The authors have declared that no competing interests exist.
Conceived and designed the experiments: TYY CLE MCH LLP. Performed the experiments: TYY. Analyzed the data: TYY LLP. Contributed reagents/materials/analysis tools: TYY CLE MCH LLP. Wrote the paper: TYY CLE MCH LLP.
The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.
Kinase inhibitor drugs represent an approximately $10 billion market in the pharmaceutical industry, and this is anticipated to expand even further over the coming decade.
A relevant companion diagnostic for kinase inhibitor pharmacodynamics needs to measure enzymatic activity, and thus substrate phosphorylation, for a targeted kinase. Protein and peptide phosphorylation by kinases has traditionally been detected using 32 P-radiolabeled ATP or antibody-based methods (such as ELISA and Western blot). These methods are reliable and well-characterized, but often are limited by concerns over waste generation (radiolabeled assays) or the requirement for phosphosite-specific antibodies, which are not always available at the scales necessary for clinical tests at a reasonable cost. To overcome these limitations, several approaches (e.g. microarray, bead-based and targeted mass spectrometry (MS) methods) have been described that detect kinase activity from relatively small amounts of cell lysate (down to ∼10 µg).
Genetically-engineered Förster resonance energy transfer (FRET) protein constructs for detecting Bcr-Abl activity in intact cells have been reported, however transfecting patient cells with sensor constructs for clinical assays is very challenging and not practical for a typical clinical laboratory. We previously reported a cell-permeable peptide biosensor for Abl kinase and its application for detecting DNA damage-related Abl kinase activation in intact cells (
The ‘reporter’ sequence, called ‘Abltide,’
Fmoc-protected amino acid monomers were purchased from Peptides International (Louisville, KY, USA). Fmoc–biotinylated lysine was obtained from Akaal Organics (Long Beach, CA, USA). The photocleavable residue (3-(2-nitrophenyl)-3-aminopropionic acid) was obtained from Lancaster Synthesis and Fmoc-protected by J. Thomas Ippoliti’s lab at the University of St. Thomas (St. Paul, MN). Other reagents were purchased from Sigma-Aldrich if not specified.
K562 cells were obtained from ATCC (Rockville, MD). Cells were routinely maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine in a 5% CO2 humidified environment at 37°C.
Peptides were synthesized via solid phase Fmoc chemistry on 50 µmol CLEAR-Amide resin using a Prelude Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with a double coupling cycle (22 min deprotection with 20% piperidine in DMF, 6×1 ml DMF wash, 2×10 min coupling with 100 mM amino acid, 90 mM HCTU/0.4 M NMM in DMF, 3×1 mL DMF wash). After synthesis, peptides were deprotected and cleaved using TFA/Water/EDT/TIS 94.5%/2.5%/2.5%/1%. After cleavage, peptides were purified to at least 90% purity using an Agilent Technologies 1200 Series HPLC system (Santa Clara, CA, USA) with a C18 reverse phase column (1×25 cm). Characterization of peptide identity and purity was performed by LC/MS (Accela/LTQ, Thermo Finnigan) with a Hypersil GOLD column (2.1×50 mm) and MALDI-TOF/TOF MS (Voyager 4800, Applied Biosystems, Foster City, CA, USA). Peptides were lyophilized and stored at −20°C before use.
