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A multiplexed quantitative MALDI MS approach for assessing activity and inhibition of protein kinases based on post-enrichment dephosphorylation of phosphopeptides by MOF-templated porous CeO2 Hongmei Xu, Meng Liu, Xiaodan Huang, Qianhao Min, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01938 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Analytical Chemistry

A multiplexed quantitative MALDI MS approach for assessing activity and inhibition of protein kinases based on postenrichment dephosphorylation of phosphopeptides by MOFtemplated porous CeO2 Hongmei Xu, Meng Liu, Xiaodan Huang, Qianhao Min*, and Jun-Jie Zhu State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China * Phone & Fax: +86-25-89681977; E-mail: [email protected]

ABSTRACT: Protein kinase is regarded as a potential target for anticancer therapeutics due to its relation to many diseases, in which more than one kinase participates in the cell signaling cascades. We herein demonstrate a multiplexed quantitative matrixassisted laser desorption/ionization mass spectrometry (MALDI MS) approach to simultaneously assess the activity and inhibition of multiple protein kinases. In this design, substrate peptides phosphorylated by kinases of interest are specifically harvested by MOF-templated porous CeO2, and consequently transformed to the dephosphorylated forms due to the phosphatase-like activity of CeO2, resulting a unique quantitative MS signal with an enhanced intensity. Based on the peak area ratios of dephosphorylated variants of the phosphorylated product to respective deuterated internal standard, the activity and inhibition of each kinase can be independently profiled. In addition to the accurate characterization of protein kinase A (PKA) activity and inhibition induced by H89, the multiplexing capability of the MS-based method allowed quantitative evaluation of the activity of Abl and Src, the two tightly associated kinases in the occurrence of chronic myeloid leukemia (CML) in a multiplexed format, exhibiting excellent orthogonality for the dual signal readout channels. Moreover, the inactivation of both Abl and Src by the inhibitor imatinib, dasatinib and ponatinib was simultaneously traced, giving a whole picture of the competition behavior between the kinases for binding therapeutic molecules. This approach holds great promise in global investigation of kinase signal pathways and high-throughput screening of effective protein kinase inhibitors.

Protein phosphorylation mediated by protein kinases is a ubiquitous post-translational modification in organism, which governs many aspects of cell signaling.1,2 Many diseases, especially cancer, have been proved to be closely linked to the dysregulation of a cascade of protein kinases, which may transmit limitless growth signals and render cell proliferation with impunity.3,4 In order to block the hyperactivation of protein kinases, a vast number of kinase inhibitors have been extensively explored as anticancer drugs.5,6 Therefore, measuring the activity and inhibition of proteins kinases in a multiplexed and quantitative format is highly significant and desirable in fundamental cell biology research, clinical diagnostics or anticancer drug development. To date, various approaches in terms of analytical technology have emerged to detect the protein kinase activity with the adoption of substrate peptides containing the kinase-specific consensus motif. Electrochemical methods, ranging from direct electrochemistry7 to photoelectrochemistry8,9 and electrochemiluminescence,10 have been extensively explored to reflect the population of phosphorylated substrates by incorporating the signal generators like enzymes, quantum dots and electroactive materials with the ligands that can specifically recognize phosphorylated residues. Another prevailing route for sensing kinase activity is fluorescence-based kinase assays, where substrate peptides combined with fluorogenic molecules,11 luminescent nanomaterials12,13 or green fluorescent protein (GFP)14 were rationally designed in an alone or collab-

orative format for signal-on/off modes upon phosphorylation on the target sites. Despite the excellent sensitivity and reproducibility of electrochemical and fluorescence approaches, the laborious procedures for labeling of electroactive and fluorescent moieties limited their applicability in simultaneous monitoring of multiple kinases. As a label-free analytical technology, mass spectrometry (MS) identifies the target analytes by directly providing molecular weight information as the reliable qualitative index. Furthermore, unlike the spectral overlap encountered in fluorescent assays, mass spectrum is able to accommodate hundreds of ion signals, offering premise for multiplexed analysis of protein kinases. However, the relatively low abundance and ionization efficiency of phosphorylated substrate peptides compared to non-phosphorylated counterparts diminishes MS interrogation sensitivity.15 Meanwhile, the neutral loss of phosphoric acid during ionization process presents a set of redundant side products with metastable peaks, thereby complicating the quantification dependent on the peak intensity of phosphorylated substrates.16 The ideal MS signal output for quantification should be unambiguous peaks exclusively assigned to each kinase-mediated phosphorylation event. In these regards, intensifying the ion signal of phosphopeptides while avoiding their further derivatization is highly desirable in quantitative MS-based kinase assay. Separation and pre-concentration of phosphopeptides prior to MS interrogation would serve as the favorable means to clarify the target products in mass spectrum. Recent work in sam-

