Integrating proteomics with electrochemistry for identifying kinase biomarkers

We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers.


Introduction
The human kinome contains over 500 identied kinases 1 that create a vast network of kinase cascades. 24][5] Therefore, kinases are important for cancer therapy at two levels: on one hand, they are promising drug targets and various kinase inhibitors are developed as anti-cancer leads. 68][9] However, a systematic study on the regulation of kinase activation and association with cancer at the proteomic network level in vivo has not been performed.1][12] The aberrant kinase could ubiquitously affect the signal transduction networks across multiple pathways.A kinase detection method capable of detecting its phenotype-dependent substrates may provide better specicity to monitor a disease related to a specic pathway.Due to their medical and biological signicance, many methods for the detection of kinase activity have been previously demonstrated.These include radiometric assays, 13 spectroscopic assays 11,12,14,15 and electrochemical methods.Electrochemical methods have the advantages of avoiding work with radioactive materials, higher detection sensitivities and consumption of lower amounts of analyte without the need for amplication systems. 16,17Diverse applications of electrochemical methods for detecting kinase activity were reported.These include the use of ferrocene labeled ATP 10 or detection with antiferrocene antibodies in an immunoarray format. 18In label-free methods, the detection is based on repulsion between the negatively charged phosphate groups and negatively charged redox species in the solution that is translated in EIS measurements to increase in the resistance to charge transfer (R CT ). 19Alternatively, there is attraction of a cationic redox species to the phosphate group on the peptide that is translated in cyclic voltammetry (CV) measurements to increase in current. 16,20We have previously reported a label-free electrochemical kinase detection method, which is based on a peptide monolayer that changes its conformation upon phosphorylation from a densely packed monolayer into a disordered one, causing a sharp drop in R CT .This approach has a built-in amplication mechanism, which is based on a reversible orderdisorder transition that the peptide monolayer undergoes due to its phosphorylation.This order-disorder transition changes the electronic properties of the layer from a good ionic insulator to an ion-permeable peptide layer and does not rely only on ionic repulsion/attraction from the phosphorylated peptide layer. 20ere we report an integrated platform combining quantitative tissue phosphoproteomics for kinase-substrate biomarker discovery and electrochemical kinase detection method for ultra-sensitive kinase-substrate biomarker validation.This can be used as a step towards individualized cancer diagnostics.Phosphoproteomic approaches have demonstrated effective identication of the abnormal protein kinase activity involved in cancer. 21,22Using NSCLC as a proof-of-concept study, labelfree quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed site-specic elevated phosphorylated proteins/peptides in NSCLC.Through sequence motif-analysis and literature mining among the 198 most dramatically up-regulated phosphopeptides, HDGF was chosen as a model protein for the electrochemical studies.A peptide derived from the HDGF phosphorylation site was coupled to a gold electrode and electrochemical impedance spectroscopy was utilized to probe its phosphorylation by ERK2 and dephosphorylation by AP.The unication of proteomics and electrochemistry enabled us both to identify the cancerrelated target sequence of the kinase and to detect the kinase levels at low concentrations.The developed method is general and could be applied to large-scale biomarker discovery and validation for other diseases such as other cancer types or neurodegenerative diseases.

Identication of over-activated phosphoproteins in NSCLC by phosphoproteomics
To identify substrate/kinase pair that are over-activated in lung cancer and potentially serve as biomarkers, we rst searched for phosphoproteins that are over-phosphorylated in lung cancer tissue in comparison with its corresponding adjacent normal tissue from the same patient.The altered phosphorylation level may represent abnormal kinase activity in this specic cancer.4][25] The phosphopeptides enriched from the IMAC were analyzed by LC-MS/MS (Triple-TOF 5600) for phosphoproteomic analysis.A total of 1451 phosphopeptides (derived from 540 phosphoproteins) were condently identied by the Mascot search engine and quantied by the IDEAL-Q soware. 26To identify kinase/ substrate pairs that are over-activated and can serve as biomarkers involved in NSCLC, the extent of over-phosphorylation of every phosphopeptide in the tumor tissue was quantitatively compared by dening: T/N ratio ¼ amount of phosphorylation in tumor tissue/amount of phosphorylation in adjacent normal tissue.The specic target phosphoprotein was selected by a series of steps (Fig. 1b).Several bioinformatics tools together with literature mining were used to search for the target kinase-substrate pairs.First, we selected phosphopeptides that had a T/N ratio >2 since we considered those to be upregulated in the tumor tissue with statistical signicance.The quantication results revealed >2-fold up-regulation of 386 phosphopeptides from 198 phosphoproteins (see details in ESI Table 1 †).Among them, 124 originate from proteins reported to be related to cancer by Metacore™ analysis.Of these peptides, 85 phosphopeptides derived from 52 phosphoproteins, underwent signicantly increased phosphorylation in the tumor tissue, i.e. $5-fold T/N ratio.
As an initial model system, we selected phosphopeptides that have only one possible phosphorylation site per peptide.Of the eight remaining proteins (Table 1), only HDGF is overexpressed (6-fold) in lung cancer, and was therefore selected. 27,28he HDGF peptide sequence is 160 DLLEDpSPKRPKEAEN 174 , with a phosphorylation site at position S165 (Table 1).We also evaluated whether HDGF was also overexpressed at the protein level.The results revealed that the protein level of HDGF also show overexpression in tumor tissue compared to adjacent normal tissue from the same patient (ESI Fig. 1 †).Therefore, detection of its kinase activity by the developed kinase assay is very important to understand the phosphorylation alteration of HDGF protein.

