Application of Nanosecond Laser Photolysis Protein Footprinting to

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Application of nanosecond laser photolysis protein footprinting to study the EGFR activation by EGF in cell Yi Zhu, Aida Serra, Tiannan Guo, Jung Eun Park, Qing Zhong, and Siu Kwan Sze J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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Journal of Proteome Research

Application of nanosecond laser photolysis protein footprinting to study the EGFR activation by EGF in cell Yi Zhu 1, #, Aida Serra 1, Tiannan Guo 1,#, Jung Eun Park 1, Qing Zhong 2, Siu Kwan Sze 1* 1

, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,

Singapore 637551 2

, Department of Pathology and Molecular Pathology, University Hospital Zürich, Zürich,

Switzerland #

, current address: Department of Biology, Institute of Molecular Systems Biology, ETH

Zurich, Switzerland * Corresponding author: Siu Kwan SZE (Email: [email protected])

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Abstract Mass spectrometry based protein footprinting emerged as a useful technology to understand protein ligand interactions in vitro. We have previously demonstrated the application of footprinting in live E. coli cells. Here we further optimized an ultrafast laser photolysis hydroxyl radical footprinting method and applied it to study the interaction of EGF and EGFR in live mammalian cells. This method used a nanosecond laser to photochemically generate a burst of hydroxyl radicals in situ in cell suspension to oxidize the amino acids on the protein surface. Mass spectrometric analysis of the thus modified peptides was interpreted to probe the solvent-accessible surface areas of the protein in its native biological state with and without EGF activation. Our footprinting data agreed with the two relevant EGFR crystal structures, indicating that this in cell laser photolysis footprinting technique is a valid approach to study the structural properties of integral membrane proteins directly in the native environment. ##place to put TOC graphic, For TOC only##

Keywords: EGFR, laser photolysis, hydroxyl radical, footprinting, mass spectrometry


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Introduction Hydroxyl protein footprinting technique is a mass spectrometry (MS)-based structural method that offers complementary data to conventional structural studies by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. This method was originally invented to map protein topology in solution by protein surface oxidation with hydroxyl (OH) radicals generated directly from water molecules by synchrotron radiation 1-4 or pulsed electron beam 5. Consequently the amino acids residing in the solvent accessible surface area (SASA) of the protein are covalently oxidized by free hydroxyl radicals. Subsequent MS analysis of the tryptic peptides from the protein is then used to identify and quantify the oxidized residues. Due to the superb and continuously improving sensitivity of MS, hydroxyl radical protein footprinting requires only small amount of proteins (ng to µg), and is achievable without complete purification of the protein of interest, which is mandatory for alternative approaches such as X-ray crystallography and NMR spectroscopy. Therefore, the footprinting approach has been extensively applied to study ligand-induced protein conformational changes 6.

Since the time frame for protein folding/unfolding kinetics ranges from microseconds to milliseconds 7, a method which generates a short pulse of OH radicals is highly desirable for accurate protein surface footprinting. Inspired by the pioneer’s work on synchrotron radiation of water molecules by M.R. Chance and coworkers 8, our group developed a pulsed-laser labelling approach for protein surface footprinting, by which the generation of hydroxyl radicals from hydrogen peroxide photolysis using pulsedlaser irradiation can be achieved in nanosecond (ns) timescale 9. Therefore it is even faster than the synchrotron method of a millisecond timescale. In this method, a high concentration of OH radical is generated via photo-dissociation of a hydrogen peroxide (H2O2) solution by a 5~7 ns pulse 266nm Nd:YAG laser. The OH radical immediately oxidizes amino acid residues located on the protein surface to produce stable covalent modifications. The half-life of OH radical is in nanosecond timescale, while the half-life of some secondary radicals generated by reacting with OH radical can be longer, most likely in the submillisecond range, and the time scale of self-quenching of OH radicals is reported to be around 100 µs 9, 10. Because of the submicrosecond time scale of the formation of OH radicals as well as their subsequent oxidation and quenching in the submillisecond range, the method has the potential to probe the solvent-accessible surface of a protein in its native state 9. In the same year, a principally similar laser flash photolysis method was developed independently and reported by Hambly and Gross 10. The method made use of a 248 nm KrF



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excimer laser to photolyze the H2O2 molecules and to generate OH radicals for fast protein footprinting. Different from our setting of Nd:YAG photolysis method, the KrF method included an OH radical quenching system to shorten the lifetime of the OH radical in the solution. The method further adopted a flow system to ensure minimal double exposure to radicals, and was named as ‘fast photochemical oxidation of proteins (FPOP)’. The FPOP method has been widely applied in structural proteomics studies 11-16.