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ple preparation for phosphoproteomic research has provided plenty of separation media qualified to purify target phosphopeptides.17,18 Unfortunately, these techniques only partially solved the aforementioned issues by enhancing the ion signal of phosphorylated substrates but remained incapable to tackle the undesired peak cluster coming from neutral loss. Among the family of phosphate affinity probes, cerium oxide was emerging as a mimic phosphatase that induces dephosphorylation after capturing the phosphate groups of macromolecules, yielding a proxy of the phosphopeptides with a mass shift of 80 Da.19-21 This natural characteristic helps to generate an intensified and unique signal for every phosphorylated substrate, laying a solid foundation for MS-based kinase activity evaluation. Recently, metal-organic frameworks (MOFs) with periodic porous structure have been proposed as the absorbent for phosphopeptides.22,23 But actually, the inner pores of MOFs remained unavailable due to the inaccessibility of peptides to ultra-small pores (~1 nm). Recruiting the MOFs as precursors, recent work has presented a myriad of MOF-derived porous or hollow metal oxide nanostructures by thermal treatment,24 which has already found their applications in energy storage,25 catalysis,26 optoelectronics and so on. Regarding the porous structure and tailorable composition, we have been evaluating the possibility of MOF-templated metal oxides as an alternative to extract biomolecules instead of MOFs. In this work, we fabricated MOF-templated porous CeO2 nanostructures as the separation medium for capturing phosphorylated substrate peptides, and also as the catalyst for facilitating the postenrichment dephosphorylation, offering unique signature peptides to indicate the degree of kinase-catalyzed reaction (Scheme 1a). Capitalizing upon the signal multiplexing ability of MS technology, we proposed a multiplexed and quantitative matrix-assisted laser desorption/ionization (MALDI) MS strategy for assessing the activity and inhibition of protein kinases. Taking advantage of MOF-templated porous CeO2, concentrated and dephosphorylated signature peptides were obtained from the products of kinase-catalyzed reaction, thus contributing to highly sensitive ion signal and easy interpreted mass spectra. Phosphopeptides whose sequence is identical with the phosphorylated substrates were adopted as the internal standards and underwent dimethyl isotope labeling with deuterated formaldehyde (CD2O), which were further combined with light-labeled phosphorylated peptides before the pretreatment with porous CeO2. The activity and inhibition of each kinase can be independently profiled in a multi-kinase regulated system, according to the MS peak area ratio of signature peptides to deuterated internal standards (Scheme 1b). Using Abl and Src kinases as typical models, we also showed the feasibility in investigating the susceptibility of highly homologous kinases in signaling pathways to one certain inhibitor, paving the way for accurate screening of kinase inhibitors as cancer therapeutic agents. EXPERMENTAL SECTION Preparation of Ce-MOF and Porous CeO2. Ce-MOF was synthesized according to previous literature27 with some modification. Briefly, 1 mmol H3BTC in 3 mL ethanol and 10 mmol CH3COONa in 10 mL ultrapure water were mixed together. Subsequently, 1 mmol Ce (NO3)3·6H2O dissolved in 20 mL ultrapure water was slowly added under vigorous stirring and the mixture was incubated for 1 h at 60 °C. The resulting precipitates were separated by centrifugation and washed several times with ethanol and water, followed by drying process at 70 °C in oven. The porous CeO2 was obtained by calcining

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Scheme 1. (a) Post-enrichment dephosphorylation of phosphopeptides mediated by MOF-templated porous CeO2 for generating unique and intensified ion signal. (b) Mass spectrometric evaluation of activity of multiple kinases by dimethyl isotope labeling and post-enrichment dephosphorylation of resulting phosphorylated substrates.

templated Ce-MOF at 650 °C in muffle furnace for 3 h with a ramping rate of 2 °C/min. Eventually, flavescent powders were obtained and suspended in water with a final concentration of 10 mg/mL. Experiments of phosphopeptide enrichment by different materials and the detection of substrate peptide phosphorylated by Abl kinase are supplied in Supporting Information. PKA Activity and Inhibition Assay by Quantification of Phosphorylated Substrate. Typically, phosphorylation of 0.5 µg/mL PKAtide (LRRASLG, Mw=771.92) was mediated by different amount of PKA in 100 µL of 50 mM pH 7.5 HEPESNaOH buffer solution containing 20 mM MgCl2 and 50 µM ATP at 30 °C. After reaction for a given time, the products were labeled by dimethyl labeling reagents containing 3 µL of 4 % (V/V) CH2O and 3 µL of 0.6 M NaBH3CN, while 0.5 µL of standard phosphorylated peptides (LRRApSLG, Mw=851.92, 0.1 mg/mL) were labeled by CD2O as the internal standard. After incubated at room temperature for 30 min, the phosphorylated products and the internal standard were pooled and diluted to 200 µL by loading buffer containing 6 % TFA in 80 % ACN. Then 20 µL porous CeO2 (10 mg/mL) were dispersed into above mixture and vibrated in a vortex for 60 min. After the supernatant was removed by centrifugation, the phosphopeptides-conjugated CeO2 nanoparticles were washed three times by 200 µL of 80 % ACN buffer containing 3 % TFA. Finally, dephosphorylated products along with the internal standard were eluted in 10 µL of 10 % NH3·H2O and then analyzed by MALDI-TOF MS. For the PKA inhibition assay, different concentrations of H-89 were incubated with a certain amount of PKA (50 U/mL) before the phosphorylation reaction. The following procedures for the phosphorylation of substrate peptide, dimethyl labeling and extraction were similar to those stated above. Procedures for quantitative analysis of PKA in cell lysates are described in Supporting Information. Simultaneous Determination of the Activity and Inhibition of Multiple Protein Kinases. To detect the activity of Abl and Src kinases, 2.5 µg/mL Abltide (EAIYAAPFAKKK, Mw=1336.60) and 1.25 µg/mL Srctide