Identication of the target kinase
0][31] ERK1/2 were shown to be potential kinases that are able to phosphorylate HDGF on serine 165 (Table 1). 25,32To verify that these kinases indeed phosphorylate the HDGF 160-174 peptide, an in vitro kinase assay was performed at both peptide and protein levels.The products of the peptide phosphorylation by ERK2 were analysed by LC-MS/MS and the phosphopeptide signal was observed at the expected mass/charge (m/z) value (607.62 for charge of 3 + ).The serial of y-fragment ions (y 10 -y 14 ) retaining the phosphate group in the MS/MS spectrum shown in Fig. 2b conrmed that S165 is the phosphorylation site by ERK2.We also performed the same in vitro kinase reaction at the protein level.The ERK kinase reaction for the dephosphorylated HDGF protein showed the same phosphorylation site at serine 165 (see details in ESI Fig. 2 †).

Assembly of the HDGF peptide on gold electrodes
The non-phosphorylated form of the selected HDGF peptide was synthesized with an N-terminal cysteine added for binding to gold electrodes (Fig. 1c, see above) and its electrochemical response to phosphorylation was examined.To determine the optimal assembly time of the peptide on the electrode, we performed in situ kinetic impedimetric measurements (Fig. 3).The frequency for the kinetic measurements was selected to be 100 Hz, because in this frequency the impedance of the peptide monolayer was signicantly higher than that of the clean gold (see Fig. 3a, inset) and not too low to be diffusion controlled.The in situ adsorption experiment (Fig. 3b) showed that the real impedance (Z 0 ) increased over time and was at a plateau aer 16 hours.We concluded that 16 hours of incubation is the optimal time for the adsorption of the peptide.The data was transferred to surface coverage (q) using the expression: with Z 0 being the real impedance at each point, Z 0 initialthe value of the clean gold and Z 0 nalthe nal value at the plateau.The adsorption isotherm was t to the equation: 33,34 (1 a The T/N ratio for each peptide refers to the amount of phosphorylation in tumor tissue/amount of phosphorylation in adjacent normal tissue.The HDGF peptide is highlighted.where q is the fractional surface coverage, k 1 and k 2 are the rate constants that were found to be k 1 ¼ 55.5 AE 0.6 M À1 s À1 and k 2 ¼ 0.858 AE 0.004 M À1 s À1 , Cthe initial concentration of the peptide solution (0.1 mM).The value of the coefficient a is 0.677 AE 0.001.The peptide formed a dense monolayer, as observed by the high R CT of the electrode aer incubation with the peptide (3400 U) compared with the impedance of the bare gold electrode (380 U) (Fig. 4).The increase in R CT is caused by a layer blocking the electron transfer from the redox species in the solution to the electrode.

ERK2-mediated phosphorylation of the HDGF peptide
ERK2-mediated phosphorylation of the peptide monolayer resulted in a decrease in R CT , showing that phosphorylation indeed took place (Fig. 5).The results were t to a Randles type circuit [R sol.(Q[R CT W])](Fig.5 inset).The results are summarized in Table 2.The parameters are the solution resistance, R sol. , the resistance to charge transfer, R CT , Q the constant phase element, and its exponent, a.
Following incubation with ERK2, the R CT decreased by 86% to 474 U.The reversibility of the phosphorylation reaction was examined by performing dephosphorylation with Alkaline Phosphatase (AP).Following dephosphorylation, the R CT increased back to 3250 Ualmost to the starting value.A reversible change is evident also in a, the exponent, changing aer phosphorylation from 0.94 to 0.90 and changing back to 0.94 aer dephosphorylation.