To progress from studying purified proteins to proteins in their native biological state in live cells, we further implemented the classical and famous Fe(II)EDTA/H2O2/ascorbate Fenton system 17 and applied the footprinting technique couple with MS in the study of integral membrane proteins directly in the live E. coli cells. With this development, we successfully monitored the voltage gating of OMPF porin of live E. coli cells. We reported that the regulation of switch on/off of the ion channel was controlled by the electrostatic field inside the channel rather than the physical structural changes of OMPF 18. This study implied the validity of applying the hydroxyl protein footprinting technique to study membrane proteins in live cells without too much effort to enrich or purify the protein of interest. However, the Fenton reaction lasts several minutes 17, 19, 20

, which does not match the typical time frame for protein folding/unfolding kinetics

ranging from microseconds to milliseconds. Therefore in this study, we tried to further implement the nanosecond laser photolysis footprinting technique to live mammalian cells to study EGF/EGFR interaction in their native environment on the plasma membrane. The EGF/EGFR protein complexes were selected as a model here because EGF binding of EGFR has been well and extensively characterized. Since the current study of EGFR was based on the method which made use of pulsed laser to photochemicaly generate OH radicals from H2O2 for protein footprinting in solution 9 and the concept that to elucidate the structural dynamics of membrane proteins by hydroxyl radical footprinting in their physiological state 18, it would be better that we look back into the two established studies and summarized the technical points here. In our first in vitro laser footprinting study, a systematic evaluation of several essential parameters including laser intensity, the number of laser shots, H2O2 concentration, and protein concentration, was performed to optimize the laser-induced footprinting method 9. Important conclusions obtained from the study included: (i), it is crucial to perform the footprinting experiment with one single pulse of laser to avoid false footprints on the protein surface, because a single laser shot could produce protein surface oxidation which can induce the protein conformational change; (ii) the H2O2 concentration has great effects on the protein oxidation in response to protein concentration changes. It was found that with increasing H2O2 concentration from 0.3% 4 ACS Paragon Plus Environment

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to 1.0%, the proportion of monooxidized apomyoglobin diminished and the number of multiply oxidized species increased; (iii), the influence of laser intensity is minimal comparing to the H2O2 concentration due to the excessive and saturated photon from the laser light source. In the second study, we implemented the classical and famous Fe(II)EDTA/H2O2/ascorbate Fenton system 17 and applied it to study the OMPF porin in E. coli cells, to investigate the voltage gating in vivo. Minor optimization was performed and the H2O2 concentration was fixed at 0.3% for the Fenton reaction to generate OH radicals. The oxidation level to the very reactive methionine (M) was generally high, while it was much lower as to the other oxidized residues in the OMPF study 18. Based on these findings, we performed the in cell footprinting of EGFR using HEK F293 cells in this study. The footprinting data reflected the conformational change of EGFR upon EGF activation. Our observations are consistent with the property of solvent accessibility of a number of relevant amino acids along the primary sequence of EGFR, which is obtained from the two crystal structures of EGFR. Therefore, we benchmarked the fast incell laser photolysis footprinting method in live mammalian cells.



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Materials and Methods Antibodies and reagents Recombinant human EGF (#GF144) was from Merckmillipore. EGF Receptor (EGFR1, #2256) mouse monoclonal antibody was from Cell Signaling Technology. EGFR mouse monoclonal antibody (sc-71032) was from Santa Cruz Biotechnology. This antibody was raised against the amino acids 985-996 of human EGFR intracellular domain. Protein G agarose beads was from Upstate. Heat-inactivated fetal bovine serum was from Hyclone. Phosphate buffered saline (PBS) was from GIBCO, Invitrogen. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Sigma. Penicillin and streptomycin were from Invitrogen. FreeStyle HEK293 Expression Medium was from GIBCO, Invitrogen. The pcDNA3-EGFR vector for expression of EGFR was kindly provided by Professor Yosef Yarden (Weizmann Institute of Science) 21. Complete protease inhibitor cocktail tablets were from Roche Diagnostics (Indianapolis, IN, USA). Sequencing grade modified trypsin for protein digestion was from Promega. HPLC grade solvents were purchased from J.T. Baker (Phillipsburg, NJ, USA). H2O2 was from Merck, Singapore. All the other chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Cell Culture and Transfection HEK293-F cells were maintained under an atmosphere of 5% CO2 in FreeStyle 293 expression medium in a shaking incubator (150 rpm) at 37°C. We determined the number of cells needed for transfection on the day before transfection and passaged cells at 6 × 105 cells/ml. On the day of transfection, we determined the density and viability of cells and ensured that the cell density was about 106 cells/ml and the viability of cells was over 90%. For expression of EGFR, F293 cells were transfected for 24 h using the corresponding pcDNA3-EGFR plasmid and 293 Fectin. We found the optimal ratio of plasmid to 293 Fectin was 1:3. Western blotting Western blotting was performed using the primary anti-EGFR mAb (EGFR1, #2256) at the dilution of 1:1000, and the secondary antibody at the dilution of 1:5000. EGF stimulation EGF was added in ice cold PBS supplemented with 50 mM glucose, 5 mM MgCl2, 8.7 mM CaCl2 at a final concentration of 100 nM 22. Prior to EGF activation, the cells were