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Analytical Chemistry (GEEPLYWSFPAKKK, Mw=1679.94) were phosphorylated by different amount of Abl and Src kinases in 20 µL of 50 mM pH 7.5 HEPES-NaOH buffer containing 20 mM MgCl2, 2 mM MnCl2, 50 µM DTT, 200 µM ATP and 0.1 mg/mL BSA. After incubated at 30 °C for 3 h, the reaction solution was digested by 2.5 µg/mL α-chymotrypsin at 37 °C for 1 h before being labeled by CH2O. Standard phosphopeptide mixture containing 0.5 µL of phosphorylated Abltide (EAIpYAAPFAKKK, Mw=1416.60, 0.1 mg/mL) and 0.25 µL of phosphorylated Srctide (GEEPLpYWSFPAKKK, Mw=1758.95, 0.1 mg/mL) were under the same treatment except that labeling reagents were replaced by CD2O. The next pretreatment procedure was the same as mentioned above. For the triplex detection of PKA, Abl and Src kinases, the only modification was that the substrate formula was changed to 1.5 µg/mL PKAtide, 5 µg/mL Abltide and 3 µg/mL Srctide. Cross reactivity experiment and orthogonality test are described in Supporting Information. For multiplexed kinase inhibition assay, three different inhibitors, imatinib, dasatinib and ponatinib were respectively incubated with a certain amount of Abl and Src kinases before phosphorylation reaction. Phosphorylation of substrate mixture, dimethyl labeling and enrichment were conducted in the same way mentioned in the preceding section. MALDI-TOF MS Analysis. For MALDI-TOF MS analysis, 1 µL of eluent and 0.5 µL of 2, 5-dihydroxybenzoic acid (DHB) solution (25 mg/mL in 70 % ACN, 1 % H3PO4) were deposited on a steel MALDI plate. Data acquisition was performed on a 4800 Plus MALDI-TOF/TOF Mass Spectrometer (AB Sciex) equipped with a Nd:YAG laser emitted at 355 nm in a positive reflection mode. MS spectrum was the average of 16 sub-spectra obtained from the edge bias of matrix spot with sum of 25 laser shots per sub-spectrum. RESULTS AND DISCUSSION Characterization of MOF-Templated Porous CeO2. The MOF-templated porous CeO2 was prepared by thermal decomposition of Ce-MOF templates, which was constructed by Ce (III) ions with the ligands of 1,3,5-H3BTC. Thermogravimetric analysis (TGA) was performed to optimize the calcination temperature. The TGA curve displayed a two-step mass loss assigned to evaporation of adsorbed or crystallization water and organic ligands at around 130 °C and 620 °C (Figure S1), and thereby the decomposition was carried out at 650 °C for 3 h in final. As shown in Figure 1a-c, porous nanoaggregates of 30 nm-sized nanoparticles were harvested with a rod-like structure after thermal annealing, which kept the similar morphology with the Ce-MOF precursor (Figure S2). Because of complete transformation from organic ligands to small gaseous molecules, concave and rough surface along with more exposed metal affinity sites appeared, which may strengthen the capability of capturing phosphopeptides and avoid the non-specific absorption originated from aromatic ligands. The dominant {220} and {111} lattice fringes in HRTEM image (Figure 1d) and detectable diffraction rings in SAED pattern (Figure 1e) all confirmed CeO2 as the only cerianite crystalline phase. X-ray diffraction (XRD) was also conducted to reflect the degree of crystalline structure transformation from the Ce-MOF precursor to porous CeO2. As shown in Figure 1f, the bulk phase of Ce-MOF matched well with the stimulated XRD patterns of La(1,3,5-H3BTC)(H2O)6 (CCDC: 290771).28 According to the XRD pattern of CeO2 (Figure 1g), the Ce-MOF was completely converted into its oxide derivatives, which was proved by its strong and incisive diffraction peaks indexed to JCPDS card 34-0394 (cerianite).

Figure 1. Characterization of Ce-MOF and porous CeO2. (a-c) SEM and TEM images of MOF-templated porous CeO2. (d, e) High resolution TEM image and selected area electron diffraction (SAED) pattern of porous CeO2. (f, g) XRD patterns of the CeMOF precursor and the derived porous CeO2 in comparison with the simulated XRD pattern of La(1,3,5-H3BTC)(H2O)6 and documented XRD patterns of JCPDS card 34-0394.