Selectivity of the assayphosphorylation with calmodulin kinase 2 (CaMK2)
To verify the selectivity of the substrate-dependent kinase assay, the same process of phosphorylation and dephosphorylation on HDGF 160-174-covered electrodes was tested with CaMK2, which is not known to phosphorylate the HDGF 160-174 sequence (Fig. 6).Aer incubation with CaMK2, the R CT decreased by only 12% from 4080 U to 3600 U.This shows that CaMK2 has much lower activity towards HDGF 160-174 and thus demonstrates the good selectivity of our assay.

Monitoring the phosphorylation by monolayer roughness changes using atomic force microcopy (AFM)
A direct and independent method to verify that the phosphorylation affects the monolayer was implemented by monitoring changes of the monolayer roughness before and aer phosphorylation.For this purpose we used an annealed gold substrate that is composed of large single crystal grains (up to several mm 2 ).The orientation of most facets on the grains  reveals the (111) plane of the fcc structure and have a triangular shape. 35Each triangle is an atomically at surface.AFM characterization of annealed gold covered with the HDGF 160-174 peptide revealed a very smooth and homogeneous surface (Fig. 7a), generally resembling the appearance of the annealed gold surface.Square scratch of the surface by the AFM tip proved that there is a relatively so, $2 nm thick, layer on top of the annealed gold (Fig. 7b), indicating very dense and seemingly homogeneous peptide coverage.
Root mean squared (RMS) roughness was calculated on small areas, 10 000-100 000 nm 2 , with a single "gold terrace" (examples in Fig. 7c).The high sampling resolution enabled detailed height distribution analysis even on small areas.At  Blue ( )the R CT of the monolayer following phosphorylation with ERK2 (R CT ¼ 474 U).Red ( )the impedance of the monolayer after dephosphorylation with AP (R CT ¼ 3250 U).Lines -fits to the equivalent circuit (inset).
Table 2 The fit values of the different circuit elements for the HDGF-derived peptide monolayer before phosphorylation, after phosphorylation with ERK2, and after dephosphorylation with AP   these areas the substrate is atomically at with RMS roughness of less than 0.2 nm.Therefore, the measured roughness originated mainly from the covering peptide layer.Sixty such areas were examined in different scans at locations with distance of few mm between them, before and aer phosphorylation.Roughness distribution of these areas is shown in Fig. 7d.The phosphorylation widens the roughness distribution and shis its peak from $0.2 (red) to higher values of $0.35 (blue).This means that the surface roughness becomes higher and less homogeneous aer phosphorylation.This could be either due to small defects in the dense arrangement of peptides on the gold surface, e.g., pinholes, or due to disturbance of the surface packing upon phosphorylation, or both.In either case this result is a clear indication for a change in the monolayer packing due to phosphorylation.

Discussion
In this work we present a novel approach for kinase related cancer research.We show that discovery and detection of overphosphorylated proteins and the relative kinases can be done in a relatively simple manner, which is very versatile.Methods for detection of phosphorylated proteins in tissues had been suggested before [36][37][38] and so were detection methods for specic kinases. 11,14,16,17We combine the two aspects and show a direct path from the tissue level to the kinase detection stage.Specically, we show that the over-phosphorylation level of proteins in tumour samples can be determined by proteomic tools.The identication of over-phosphorylated residues in a target protein leads to kinase identication and the use of a peptide containing the phosphorylation site in an electrochemical assay provides a powerful tool for kinase detection.
Our detection method is unique, because it is label-free and very simplethe biosensor is composed of only the peptide and a simple gold macro electrode.[41][42] Tissue phosphoproteomics identied over-activated kinasesubstrate pairs in NSCLC Protein kinases are highly attractive targets for therapeutic intervention.Therefore, identication of their pathway-associated phosphorylation substrates may generate biomarkers to aid either the disease detection or the drug development process.The selection of the kinase/substrate target is the most crucial aspect of drug target identication.Proteomics technology is a powerful tool for discovering aberrant protein expression and post-translational modication associated with disease.The polyMAC has been applied to tissue phosphoproteomics in transgenic mouse model of HER2 positive breast cancer and identied potential therapeutic targets. 36Phosphoproteomics has also been applied to directly identify kinase inhibitor targets in cancer cell lines and mouse xenogras. 43hough these studies show the promise of phosphoproteomics to provide new targets for designing anticancer therapies, most current approaches are not accessible to individualized human tissue analysis yet.This advantage of our approach is critical since it enables to reect the human microenvironment as well as the difference between individual patients.
To our knowledge, our study is the rst report of an individualized phosphoproteomic analysis on a pair of cancerous and adjacent normal tissues from the same patient.By our sensitive label-free quantitative phosphoproteomic strategy, the results revealed identication of over one thousand phosphorylation substrates in the NSCLC tumour tissue.Hundreds of abnormally activated phosphorylation substrates enriched in human lung cancer tissue served as starting point to search for their putative kinases by motif-based informatics analysis.By comparing to adjacent normal tissues, we could largely reduce the individual difference between samples.Through the logical dissection of the phosphoproteomics data by bioinformatics tools, the HDGF peptide and the corresponding ERK2 kinase were studied by the electrochemical impedance spectroscopy method.In addition to HDGF, dozens of up-regulated phosphoproteins identied by our platform had dramatic change in expression in the tumour tissue compared to the normal tissue and also passed our criteria since their function is related to cancer.With the added advantage of multiplexed phosphopeptide enrichment by automatic IMAC protocol, the phosphoproteomic method described herein is an efficient strategy for identifying the full panel of substratekinase biomarker pairs and can be applied in a large cohort of clinical samples.