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washed twice with PBS and incubated with the EGF solution with gentle agitation for 1 h at 4°C. Flash oxidation of living cells using a nanosecond pulsed laser Three groups of cell samples were included in this study. The first was the control group (abbreviated as ‘Ctrl’) containing cells without EGF stimulation or oxidation. The second was the normal group (abbreviated as ‘Norm’), from which cells were resuspended in PBS for laser photolysis. The third was the EGF activation group (‘EGF’). Cells from this group were stimulated by EGF first, spun down to remove free EGF and further washed with ice-cold PBS one time, and were then resuspended in ice-cold PBS for laser irradiation. Each samples was prepared in triplicates. Cells were then oxidized with OH radicals generated by exposing the suspension to a pulse Nd: YAG laser (LOTIS TII LS-2134UTF, Minsk, Belarus) operated at 266 nm and 30 mJ with one single pulse of laser strictly 9. The 12-well plate was held horizontally, and each well opening was properly aligned with the laser beam with beam size expanded to the size of the cross-section area of cell culture well with an optical system, as was shown in Figure 1. Immediately after one laser shot, catalase was added to remove the residual H2O2 according to the manufacturer’s protocol. Cells were collected subsequently by spinning down at 500 g, 4 °C for 10 min. We performed five independent replicates of the experiment (Table S1.1). Immunoprecipitation (IP) Cells were lysed in lysis buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% (v/v) NP-40, supplemented with protease inhibitors cocktail. Cell debris was removed by spinning down at 13,000×g, 4 °C for 15 min. The supernatant was pre-cleared with protein G beads. EGFR antibody (sc-71032) was added to the precleared supernatant and incubated with end-to-end rotation in 4 °C for 3 h followed by further incubation with the protein G beads for 1 h. Before elution, beads were washed with ice-cold lysis buffer for five times. Bound proteins were eluted with 3×150 µl of glycine-HCl (pH 1.9). Protein samples were subsequently subjected to SDS page for resolving. In-gel digestion EGFR protein bands from the SDS-PAGE were cut out. The glycans were enzymatically released from the gel bands by incubation with PNGase F (500 units/µL) in 25