The nitrogen adsorption-desorption experiment reveals a favorable Brunauer–Emmett–Teller (BET) surface area (68.4 m2g-1) and a type IV isotherm (Figure S3a). As shown in the Barrett-Joyner-Halenda (BJH) pore size distribution curve (Figure S3b), several peaks were found to be located in mesopore (2.5 nm) and macropore (45 nm and 85 nm) regions, corr-esponding to the pore structures formed by the stack of CeO2 nanoparticles. X-ray photoelectron spectroscopy (XPS) was performed to confirm the elemental composition and valence states of Ce element, which are closely related to the catalytic activity of porous CeO2. In addition to contaminant carbon species, characteristic peaks derived from O 1s, Ce 3d and Ce 4d electrons indicated the formation of cerium oxide (Figure S4). In the deconvoluted high-resolution XPS spectrum of Ce 3d, the peaks at 882.5, 888.2, 898.1, 900.8, 907.3 and 916.5 eV respectively assigned to v, v″, v ‴, u, u″ and u‴ were all attributed to Ce (IV),29 implying that the valence state was fully shifted from Ce(III) to Ce(IV) along with the transformation from the Ce-MOF precursor to porous CeO2 during thermal annealing. Enrichment and Dephosphorylation of Phosphopeptides by Porous CeO2. For MS-based kinase assays, separation and enrichment of the resulting phosphopeptides from unreacted substrates is an effective way to intensify the ion signal of products and consequently improve the detection sensitivity. In the beginning, we investigated the capability of porous CeO2 for phopshopeptide enrichment in comparison with the Ce-MOF precursor, commercial CeO2 and TiO2 nanoparticles. As the conventional separation probe for capturing phosphopeptides, commercial TiO2 nanoparticles extracted 3 detectable phosphorylated peptides (m/z 2060.96, 2555.03 and 3120.90) from 16 pmol tryptic digest of β-casein, as observed in the MALDI-TOF mass spectrum (Figure 2a, detailed peptide sequences are listed in Table S1). However, neutral loss ions (M-HPO3 and M-H3PO4) for the phosphopeptides β1 (m/z 1980.02 and 1962.95) and β2 (m/z 2456.98)



Figure 2. MALDI-TOF mass spectra of tryptic digest of β-casein (16 pmol) treated with (a) commercial TiO2 nanoparticles, (b) CeMOF precursor, (c) commercial CeO2 and (d) porous CeO2. Pound signs (#) represent dephosphorylated products of corresponding phosphopeptides.

with considerable abundance were also visualized in the MS spectrum, which complicated the ion intensity-based peptide quantification. In addition, the tetra-phosphorylated peptide β4 exported extremely low ion signal in the positive ionization mode, due to its highly negative charge state. To avoid counting undesired neutral loss by-products for quantification, we turned to exploit cerium-related compounds to first capture phosphopeptides via Lewis acid-base interaction and then produce dephosphorylated peptides by hydrolyzing phosphate esters, which is termed post-enrichment dephosphorylation here. After the treatment with cerium-based materials, CeMOF precursor, commercial CeO2 and porous CeO2, all the four phosphopeptides, including the tetra-phosphorylated peptides β3 and β4, were harvested and observed in the form of dephosphorylated fragments in MS spectra (Figure 2b-d). Notably, the Ce-MOF precursor also co-extracted a vast number of non-phosphopeptides that disturbed the spectrum interpretation, presumably ascribed to the nonspecific absorption on organic ligands in MOFs (Figure 2b). Because of the small pore size (~ 1 nm) and insufficient exposure of cerium ions in Ce-MOF structure, the dephosphorylated products exhibited lower MS intensity than those obtained with porous CeO2 (Figure 2d). For the commercial CeO2 nanoparticles, the MS profile similar to Ce-MOF precursor was obtained (Figure 2c), probably due to the less active surface area in comparison with MOF-templated porous CeO2. With this consideration, we selected MOF-templated porous CeO2 as the separation and catalytic medium for executing post-enrichment dephosphorylation of phosphorylated substrates that reflect kinase activity. To test the applicability of porous CeO2 in extraction of products from a real kinase-mediated phosphorylation reaction solution, Abltide was employed as a model substrate peptide phosphorylated by Abl kinase and subjected to the pretreatment with porous CeO2. From MALDI-TOF mass spectrum of the reaction solution incubated for 1 h (Figure 3a), we can find a barely visible peak at m/z 1417 (S/N=8.08) derived from the phosphorylated Abltide (P-Abltide), in contrast to the predominant peak at m/z 1337 coming from the unreacted Abltide. In comparison, porous CeO2 picked out the resulting phosphopeptides and removed the phosphate group to yield a signal at m/z 1337 with an almost 10-fold improved S/N ratio