Substrate-dependent kinase monitoring by an electrochemical assay
To provide a facile and sensitive assay to monitor the substratedependent kinase activity, we have devised an electrochemical assay using peptide-coated gold electrodes.The peptide assembly on the electrode is carried out in two stepsa fast adsorption process, beginning right at the moment of peptide injection, followed by a second, slower process.The rate constants show that the rst adsorption process lasts until about two thirds of the adsorption sites are bound.Then the second process, which is likely to be an interaction between the peptides in the solution and the adsorbed peptide layers, takes place and slows down the adsorption of more peptides onto the surface.We interpret this barrier in terms of increasing peptide-peptide electrostatic and steric repulsion with increasing coverage.Aer the adsorption, the resulting monolayer was dense, as observed by the high R CT values obtained compared with other studies. 10,16Previously, high initial R CT gave us better sensitivity in this system.The introduction of the negatively charged phosphate groups into a denser monolayer causes a more prominent change, resulting in a larger change in the R CT following phosphorylation.This is evident in Fig. 5, where there is a sixfold decrease in the R CT aer phosphorylation.
A decrease in R CT can be caused by many reasons, among them detachment of the peptides from the monolayer.We showed that the process is reversible, and harmless to the monolayer, by performing the dephosphorylation reaction with AP.AP removes the phosphate groups from the peptide, resulting in free hydroxyl groups.We observed that following dephosphorylation, the R CT of the peptide monolayer increased again, practically to the same starting value as of the native peptide.This reversible change may also indicate a change in the properties of the monolayer.Upon phosphorylation, the order of the monolayer is disrupted and more water molecules penetrate into the monolayer, facilitating the passage of the redox species and making it more conductive.The larger hydration layer of the phosphate group relative to the hydroxyl group most likely also inuences the dielectric properties of the monolayer and therefore the diffuse layer of the electrode.
Fitting the plots to the equivalent circuit shown in Fig. 5 provided us with additional information about the changes on the surface.The reversible change in a, the exponent of the constant phase element, indicates a change in the homogeneity of the surface. 44,45The values of the parameter a are usually between 0-1, 1 indicating a perfect capacitor, and 0a perfect resistor.For at metal electrode the value is close to 1.The decrease in a indicates roughening of the surface upon phosphorylation.This is in agreement with our model and the AFM results, showing the reversible formation of pinholes upon phosphorylation of the monolayer.