Three independent replicate experiments were performed for the time course with Western blot detection. Three side-by-side replicate experiments with just one time point (5 min) were performed for the MRM analysis. K562 cells were cultured to log phase growth and seeded to 5×106 cells/ml in a six-well plate (3 mL per well). When necessary, cells were pre-incubated with imatinib (10 µM) for 1 h at 37°C followed by incubation with three treatments: 25 µM peptide (dissolved in PBS), 25 µM peptide +10 µM imatinib, and 25 µM peptide +1 µM pervanadate (prepared by reacting a solution of sodium orthovanadate with H2O2, followed by heating at 95°C to degrade excess H2O2). At the indicated time points (5, 30, 60 min), aliquots (1 mL) were collected and centrifuged (2200 rcf, 1 min, 4°C) to remove excess media. To collect any remaining cells in the wells from the final aliquot, all wells were washed with phosphate buffered saline (PBS, 400 µl) and these washes were combined with the collected cells. Cells were suspended in PBS (1 ml) to wash away excess peptide, centrifuged again (2200 rcf, 1 min, 4°C), and lysed using Phosphosafe Extraction Reagent (Novagen) supplemented with EDTA and protease inhibitor cocktail (Roche). Cells were immediately flash-frozen in liquid nitrogen, thawed on ice for 15 min, vortex mixed, and centrifuged to clarify (16,000 rcf, 15 min, 4°C). The supernatant was collected, measured for total protein concentration using the BCA assay (ThermoFisher Pierce, Rockford, IL), flash frozen again and stored at −80°C until use.
Biosensor peptide from samples generated as described above (in the Cell-based biosensor assay section) was captured using streptavidin-coated MagneSpheres (Promega Corporation, Madison, WI). The beads (20 µl) were prepared by washing with 0.1% Octyl-β-glucoside/PBS (3×150 µl). K562 cell lysates (200 µg total protein) were incubated with the beads on a shaker (600 rpm, 60 min). Beads were captured using a MagnaBot 96-well magnetic capture device (Promega) and washed with 0.1% Octyl-β-glucoside/PBS (3×150 µl) and deionized water (3×150 µl). Peptide was eluted using 15 µL sample buffer (ACN/H2O/TFA, 50%/50%/0.1%). 0.5 µL from each sample was co-spotted with α-cyano-4-hydroxycinnamic acid (10% w/v) containing ammonium dihydrogen phosphate (5 mg/ml),
Samples of equal protein content (100 µg/lane) were diluted into Laemmli buffer and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membrane and analyzed by Western blotting. Membranes were split at the 15 kD mark and blocked in 3% milk in TBS-T overnight at 4°C, followed by blotting with the indicated antibodies in 3% milk/TBS-T. The bottom membrane was blotted with: DyLight-649 labeled Streptavidin (1∶1000, ThermoFisher Pierce) to detect total biosensor; 4G10 α-phosphotyrosine antibody to detect the phosphorylated biosensor. Upper section of the membrane was blotted with: α-phospho-Abl (Y245) (1∶1000, Cell Signaling), α-phospho-STAT5 (Y694) (1∶5000, Abcam) and α-phospho-CrkL (Y207) (1∶1000, Abcam) to detect phosphorylation of endogenous sites in the Bcr-Abl signaling pathway. α-β-tubulin (1∶100,000, Millipore) was used as a loading control. Blots were incubated with IR-dye-labeled secondary antibodies (Rockland Immunochemical) in 3% milk/TBS-T (1∶10,000). Signals of immunoblots were visualized using the Odyssey system (LiCOR Biosciences, Lincoln, NE), quantified using densitometry with Quantity One (Bio-Rad), and analyzed with GraphPad Prism software.
Aliquots (18 µg each) of cell lysate samples were processed to separate proteins from lipids.
Tryptic peptides were separated on a nano-LC/MS system which included Agilent 1100 Series capillary and nano flow pumps, micro-well plate sampler with thermostat, and Chip Cube MS interface on the Agilent 6410 Triple Quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA). The peptides were loaded at 3 µl/min on an Agilent chip containing a 40 nl enrichment column packed with Zorbax 300SB-C18 5 µm material. The enrichment column was switched into the nano flow path after 5 min, and peptides were separated with an analytical column (0.75 µm×150 mm) packed with C18 reverse phase ZORBAX 300SB-C18 5 µm material at a flow rate of 0.3 µl/min. The chip is coupled to the electrospray ionization (ESI) source of the triple quadrupole mass spectrometer. The peptides were eluted from the column using a linear gradient of increasing acetonitrile. For the first 5 min, the column was equilibrated with 5% acetonitrile/95% water/0.1% formic acid (mobile phase A) followed by a linear gradient of 5%–15% B (100% acetonitrile/0.1% formic acid) in 10 min, 15–22% B in 30 min, and 22–100% B in 35 min. The column was washed with 100% B and then equilibrated with A before the next sample was injected. Blank injections were run between samples to avoid carryover.