Figure 3. MALDI-TOF mass spectra of the substrate Abltide (1.16 µg/mL) phosphorylated by 0.2 µg/mL of Abl kinase (a) before and (b) after treatment by porous CeO2. Phosphorylated Abltide and its dephosphorylated product are denoted as PAbltide and D-Abltide, respectively. (Figure 3b). To exclude probable contribution from unreacted Abltide to this ion signal, Abltide solution incubated without Abl kinase was treated with porous CeO2 as control. The MS spectrum showed that no signal located at m/z 1337 was detected in the eluent (Figure S5), demonstrating the peak at m/z 1337 was fully originated from dephosphorylation of PAbltide other than nonspecific adsorption of the remaining Abltide. As compared to direct MS interrogation of reaction solution, porous CeO2-mediated post-enrichment dephosphorylation effectively eliminated the interference from highly abundant substrates and significantly enhanced the quantitative ion signal of phosphorylated peptides. Besides, specificity test was performed by replacing Abltide with a negative control peptide (EAIAAAPFAKKK, Mw=1244.73). As shown in Figure S6, no phosphorylated peptide was observed, indicating specific recognition of substrate peptide by Abl kinase. Quantitative Evaluation of PKA Activity and Inhibition. Stable isotopic labeling is an ideal strategy to create an internal standard for the target molecule, with identical properties during sample processing and ionization, except for the only difference on the mass to charge ratio that can be recognized by mass analyzer. In this context, the inexpensive dimethyl labeling strategy was used to quantify the concentration of substrates phosphorylated by protein kinase. As illustrated in Scheme 1b, the substrate peptide phosphorylated by protein kinases and a known amount of the standard phosphopeptide with same sequence were respectively labeled by CH2O and CD2O, and were pooled for phosphopeptide extraction with porous CeO2. Due to the phosphate affinity and mimic phosphatase activity of CeO2, the enriched phosphopeptides containing light-labeled phosphorylated substrates and heavylabeled internal standards were detected as dephosphorylated forms. The activity and inhibition of protein kinase can be evaluated by quantification of phosphorylated substrates based on the varied ion intensity ratio of the signature peptides to deuterated internal standards. In this work, the reaction between PKA and its substrate PKAtide was adopted to demonstrate the feasibility of proposed strategy. As shown in Figure 4a, compared with the dephosphorylated signature peptide (DPKAtide, LRRASLG, m/z 772), the product peptide and internal standard (LRRApSLG) which experienced dimethyl labeling and CeO2-mediated post-enrichment dephosphorylation presented an intensive peak pair at m/z 800 and 804 in the spectrum. Hence, the activity and inhibition of PKA could be assessed by the signal intensity ratio of the doublet. To verify the accuracy in quantification of phosphorylated PKAtide, a calibration curve was plotted by recording the peak


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Figure 4. (a) Representative MALDI-TOF mass spectra of PKAtide (0.5 µg/mL) phosphorylated by 50 U/mL PKA (i) before and (ii) after treatment with porous CeO2. (iii) The resulting phosphorylated PKAtide was combined with internal standard and subjected to dimethyl labeling and post-enrichment dephosphorylation. (b) Normalized MALDI-TOF mass spectra of PKA inhibition assay in the presence of different concentrations of H-89 (0.05 nM to 15 µM). The red line refers to blank control without H-89. Experimental condition of PKA inhibition assay: PKAtide, 0.5 µg/mL; ATP, 20 µM; internal standard, 0.5 µL of 0.1 mg/mL P-PKAtide. Phosphorylated PKAtide and dephosphorylated products are respectively denoted as P-PKAtide and D-PKAtide, and light- and heavy-labeled dephosphorylated products are denoted as L-D-PKAtide and H-D-PKAtide. (c) The relationship between peak area ratio of the doublet (m/z 800 and 804) and the concentration of PKA in diluted cell lysates. Inset shows a linear correlation of intensity ratio to the logarithmic concentration of PKA in the range of 2−50 U/mL.

area ratio with varied light-labeled phosphopeptide and the constant heavy-labeled internal standard (0.5 µL of 0.1 mg/mL P- PKAtide). As shown in Figure S7, the peak area ratio was linearly correlated with the concentration ratio over the range from 0.05:1 to 1:1, and the regression equation was represented as AH/AD=1.1CH/CD-0.033 with the R-squared value of 0.99. Enc -ouraged by the good feasibility in quantification of phosphorylated PKAtide, we next investigated the inhibition of PKA by a well-recognized inhibitor H-89 using the above calibration curve. The inhibition experiment was carried out by incubating 50 U/mL PKA with a varying amount of H-89, which subsequently catalyzed the phosphorylation of PKAtide. As the concentration of H-89 rose from 0.05 nM to 15 µM, the yield of phosphorylated PKAtide was gradually suppressed, as indicated by the step-by-step lowered intensity of MS peak located at m/z 800 (Figure 4b). The dose-dependent inhibition curvewas plotted with the IC50 value calculated as 149 nM (Figure S8), which was comparable to previous reports.30 Quantification of PKA in Cell Lysates. In addition, to demonstrate the applicability of kinase assay in real biosamples, MCF-7 cells were stimulated by forskolin and IBMX and lysed to examine the endogenous PKA activity. The substrate PKAtide was phosphorylated by 10-fold diluted cell lysates and then enriched and dephosphrylated by porous CeO2. As illustrated in Figure S9a, the phosphorylated products were found as its dephosphorylated form with a mass peak situated at m/z 772, which disappeared in the MS spec-