Biological implications
HDGF 27,46 is a secreted protein 46,47 that stimulates growth of several cell types including smooth muscle cells, 48 endothelial cells 49 and lung epithelial cells. 20,21,36,51HDGF was previously linked to lung cancer and specically to poor clinical outcome.It was shown to cause tumorigenesis in vivo. 52Exogenously supplied HDGF was found to enhance the invasiveness of breast cancer. 47nhibition of HDGF using antibodies showed anti-tumor activity. 50The observation that HDGF is over-phosphorylated in NSCLC tissues is in agreement with previous studies, which showed that the S165A mutation in HDGF prevented the secretion of HDGF.The S165D mutation, which introduces a negative charge as the phosphorylation does, caused secretion of HDGF. 46herefore, the phosphorylation on HDGF S165 is a valid target for cancer therapy based on kinase inhibitors.
ERK1/2, also known as Mitogen Activated Protein Kinases 3/1 (MAPK3/1), are part of the MAPK/ERK pathway that communicates signals from the cell surface receptors to the nucleus. 2,54][55] They promote cell proliferation and metastasis. 2 Over-activation of ERK1/2 is common in cancer, including NSCLC, 9,41,56 prostate and breast cancers. 57,58Overexpression of HDGF causes activation of ERK1/2. 59Residues 8-17 on HDGF are a possible MAPK docking motif with 100% similarity to the consensus sequence. 46We show that HDGF S165 is a likely substrate of ERK2, and therefore inhibition of ERK2 could possibly inhibit the secretion of the phosphorylated HDGF, thus decreasing its tumorigenic activity.

Conclusions
In this study we combined mapping of over-phosphorylated proteins in tumor tissues, quantication of the extent of over-phosphorylation that points to abnormal kinase activity in the tissue and the detection of a kinase that is likely to phosphorylate the substrate peptide sequence on the selected target protein.Using the model system of NSCLC, we demonstrated a sensitive integrated approach for targeted substrate-kinase pair selection based on differential phosphoproteomic proling between cancer and adjacent normal tissues.This provides the advantage of individualized analysis as well as a generic platform to be applied to a specic type of cancer.The phosphorylating kinase can be predicted by bioinformatics tools and veried by phosphorylation in vitro and MS/MS sequencing to conrm the phosphorylation site.The developed label-free electrochemical assay then provides rapid and sensitive quantication of the substrate-dependent kinase activity without the requirement for specic antibodies or electrochemical labels.Therefore our methodology is general and could be applied for a panel of substrate-kinase biomarkers.Integrated approaches for the mapping of phosphorylation sites and prediction of their corresponding kinase have been previously suggested. 60Our method shows, however, a simpler implementation from the patient to the detection stage.It can be used for nding new drug targets, for mapping over-phosphorylation in specic cancer types and matching specic conditions for a specic overactive kinase, thus facilitating the design of specic kinase inhibitors as personalized drugs.signaling).By bead mill homogenizer (Precellys 24 Bead Mill Homogenizer, Bertin Technologies, MD), the pre-cleaned 50 mg tissue samples were put into one tube contain 10 beads in 200 mL lysis buffer.The tissues were crashed in 20 seconds at 5500 rpm under 4 C.The protein concentration was determined by BCA protein assay kit.
The protein samples from tissue samples were subjected to gel-assisted digestion. 23The protein sample was xed into a gel directly in the Eppendorf vial with acrylamide/bisacrylamide solution (40%, v/v, 29 : 1), 10% (w/v) ammonium persulfate, 100% N,N,N 0 ,N 0 -tetramethylenediamine by a 14 : 5 : 0.7 : 0.3 ratio (v/v).The gel was cut into small pieces and washed several times with 25 mM TEABC containing 50% (v/v) ACN.The gel samples were further dehydrated with 100% ACN and then completely dried by vacuum centrifugation.Trypsin was then added into gel for proteolytic digestion (protein : trypsin ¼ 50 : 1, g g À1 ) in 25 mM TEABC with incubation overnight at 37 C. Digested peptides were extracted three times with 5% (v/v) FA in 50% (v/v) ACN for 30 min, dried completely by vacuum centrifugation at room temperature.

Phosphopeptide purication by IMAC
Phosphopeptide purication was performed using an IMAC protocol. 24,25The in-house-constructed IMAC tip was capped at one end with a 20 mm polypropylene frits disk enclosed in a tipend tting.The tip was packed with 20 mg of Ni-NTA silica resin.First, Ni 2+ ions were removed with 50 mM EDTA in 1 M NaCl.The tip was then activated with 100 mM FeCl 3 and equilibrated with loading buffer (6% (v/v) acetic acid (AA) at pH 3.0) prior to sample loading.Tryptic peptides were reconstituted in loading buffer and loaded onto the IMAC tip.Aer successive washes with 6% (v/v) AA, 25% ACN, and 6% (v/v) AA, the bound peptides were eluted with 200 mM NH 4 H 2 PO 4 .The eluted peptides were desalted using reverse phase-Stage Tips(SDB-XC). 61