Product ion scans were run on the triple quadrupole instrument and analyzed using Skyline software
We based the peptide biosensor assay for Bcr-Abl/Abl kinase activity on our previously reported methodology,
Bcr-Abl activity was detected using the biosensor peptide in K562 cells at 5, 30 and 60 min (N = 3). Peptide and phosphopeptide were detected below the 15 kD molecular weight marker by overlaid signal from IR-dye-labeled streptavidin (showing biotinylated peptide) and Western blot for antiphosphotyrosine (4G10). Phosphorylated endogenous proteins Abl, STAT5 and CrkL, as well as β-tubulin as a loading control, were detected as described in the Materials and Methods. Additional replicate blots shown in the supporting information. Quantification of total peptide (streptavidin bands) and phosphopeptide (4G10 bands) via raw data from integration of fluorescence intensity at the MW observed for the biosensor (without background subtraction) is shown (B) to illustrate degradation of the peptide in the presence of phosphatase inhibitor, which may have contributed to the decrease in phosphopeptide signal over time (C). White = peptide alone, grey = inhibition by Gleevec (imatinib/IM), black = phosphatase inhibitor sodium pervanadate (PV). Error bars represent SEM from three independent experiments.
To test this, we exploited the biotin affinity tag to capture peptide from lysate generated after 5 min incubation and analyzed it by MALDI-TOF/TOF mass spectrometry. Within just 5 min of exposure to cells (as well as approximately 2–3 min additional processing time for cell harvesting), essentially no intact peptide was observed, even for samples treated with peptide alone (
After just 5 min of incubation with cells and ∼2–3 min sample handling time, no intact peptide biosensor (Mw: 4753.5) remains. Detected ions suggest that the biosensor peptide underwent C-terminal degradation in K562 cells, leaving the N-terminal “reporter” region unperturbed. Representative degradation products are listed in the sequence table (C) and highlighted in the MALDI-TOF spectrum (B).
We pursued MRM as a strategy to achieve better sensitivity in the context of trypsin-digested whole cell lysate from cells incubated with the biosensor peptide. A key advantage of MRM is the ability to detect and reproducibly quantify targeted peptides and phosphopeptides
Synthetic versions of each peptide were added into trypsin-digested K562 cell lysate and analyzed by MRM. Extracted ion chromatograms for each were generated (A), integrated and plotted against input peptide to establish the calibration curve (B). Solid lines represent the 540604 transition from the unphosphorylated peptide EAIYAAPFAK; dashed lines represent the 580480 transition from the phosphorylated derivative EAIpYAAPFAK. Error bars (which are too small to be visible on the graph) represent standard deviation (SD) of two replicate analyses.
Parent ion | EAIYAAPFAK | Parent ion | EAIpYAAPFAK | ||||||
(M+2H+2540.8 | CID (V) | LOD (fmol) | LOQ (fmol) | Average CV (mean±SD) acrossconcentration range | (M+2H)+2 580.9 | CID (V) | LOD (fmol) | LOQ (fmol) | Average CV (mean±SD) across concentration range |
|
120.0 | 0.5 | 0.5 | 7±4% |
|
80.0 | 0.5 | 5 | 4±4% |
|
120.0 | 0.5 | 1 | 7±10% |
|
80.0 | 5 | <10 | 3±2% |
Lower limit of detection (LOD) represents 3 SD from background (cell lysate tryptic digest alone). Lower limit of quantification (LOQ) represents 10 SD from background. Reproducibility for detecting each transition from two technical replicates across the calibrated range (5–250 fmol) is reported as the average of the CV for each transition at each concentration.