trum of the lysates blank without PKAtide (Figure S9b). Successful detection of PKA-phosphorylated substrates in cell lysates was further confirmed by the ion signal from dimethyl labeled signature peptides (L-D-PKAtide, m/z 800) after extraction (Figure S9c and 9d). Furthermore, the reliability of quantitative approach for protein kinase in complex biological samples was also evaluated by the determination of the spiked PKA in cell lysates. Figure 4c depicted the elevated MS peak area ratio (AH/AD) with the increase of kinase concentration, which showed a good linear relationship with the logarithm of kinase concentration from 2 to 50 U/mL. The regression equation was represented as AH/AD =0.066CPKA+0.022 with the Rsquared value of 0.99. Different concentrations of PKA were spiked in diluted cell lysates with the protein content of 1 µg/mL, and the recoveries were distributed from 96.4% to 106.4% (Table S2), thus proving the good applicability and reliability of the protein kinase assay in real samples of biological complexity. Simultaneous Quantitative Evaluation of Activity of Multiple Protein Kinases. The cell signaling cascade implicates a myriad of phosphorylation events regulated by more than one protein kinase, among which one may act as the substitute, substrate or activator of another one.31,32 In this regard, developing a multiplexed protein kinase activity assay is significant in understanding the functions of a vast array of protein kinases in cell signaling pathways. On the other hand, due to crosstalk of kinases and pathways reprogramming, resistance of drugs is still a challenging problem in the development of protein kinase inhibitors.33 Therefore, simultaneously assessing the inhibitor-induced inactivation of correlated protein kinases would profile the competition kinetics of multiple kinases for binding the therapeutic molecules and provide an insight into screening of multi-targeted inhibitors for deregulated protein kinases. As a typical case, Src and Abl kinases are known as relational couples in the occurrence of chronic myeloid leukemia (CML) and their active forms resemble one another. To demonstrate practical application of the proposed method in activity estimation of multiple protein kinases, Abl and Src kinases were selected to mix and catalyze the phosphorylation of their respective substrates (Abltide: EAIYAAPFAKKK, Srctide: GEEPLYWSFPAKKK) in one reaction vial, and the phosphorylated peptides were captured, dephosphorylated by porous CeO2 and analyzed by MALDI-TOF MS. As opposed to the predominant peaks corresponding to unreacted substrates (Abltide and Srctide) and their sodium adducts in the peptide mixture, two unambiguous ion signals of dephosphorylated signature peptides reflecting the degree of phosphorylation were distinctly observed in one spectrum (Figure 5a, b). Considering the incompleteness of dimethyl labeling reaction at rather abundant lysine residues of Abltide and Srctide, we utilized α-chymotrypsin, a protease specific for aromatic residues, to truncate the as-phosphorylated peptides to shorter segments (denoted as tru-P-Abltide EAIPYAAPF and tru-PSrctide GEEPLPYW) with the only N-terminal amine. After αchymotryptic digestion and dimethyl labeling, the peptide mixture was combined with deuterated internal standards and subjected to CeO2-mediated enrichment and dephosphorylation. The MALDI-TOF mass spectrum revealed two groups of twin peaks with mass difference of 4 Da referring to Abl and Src kinases, with the peak area ratio as the quantitative basis for phosphorylated Abltide and Srctide (Figure 5c). Since the two kinases possess a high degree of similarity in



Figure 6. (a) MALDI-TOF mass spectrum of enriched peptides phosphorylated by PKA, Abl and Src in K562 cell lysates (the protein concentration was 40 µg/mL). (b) MALDITOF mass spectrum of enriched phosphopeptides after dimethyl labeling and internal standard addition. (c) The peak area ratio of doublets of PKA, Abl and Src in response to different stimulations.

Figure 5. Representative MALDI-TOF mass spectra for evaluation of multiple kinases activity. MALDI-TOF mass spectra of Abltide (2.5 µg/mL) and Srctide (1.25 µg/mL) phosphorylated by Abl (3 µg/mL) and Src (5 µg/mL) kinase (a) before and (b) after treatment with porous CeO2. (c) Mass spectrum of kinasephosphorylated Abltide and Srctide mixed with respective internal standards (1.25 µg/mL P-Abltide and 0.625 µg/mL P-Srctide) after α-chymotryptic digestion, dimethyl labeling and postenrichment dephosphorylation. (d) Mass spectrum for triplex detection of PKA, Abl and Src kinases by the proposed method. The inset shows the relationship between respective peak area ratios with different amounts of kinases. The concentration of PKA, Abl and Src in the kinase mixture was 25 U/mL, 1.32 µg/mL and 5 µg/mL, and then expanded 1.5- and 2.0-fold. Pound signs (#) represent sodium adducts of substrate peptides, and phosphorylated Abltide and Srctide and their dephosphorylated products are denoted as P-Abltide, P-Srctide, D-Abltide and DSrctide respectively. Light and heavy labeled dephosphorylated products of Abltide and Srctide truncated by α-chymotrypsin are donated as L-D-tru-Abltide, H-D-tru-Abltide, L-D-tru-Srctide and H-D-tru-Srctide.