Mass spectrometry analysis
Puried phosphopeptides were reconstituted in buffer A (0.1% FA in H 2 O) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS).The TripleTOF 5600 system (AB SCIEX, Canada) equipped with a nanoACQUITY UPLC (Waters Corporation, Milford, MA, USA) was employed for nanoLC-MS/ MS analysis.The samples were loaded and separated on a 15 cm column with 100 mm inner diameters, packed in-house with 3 mm C18 particles (Dr Maisch, Ammerbuch, Germany).The LC mobile phase system consists of water with 0.1% acetic acid (buffer A) and acetonitrile with 0.1% acetic acid (buffer B).The separation gradient of buffer B was up to 80% in 120 min with ow rate of 500 nL min À1 in nanoACQUITY UPLC system.The TripleTOF 5600 was tted with a Nanospray III source.Data was acquired using an ion spray voltage of 2.5 kV, curtain gas of 20 PSI, nebulizer gas of 15 PSI, and an interface heater temperature of 150 C. For information dependent acquisition (IDA), MS survey scan range was m/z 300-1500 acquired in 250 ms.The top 10 precursor ions were selected if exceeding a threshold of 100 counts per second (counts per s) in each MS survey scan, and subsequent 10 MS/MS scans were performed for 200 ms each.Collision energy setting of 40 AE 15 eV was applied to all precursor ions for collision-induced dissociation.To minimize repeated scan, dynamic exclusion was set for 6 s and then the precursor was refreshed off the exclusion list.