Transitions chosen for quantitative analysis.
To analyze biosensor phosphorylation, K562 cells were incubated with the biosensor for 5 minutes either alone or in the presence of Bcr-Abl inhibitor (imatinib) or phosphatase inhibitor (pervanadate) as described above. 1 µg of each cell lysate was analyzed using the MRM method described above. MRM signal data were extracted as chromatograms and the substrate peptide and its phosphorylated derivative were identified by the presence of both transitions in their respective peaks at the retention time expected for these analytes from the calibration curve analyses. Some background peaks were observed in each extracted chromatogram (
Full chromatograms are shown in row (A). Expanded chromatograms for the region in which EAIYAAPFAK and EAIpYAAPFAK are expected to elute (based on the calibration curve) are shown in row (B), and show that the relevant peaks had signal for both transitions from each analyte.
Total peptide detected is represented by each full bar in (A), with the white portion representing the fraction of phosphopeptide and the gray fraction representing the fraction of unphosphorylated peptide. % phosphorylation is shown in (B). Error bars in both represent SEM from three replicate biological experiments.
All peptides were detected at levels above both their LOD and LOQ (
Peptide only | Peptide+imatinib | Peptide+pervanadate | ||||||||||
Unphospho (fmol) | Phospho (fmol) | Total (fmol) | % phospho | Unphospho(fmol) | Phospho(fmol) | Total (fmol) | % phospho | Unphospho(fmol) | Phospho(fmol) | Total (fmol) | % phospho | |
|
23±5 | 4±0.9 | 28±5 | 17±6 | 36±15 | ND | N/A | 0 | 14±0.6 | 17±12 | 31±13 | 52±16 |
|
23% | 20% | 16% | 38% | 42% | N/A | N/A | N/A | 5% | 71% | 42% | 28% |
The amounts of analyte detected (±SD) per µg of digested sample are given in fmol. Values represent the mean of three replicate biological experiments.
Taken together, these results demonstrate that we can accurately and reproducibly detect Bcr-Abl biosensor peptide phosphorylation and inhibition in an intracellular assay. Using K562 cells as a human CML model system, we showed substantial improvements in the lower detection limits for the assay read-out compared to our previous detection strategies. The amount of total sample analyzed (1 µg) is equivalent to approximately 15,000 cells, indicating that we can achieve several orders of magnitude improvement in sensitivity compared to Western blot or MALDI-TOF detection.
In this method development work, we demonstrated a peptide biosensor-based assay for monitoring Bcr-Abl kinase activity and inhibition in intact, live cells using MRM detection with femtomole sensitivity and good reproducibility. SRM and MRM have long been established as clinical tools for monitoring small molecule metabolites, and are becoming more and more popular for analysis of protein biomarkers from blood samples. Peripheral blood mononuclear cell (PBMC) and serum samples are routinely prepared by pathology laboratories and can either be sent to contract facilities for MRM/SRM analysis or even analyzed in-house, since many hospital labs now have the necessary instrumentation and expertise. Therefore, it should be possible to establish MRM-based biosensor assays as companion diagnostics for kinase inhibitor therapy. Since mass spectrometry is also capable of detecting many analytes simultaneously, expansion of the suite of peptide biosensors to additional kinase targets for multiplexed analysis in CML and other leukemias may allow this strategy to be used in the future to analyze signaling profiles and drug sensitivity for individual patients, enabling personalized assessment of the therapeutic options from available kinase inhibitors.
A compiled supporting information document containing additional data is available in PDF format.
(PDF)
Raw EIC data from MRM analyses is available as
(ZIP)
A table of calculations for all MRM experiments is available as
(XLSX)
We thank Victoria Hedrick (Purdue Proteomics Facility) for technical assistance with sample preparation and analysis. We are also grateful to G. Marc Loudon (Medicinal Chemistry and Molecular Pharmacology, Purdue University) for helpful discussions on the mechanisms and possible intermediates for the photo-fragmentation reaction.