sequence (47%) and structural scaffolds,34 the possibility of cross reaction between one kinase and the other’s substrate should be taken into consideration. As demonstrated in Figure S10, there was no ion signal on behalf of phosphorylated Srctide under the catalysis by only Abl kinase, and vice versa, confirming exclusive recognition between kinase and target peptide. Orthogonality between the dual channels of Src and Abl kinase influences the reliability and accuracy for assaying the activity of individual kinase. In principle, because of the intermolecular competition for ionization, concentration change of co-existing molecules leads to suppression or enhancement of the target ion signal in mass spectrum. In this approach, the peak area ratio of phosphorylated substrate peptides to its deuterated internal standard is supposed to be irrespective of the disturbance from external circumstance, due to their theoretically identical behavior during pretreatment and ionization. The orthogonality was tested by measuring the

peak area ratios of light-labeled tru-P-Abltide to heavy-labeled internal standard (denoted as Abl AH/AD) varying with different amounts of P-Srctide. The degree of disturbance could be simply represented as the slope of fitting curve, plotted by the Abl AH/AD under perturbation of increasing P-Srctide against the value without perturbation (Figure S11). The closer the slope was to 1, the more orthogonal the two channels were. The results showed that little effect of extra P-Srctide (0.375 and 0.625 µg/mL) on the Abl AH/AD with the slopes fitted as 0.98 and 1.13 (Figure S11a). Likewise, interference of PAbltide (1.25 and 2.5 µg/mL) to the Src AH/AD was expressed by linear fitting curves with slopes of 1.00 and 0.97 (Figure S11b), further confirming the orthogonality between Abl and Src kinase activity channels in this multiplexed MS-based assay. The satisfactory orthogonality allows us to plot calibration curves by gathering the peak area ratios corresponding to Abl and Src kinases in the same spectrum. As illustrated in Figure S12, regression equations for Abl and Src kinases are represented as AH/AD=0.98CP-Abltide/CIS-Abl-0.013 and AH/AD=0.99CP-Srctide/CIS-Src-0.056, with the R-squared values of 0.999 and 0.995, respectively. Next, we further demonstrated the multiplexing capability of this approach by triplex detection of PKA, Abl and Src kinases. As shown in Figure 5d, three doublets of phosphorylated substrates and internal standards respectively corresponding to PKA, Abl and Src kinases were explicitly recognized in a mass spectrum-resolved manner at the same time. As can be found in Figure 5d inset, the MS peak area ratio indicative of each kinase activity was linearly increased in parallel with the synchronously enlarged kinase concentrations, reflecting the ability of this multiplex method to simultaneously output quantitative information on each of the kinases. Human erythroleukemic cell line K562 was chosen to detect the endogenous multiple protein kinases in cell extract and the results were shown in Figure 6. Similarly, dephosphorylated products were clearly observed (Figure 6a) and each kinase activity could be easily assessed (Figure 6b) after introducing dimethyl-labeled signature peptides. The control experiments indicated that the signature peptides were indeed derived from the substrate peptides phosphorylated by intracellular kinases (Figure S13). After stimulated by 0.2 mM Na3VO4, a tyrosine phosphatase inhibitor, all kinase activity was increased and Src kinase showed a superior improvement (1.44 fold) compared to Abl (1.15 fold) and PKA (1.21 fold), while forskolin and IBMX exhibited exclusive activation to


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Figure 7. Dose-response curves of the kinase inhibitor (a) imatinib, (b) dasatinib and (c) ponatinib determinated by the proposed method. Reaction mixtures for the kinase inhibition assay in the presence of imatinib or dasanitib: Abltide, 5 µg/mL; Srctide, 3 µg/mL; Abl kinase, 1.1 µg/mL; Src kinase, 5 µg/mL; ATP, 200 µM. Reaction mixtures for the kinase inhibition assay in the presence of ponatinib: Abltide, 2.5 µg/mL; Srctide, 3 µg/mL; Abl and Src kinase, 5 µg/mL; ATP, 200 µM.