Data processing, phosphoprotein identication and quantitation
Raw MS/MS data from the TripleTOF 5600 were transformed to mgf-les using the soware AB SCIEX MS Data Converter (AB SCIEX, Canada).The mgf-les were searched using Mascot against the Swissprot database (Homo_sapiens) with the following settings: the fragment ion mass tolerance was set at 10 ppm, and the parent ion tolerance at 0.1 Da; for phosphopeptide search, phosphorylation (STY) and oxidation (M) were specied as variable modications; for protein search, only oxidation (M) were specied as variable modications.Peptides were considered identied when their Mascot Signicance threshold is set as p < 0.05.
To evaluate the protein identication false discovery rate (FDR), we repeated the searches using identical search parameters and validation criteria against a randomized decoy database created by Mascot.For phosphorylation site determination, we reported the difference in Mascot scores of peptides with top two assignments on different phosphorylated sites (delta score). 62If the delta score is greater than 5, the top ranked phosphorylated site is considered condently determined.For delta score less than 5, the phosphorylated site assignment is considered ambiguous.
The quantitative analysis of phosphopeptide was performed by IDEAL-Q. 26The raw data les from the TripleTOF 5600 were transformed to mzML format using the AB SCIEX MS Data Converter.The search results in MASCOT were exported in eXtensive Markup Language data (.XML) format.Aer data conversion, the condent peptide identication results (p < 0.05) from each LC-MS/MS run were loaded and merged to establish a global peptide information list (sequence, elution time and mass-to-charge).Alignment of elution time was then performed based on the peptide information list using linear regression in different LC-MS/MS runs followed by correction of aberrational chromatographic shi across fragmental elutiontime domains.To increase correct assignment, the detected peptide peaks were validated by the SCI validation using the three criteria: (a) signal-to-noise (S/N) ratio > 3, (b) accurate charge state and (c) correct isotope pattern.To calculate relative peptide abundance, the tool performs reconstruction of extracted ion chromatography (XIC), and calculation of XIC area.The fold-change of a given phosphopeptide was calculated by the ratio of relative peptide abundance between different samples.
In vitro phosphorylation of HDGF 160-174 in vitro kinase reaction 5 mL of 0.11 mM HDGF 160-174 solution (1 mg) were mixed with 5 mL of 2Â kinase reaction buffer from Promega ADP Glo kinase assay (160 mM Tris-HCl, 80 mM MgCl 2 , 0.4 mg mL À1 , 0.2 mM DTT), 5 mL of 250 mM ATP solution (Promega), and 4 mL of ERK2 as kinase (p42 MAP Kinase, New England Biolabs Inc.).The mixture was allowed to react for 4 hours at 30 C under constant shaking.Aerwards, the reaction was stopped by cooling on ice and acidifying the solution with TFA (0.5% v/v nal concentration).The mixture was desalted using reverse phase-Stage Tips (SDB-XC) 61 and the phosphopeptides were enriched using a Fe-IMAC protocol previously described. 24,25ectrochemical characterization of phosphorylation Gold electrodes preparation.Polycrystalline bulk gold electrodes with a surface area of 4 mm 2 were used for electrochemical measurements (CH instruments).These electrodes were hand-polished on micro-cloth pads (Buehler, Lake Bluff, IL) with de-agglomerated alumina suspension (Buehler) of decreasing particle size (1.0 and 0.05 mm) using a homemade polisher.Aer polishing the electrodes were sonicated in triple distilled water (TDW) for 15 min followed by additional sonication in EtOH solution (50% v/v) for 10 min.The washed gold electrodes were then cleaned with warm HNO 3 (70%) for 20 min.The electrodes were then rinsed with TDW and cycled from À0.4 to 1.6 V in 0.5 M H 2 SO 4 at 0.1 V s À1 until stable reproducible cyclic voltammograms were obtained representing the electrochemical reduction of an AuO monolayer.
Electrode surface modication.The peptide used in the monolayer experiments was HDGF 160-174 with an added Cterminal cysteine for attachment to the gold surface (CDLLEDSPKRPKEAEN) (Biomatik, Canada).Treated gold macroelectrodes were immersed into prepared HDGF 160-174 peptide solution (0.1 mM) and were kept for 16 hours at 25 C. Aer removal, the electrodes were rinsed three times in TDW and dried under nitrogen gas stream.Electrochemical characterization was preformed directly aer the electrodes were washed.The peptide solution was prepared by dissolving the peptide in weakly basic TDW.
Kinase catalysed phosphorylation.Peptide modied gold electrodes were reacted with Glutathione S-transferase (GST)tagged ERK2 (New England Biolabs).Kinase mediated phosphorylation was performed by dripping of kinase solution on modied electrodes surfaces at 37 C. Reaction medium (3 mM kinase in a nal reaction volume of 40 ml) 80 mM Tris-HCl pH ¼ 7.5, 40 mM MgCl 2 , 0.2 mg mL À1 BSA and 100 mM ATP.The reaction was initiated with the addition of the freshly prepared protein kinase solution and stopped by rinsing, aer 30 min.Electrodes were washed multiple times in TDW, followed by a brief sonication in TDW.
Phosphorylation with CaMK2 was done with CaMK2 (New England Biolabs) in a reaction medium containing 3 mM kinase in a nal reaction volume of 40 ml) including 80 mM Tris-HCl pH ¼ 7.5, 40 mM MgCl 2 , 0.2 mg mL À1 BSA, 2.4 mM calmodulin, 4 mM CaCl 2 and 100 mM ATP.
Phosphatase catalysed dephosphorylation.Hydrolysis of surface graed phosphorylated peptides was performed in the presence of AP, 0.1 Units in a nal reaction volume of 40 ml, for 30 minutes at 37 C. 1 Unit AP was dissolved in a 1 ml of tris borate buffer, (pH 8.5), containing 10 mM MgCl 2 .(One unit (U) is dened as the amount of enzyme that can hydrolyze 1 mmol of phosphorylated peptide in a total reaction volume of 1 ml in 1 min at 37 C).
Electrochemical characterizations.Electrochemical measurements were taken following each modication step.EIS measurements were performed using an Autolab PGSTAT12 digital potentiostat (EcoChemie BV, Utrecht, The Netherlands) connected to NOVA soware.A conventional three-electrode cell was employed, with a peptide modied gold electrode as working electrode, and a standard Ag/AgCl reference electrode (Metrohm) with a KCl concentration of 3 M.The EIS measurements solution contained 5.0 mM K 3 [Fe(CN 6 )], 5.0 Mm K 4 [Fe(CN 6 )], and 0.1 M of KCl as supporting electrolyte.The spectra were recorded at a frequency range of 0.1 Hz-10 kHz with an amplitude of 10 mV, at the formal potential of the redox couple vs. Ag/AgCl electrode.The peptide adsorption was done over 15 hours.A three electrode cell, containing a clean gold electrode, was lled with a solution of 0.10 M phosphate buffer, pH ¼ 8, that was also used as the supporting electrolyte, with added redox species: 5.0 mM K 3 Fe(CN 6 ) and 5.0 mM K 4 Fe(CN 6 ).The measurement was started, and aer it reached a stable signal, a concentrated peptide solution was injected into the cell, to a nal peptide concentration of 0.1 mM.Data points were measured every 25 s, at 100 Hz.The data was t to an appropriate equivalent circuit using NOVA soware.