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mM NH4HCO3 at 37 ºC overnight. Samples were reduced with 10 mM dithiothreitol in 25 mM NH4HCO3 at 56 ºC for 1 h, followed by alkylation with 55 mM iodoacetamide in 25 mM NH4HCO3 solution at room temperature in dark for 45 minutes and digested by sequencing grade modified trypsin (Promega Corporation, Madison, WI. USA) in a 1:100 mass ratio (trypsin: protein) at 37 ºC overnight. LC-MS/MS analysis The LC-MS/MS analysis was done in an LTQ-FT Ultra mass spectrometer (Thermo Electron) coupled with a prominenceTM HPLC unit (Shimadzu). The tryptic peptides were reconstituted to 100 µl of 0.1% formic acid (FA) in HPLC grade water. The samples were then injected by an autosampler and on-line desalted in a Zorbax peptide trap (Agilent, Pola Alto, CA). The peptide separation was performed in a home-packed C18 column (75µm inner diameter×10cm, 5µm particles, 300 Å pore size, Michrom BioResources, Auburn, CA, USA). Buffer A (99.9% H2O, 0.1%FA) and buffer B (80% ACN, 0.1%FA) were used for the LC gradient. The 90-min gradient was ramped from 5% ACN to 30% ACN in 65 min, then to 60% in 10 min and to 80% ACN over 2 min and then was kept at 80% ACN for 3 min and ramped back to 5% ACN for the last 10 min. The sample was ionized into the mass spectrometer through an ADVANCE™ CaptiveSpray™ Source (Michrom BioResources) with an electrospray potential of 1.5 kV operating at a flow rate of about 200nl/min after a splitter. The nitrogen gas flow was set at 2, ion transfer tube temperature at 180°C and collision gas pressure at 0.85 mTorr. The LTQ-FT Ultra was set to perform data acquisition in the positive ion mode as described previously 23. Briefly, a full MS scan (350-2000 m/z range) was acquired in the FT-ICR cell at a resolution of 100,000 and a maximum ion accumulation time of 1000 msec. The AGC target for FT was set at 1e+06 and precursor ion charge state screening was activated. The linear ion trap was used to collect peptides and to measure peptide fragments generated by collision-activated dissociation (CAD). The default AGC setting was used (full MS target at 3.0e+04, MSn1e+04) in linear ion trap. The 10 most intense ions above a 500 counts threshold were selected for fragmentation in CAD (MS2), which was performed concurrently with a maximum ion accumulation time of 200 msec. Dynamic exclusion was activated for the process, with a repeat count of 1 and exclusion duration of 20 s. Single charged ion is excluded from MS/MS. Isolation width was 2 Da, and default charge state was 2. For CAD, normalized collision energy was set to 35%, activation Q was set to 0.25, and activation time 30 ms. Spectra were acquired in centroid format in raw data files with XCalibur (version 2.0 SR2).


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Bioinformatics analysis Protein database search against a restricted database with 174 entries including integrin and epidermal growth factor isoforms (Data S1) was carried out using PEAKS Studio 24 version 7.5 (Bioinformatics Solutions, Waterloo, Canada) with a precursor ion tolerance of 10 ppm and fragment ion tolerance of 0.8 Da. Enzymatic cleavage by trypsin was selected, with maximum 3 missed cleavages allowed. Carbamidomethylation (C) was enabled as fixed modification. PEAKS PTM algorithm 25 was used to identify variable post-translational modifications and 1% false discovery rate (FDR) adjustment was applied. Additionally, three types of oxidation were manually included as variable modifications: the oxidation with a mass shift of +15.99 Da at CDFHKMNPRWY, the oxidation with a mass shift of +13.98 Da at EIKLPQRV and the oxidation with a mass shift of +31.99 Da at CFMWY 26. Extracted ion chromatograms (XIC) at 10 ppm were obtained using an in-house program and used to compute the oxidation levels of the oxidized peptide products of each tryptic peptide arising from hydroxyl radicals. The oxidation efficiency was computed following the equation below as described previously 18, where A0 indicates the area under curve of extracted ion chromatography (XIC) for the nonoxidized peptide ions, while Ai (i=1, 2… ) represents the area under XIC curve for the oxidized counterparts.

Oxi(i ) % =

Ai × 100% A0 + ∑ Ai i

Calculation of solvent-accessible areas of amino acids Exact solvent accessible surface area (SASA) of molecular surface for macromolecules was calculated by the program Surface Racer 5.0 27 with a probe radius of 1.4 Å based on the crystal structures of EGFR, 1NQL for tethered monomer, and 1IVO for an extended dimer. The schematic overview of the workflow is shown in Figure 1 below.