PKA (1.5 fold). Therefore, the developed multiplexed quantitative approach can be used to determine the activity of multiple kinases in real biological samples. Simultaneous Profiling of the Inhibition of Abl and Src Kinases by Anticancer Therapeutics. Bcr-Abl oncoprotein has proved to be a hallmark of CML, while presenting an attractive drug target for the treatment.35 As an ATP-competitive inhibitor, imatinib was the preferred therapeutic agent for CML. However, most advanced-phase CML patients show resistance and clinical relapse on imatinib therapy.36 Recent reports have explained that the up-regulated Src family proteins might partially substitute for Abl kinase activity to induce relevant signaling pathways and thus result in disease progression and resistance in patients treated with imatinib.37,38 Therefore, screening of dual-specific Abl/Src inhibitors is a promising strategy to overcome imatinib resistance in CML therapy. With the ability of simultaneously presenting the activity of multiple kinases, our proposed methodology offers an ideal platform for monitoring the activity fluctuation of Abl and Src kinases in the presence of inhibitors, thereby potentially creating a new toolkit for the selection of dual inhibitors. In this study, different generations of kinase inhibitors ranging from imatinib, dasatinib to ponatinb were tested for investigating the susceptibility of Abl and Src kinases to various inhibitors. Each inhibitor was separately incubated with the mixture of Abl and Src kinases, and the concentrations of phosphorylation products were also obtained in the same way stated above. In the dose-response curves of a certain inhibitor constructed for Abl-Src binary system, we defined IC50Abl vs Src as the concentration of the inhibitor where the Abl kinase activity is reduced by half in the presence of Src. As shown in Figure 7a, concentration-dependent inhibition curves of imatinib to both Abl and Src kinases were plotted. The IC50Abl vs Src and IC50Scr vs Abl values of 26.46 and 147.0 µM implied the preference of imatinib for targeting Abl kinase rather than Src kinase, which is rationalized by overexpression of Src kinase in imatinibresistant patients. Differing from the inhibitory potency obtained by incubating the inhibitor with single kinase, the multiplexed kinases activity assay provides a more genuine profile of discriminative efficacy on the two kinases, enforcing multitargeted drug screening in a single vial. From another perspective, most kinase inhibitors obey the competitive inhibition principle that binding of an inhibitor impedes binding of ATP molecules on the active sites.39 Given the coexistence of Abl and Src kinases in the reaction mixture, it is anticipated that there must be a trade-off between the kinases for capturing inhibitor molecules. As displayed in the inhibition double-

curve, Src kinase reactivity is not affected until imatinib accumulates to 31.62 µM, while the inhibition to Abl kinase has reached up to above 60%. This means the presented dosedependent double-curve sketches the competitive behavior of Abl and Src kinases for binding small molecule drugs, which is crucial to shed some light on the imatinib’s binding specificity39 but unfortunately cannot be described by the single kinase measurement. As an alternative of imatinib, dasatinib is currently used in the case of imatinib therapy failure.40 Figure 7b shows similar response of the two kinases to dasatinib with comparable IC50 values, coinciding with the reported function of dual-specific Abl/Src inhibition.41 The nanomolar concentration level also manifests its higher blocking potency than the previous generation imatinib. Similarly, another dualspecific inhibitor ponatinib exerts unbiased influence to Abl and Src kinases with close IC50 values and almost overlapped curve traces (Figure 7c). These performances indicate that the multiplexed quantitative MALDI MS approach affords a powerful tool for profiling the drug binding specificity to highly homologous kinases and screening effective multi-targeted kinase inhibitors. CONCLUSIONS In summary, a multiplexed quantitative MALDI MS approach has been developed for simultaneously monitoring the activity and inhibition of multiple protein kinases with the aid of dimethyl isotope labeling strategy and post-enrichment dephosphorylation based on MOF-templated porous CeO2. By virtue of the admirable properties of porous CeO2 in both enrichment and dephosphorylation of phopshopeptides, kinasephosphorylated products can be concentrated from reaction solution and transformed into the dephosphorylated forms, giving a distinct quantitative index instead of having to consider the extra metastable signals from neutral loss. Depending on the purified signal of phosphorylated substrate as well as the deuterated internal standard, we have successfully assessed the activity and inhibition of PKA even in MCF-7 cell lysates. More importantly, thanks to the multiplexing capability of the MS-based method, we were able to quantitatively evaluate the activity of multiple protein kinases with favorable orthogonality in a single run. In addition to the simultaneous determination of Abl and Src kinase activity, the competition behavior of Abl and Src kinases for binding different therapeutic inhibitors have been visualized by profiling the inhibition of the kinase pair caused by imatinib, dasatinib or ponatinib. It is our hope that the multiplexed quantitative MALDI MS approach for phosphorylation recognition will facilitate understanding



the correlation between homologous kinases in cell signal pathways and screening effective multi-targeted kinase inhibitors.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary experimental regents, TGA curve, TEM and SEM images of Ce-MOF, nitrogen adsorption−desorption experiment and XPS characterization of porous CeO2, MALDI-TOF mass spectra of non-phosphorylated Abltide after pretreatment with porous CeO2, specificity test of Abl kinase, inhibition curve of H89, MALDI-TOF mass spectra of labeled substrate peptide phosphorylated by PKA in MCF-7 cell lysates, cross reactivity assay, orthogonality assay, calibration curves for the quantification of PPKAtide and for simultaneous quantification of P-Abltide and PSrctide, MALDI-TOF mass spectra of K562 cell lysates, sequence information of phosphorylated peptides enriched from the digests of β-casein and the results of the spike and recovery test of PKA in cell lysates (PDF).

AUTHOR INFORMATION Corresponding Author * Phone & Fax: +86-25-89681977; E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript

ACKNOWLEDGMENT We gratefully appreciate the support from the National Natural Science Foundation (Grant Nos. 21622505, 21575061, and 21335004), and the Fundamental Research Funds for the Central Universities (020514380167 and 020514380141).

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