AFM characterizations
The substrates for AFM scans were freshly prepared annealed gold with atomically at terraces. 35A HDGF 160-174 peptide layer was deposited by submerging the substrate immediately aer annealing in peptide solution (0.1 nM) overnight.The samples were then washed with TDW and dried in a ow of nitrogen gas.AFM scans were performed with a Smart-AIST system (AIST-NT, SmartSPM™ 1000) using Olympus tips (OMCL-RC800PSA, Olympus Optical Co. Ltd) with cantilever force constant of 0.76 N m À1 and tip radius of 15 nm.To reduce surface damage, the scanning mode was true non-contact -veried by using tip-surface distance which was larger than the oscillation amplitude and with positive phase shi.Several macroscopically distant locations on each sample were scanned to eliminate "local effects" and at least ve images of 1 mm Â 1 mm were taken at each position.Scratches were done with 200 nN force.The scanning resolution was 1 pixel per nm.Image processing and analysis were done using WSxM 5.0 develop 7.0 soware. 63

Fig. 1 A
Fig. 1 A strategy for selecting the target HDGF-kinase pair assay.(a) Identification of HDGF by quantitative phosphoproteomics analysis of 66 year-old male patient tissues.A paired tissue analysis of the tumor tissue and the adjacent normal tissue samples were subjected to gel-assisted digestion, purification for phosphopeptides by automated pH/acid-controlled IMAC, and triplicate LC-MS/MS analysis.Bioinformatics analysis by IDEAL-Q software was used to process the quantitation result for peptide-kinase pair selection; (b) a decision tree for selecting the target peptide for synthesis.T/N ¼ amount of phosphorylation in tumor tissue/amount of phosphorylation in normal tissue; (c) design, synthesis, and electrochemical detection of kinase-promoted phosphorylation in HDGF peptide.

Fig. 2
Fig. 2 (a) HDGF phosphopeptide sequence with b-and y-ion assignments observed in MS/MS spectrum.A series of y-ions (y 10 -y 14 ) containing the phosphate group.(b) The MS/MS spectrum of the HDGF phosphopeptide.The arrows indicate the ions containing the phosphate group.The observed m/z is 607.62 with charge of 3+ which corresponds to the HDGF phosphopeptide peak.

Fig. 3
Fig. 3 (a) Bode plots of a clean gold electrode (black square) and the same electrode after peptide adsorption overnight (red circle).Inset: the difference in the real impedance (Z 0 ) between the peptide coated electrode and the clean gold electrode (DZ 0 ).(b) Adsorption of HDGF 160-174 onto a clean gold electrode.Relative surface coverage (q) was plotted against time (-).The curve was fit to a second order reaction kinetic equation (red line).

Fig. 4
Fig. 4 Nyquist plot of the HDGF 160-174 monolayer on the electrode (black square), compared with that of a bare gold electrode (red circle).Lines -fits to the equivalent circuit [R(C[RW])] (see, Fig. 5 inset).The monolayer was created by incubating a weakly basic 0.1 mM solution of the peptide on a clean gold electrode for 16 hours.EIS spectrum was obtained at a frequency range of 0.1 Hz-10 kHz with amplitude of 10 mV, at the formal potential of the redox couple vs. Ag/AgCl electrode (0.17 V).

Fig. 5
Fig.5Reversibility of the phosphorylationdephosphorylation process.Black ( )the R CT of the peptide monolayer (R CT ¼ 3400 U).Blue ( )the R CT of the monolayer following phosphorylation with ERK2 (R CT ¼ 474 U).Red ( )the impedance of the monolayer after dephosphorylation with AP (R CT ¼ 3250 U).Lines -fits to the equivalent circuit (inset).

Fig. 6 A
Fig.6A phosphorylation process with CaMK2.Black (-)the R CT of the peptide monolayer (R CT ¼ 4080 U).Red (C)the R CT of the monolayer following phosphorylation with CaMK2 (R CT ¼ 3600 U).Blue (:)the R CT of the monolayer after phosphorylation with ERK2 (R CT ¼ 390 U).Lines -fits to the equivalent circuit.

Fig. 7
Fig. 7 (a) HDGF 160-174 layer on top of annealed gold substrate.The layer looks very smooth and uniform.The inset is a cross section along the green line (on single gold terrace) with RMS roughness of $0.3 nm.(b) Square scratch in the peptide layer.The inset is a profile along the green line, showing a $2 nm height difference between scratched and non-scratched areas.(c) An example of several small flat areas for roughness analysis on a single gold terrace.(d) Roughness distribution of $60 areas at macroscopically distant scans before and after phosphorylation.

Table 1
A list of phosphoproteins and their phosphorylation sites that show increased levels in lung cancer tissue compared to adjacent normal tissue a