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Results and Discussion Establishment of the laser photolysis footprinting of EGFR in live mammalian cells We applied the previously established laser-induced hydroxyl radical footprinting method to the live F293 cells, and adjusted the following experimental parameters based on the in vivo study of OMPF 9, 18, 26 : (i) We used only one single laser shot for photolysis; (ii) H2O2 concentration was increased to 1% to achieve an overall higher level of oxidation; (iii) cell density was adjusted to 1×107 / mL; (iv) The laser intensity was set at 30 mJ. The effective photon density was similar to our previous study after laser beam expansion using optics to cover the cell culture well. For each sample, the cell suspension was aliquoted into a 12-well plate (three plates for each group, respectively), with each well containing 1.5 mL of cell suspension with the density around 1×107 /mL. H2O2 was added to the cell suspension to a final concentration of 1% before laser irradiation. To maintain photon density over the larger irradiation area on the 12well plate, we used a more powerful laser (30mJ) compared to the lower power laser (1mJ) used in our previous experiment 9. As the laser beam was a highly collimated parallel light source which could not illuminate the whole cross section area of the cell culture well, we used an optical system to expand the laser beam size by ~30X to obtain similar photon density used in our previous experiment 9, as shown in Figure 1. Due to many interdependent parameters for optimal in cell footprinitng, we acknowledge that the current experimental settings for the laser footprinting in live cell have room for further optimization, for examples, the cell density and H2O2 concentration as well as the OH quenching method could be optimized. Our method may be integrated into the recently reported microfluidics methodology for in-cell FPOP footprinting 28, 29. Detection of oxidized peptides from EGFR ectodomain by LC-MS/MS analysis Prior to MS analysis, an IP step was performed to enrich EGFR protein from whole cell lysates, and the enriched eluates were further resolved by SDS-PAGE to enrich the EGFR protein, as shown in Figure 2. Considering that the large extracellular domain of EGFR, including the EGFR antibody binding motifs, can be modified by oxygen atom after oxidation leading to decreased binding affinity to its antibody, we adopted an antibody against amino acids 985-996 from the intracellular domain of EGFR in this step. Interestingly, we observed in the Western blotting bands that the expression of EGFR in the three groups of cells are comparable (Figure 2A), but the IP enrichment efficiency and 10 ACS Paragon Plus Environment

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the protein yield decreased in the Norm and EGF samples (Figure 2B), probably due to EGFR was truncated by protein backbone cleavage by the intensive OH radical. It could also due to the decreased binding affinity of EGFR after OH radical oxidation occurred inside the cell because H2O2 is plasma membrane permeable but the quenching reagent catalase is not, suggesting the need for alternative quenchers. Our observation agreed with the report showing the decreased binding of a protein to its target when it is oxidatively modified 30. In this study, tandem MS was used to sequence the oxidized EGFR and to locate the oxidized amino acid residues. In general, the oxidation to most amino acids produces a hydroxyl group at their side chains and the formation of alcohol group as major products, resulting in +15.99 Da mass increases. However, in amino acids with aliphatic side chains, the minor products with the formation of carbonyl (aldehyde/ketone) group, with mass increases of +13.98 Da, are sometimes observed along with the relevant major products. Products with multiple oxygen additions (+31.99 Da) can also be generated, especially to those amino acid residues with higher reactivity, i.e., amino acids with aromatic side chains and sulfur-containing side chains 4, 18. While more variant oxidations may also happen, due to the limit of mass spectrum search space, we included only these three most frequent modifications, +13.98 Da, +15.99 Da, and +31.99 Da in this study. Oxidized peptides were detected by the increased peptide mass with an integral multiple of +13.98 Da, +15.99 Da, and +31.99 Da, and the modified sites were then confirmed by manually inspection of the mass spectra (Data S2). The relative abundance of oxidized counterparts of the tryptic peptide were evaluated based on the area under curve of the respective XIC chromatography (Figure 3). We detected 81% of the whole EGFR protein sequence consistently in all three groups of samples (Ctrl, Norm, and EGF), suggesting good reproducibility of our protocol. The spectra for the modified peptides were further manually curated. Only peptides with XIC chromatographic peak area above 6,000 from the extracellular domain of EGFR were included for calculation of oxidation levels. Thirty tryptic peptides from the ectodomain of EGFR were identified and quantified (Table S1). Among them, eleven peptides were measured with relatively high and comparable oxidation levels across all three groups, thus considered as the oxidative background (Table S1.2). We found these peptides contain high numbers of reactive amino acids such as M, F, Y, P, L, V etc. Nine peptides were found to be un-oxidized (Table S1.3), with the oxidation extent from 0 to 8% for each peptide, and showing no difference for the same peptide among all three conditions. These peptides are either from the cysteine rich 11 ACS Paragon Plus Environment


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domain with very compact internal structure or are intrinsically hydrophobic, thus less accessible to hydroxyl radicals (Table S2). Ten peptides responded to the oxidative OH radicals and showed interesting pattern upon EGF treatment, as was shown in Table 1, Table S1.4-S1.6. We calculated the p value for each of the ten peptides to determine the significance of the relevant oxidation level changes (Table S1.7). Three peptides showed no change in oxidation level upon EGF activation (Table S1.6). Seven peptides showed increase/decrease to their oxidation levels (Figure 4A), and their p values were below 0.23, among which two peptides showed significant changes (p value less than 0.05) (Table S1.7). There are several reasons for the not so significant changes as indicated by relatively greater p values (

Application of Nanosecond Laser Photolysis Protein Footprinting to

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