S8). Even low-abundance AKT1 at ~3700 copies per cell was also detected with the average S/N ratio of 7 (Table S9), consistent with cLC–SRM measurements of MCF7 cell lysates (Figure S11). When the cell number was increased to 100, the endogenous SRM signal of AKT1 peptide was increased accordingly with the average S/N ratio of 28 (Table S9). A majority of previously identified important EGFR pathway proteins (22 out of the total 32 proteins)⁴³ were reliably detected and quantified by cLC–SRM with the median CV of 12.7%, with the exception of extremely low-abundance negative feedback regulators and known low-expression EGFR protein in MCF7 cells (Figure S12 and Table 2). These results suggests that cLC–SRM can be used to study signaling pathways in small numbers of mammalian cells. Significantly, the endogenous peptide from low-abundance SOS1, present at only ~3000 copies per cell, was detected in 100 MCF7 cells with the average S/N ratio of 9 (Table S9), suggesting ~500 zmol of absolute sensitivity of cLC–SRM. This further confirms that cLC–SRM can provide ~100–1000 zmol of detection sensitivity. We also note that the detectability of endogenous peptides shows a strong positive correlation with the total number of protein molecules (Tables 2 and S8).