Quantitation of N6-Formyl-Lysine Adduct Following

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Quantitation of N6-Formyl-Lysine Adduct Following Aristolochic Acid Exposure in Cells and Rat Tissues by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method Yao Zhao, Chi Kong Chan, K. K. Jason Chan, and Wan Chan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00272 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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Chemical Research in Toxicology

Quantitation of N6‑Formyl-Lysine Adduct Following Aristolochic Acid Exposure in Cells and Rat Tissues by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method

Yao Zhao†, Chi-Kong Chan†, K. K. Jason Chan†, and Wan Chan†,‡,*

† Department of Chemistry and ‡ Division of Environment and Sustainability, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

* Correspondence author. Phone: +852 2358-7370; Fax: +852 2358-1594; E-mail: [email protected]

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ABSTRACT

N6-formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational N6-formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC−MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentrationdependent formation of FLys in cells exposed to formaldehyde and Fe2+-EDTA, an OH radicalmediated oxidant. The study reveals formaldehyde and Fe2+-EDTA produced FLys at a frequency of 20.2 and 4.1 per 104 lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed E. coli cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time- and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic N6formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of N6-formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.

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INTRODUCTION

Understanding the molecular basis of how cellular damage originates is the key to combating cancer and other degenerative diseases. Oxidative stress and inflammation are two of the most common causes of cellular damage, both resulting in the production of reactive electrophilic agents that are known to attack cellular molecules, including DNA, RNA, and proteins to produce secondary damage products.1 These damages are linked to the pathophysiology of cancer and many other human diseases.1 Among the cell-damage related modifications to biomolecules, carbonylation of amino acid residues in proteins has become established as a key biomarker for oxidative stress and tissue pathology.2,3 For example in the process of lipid peroxidation, it was discovered that two reactive aldehydes (formaldehyde and malondialdehyde) were produced and formed covalent adducts with cysteine and lysine residues of proteins.4,5 Studies further revealed that in cells exposed to oxidative conditions, formaldehyde would also condense with lysine residues to give N6-formyl-lysine (FLys, Figure 1) as an abundant and stable biomarker.5 Furthermore, data showed strong correlations between FLys formation and DNA damage.6

Recently, oxidative cellular damage pathways were linked to the toxicology of aristolochic acids (AAs, Figure 1), toxic natural products from the Aristolochia plants.7 These plants have been widely used as herbal medicine or in formulations of proprietary Chinese medicine products.8 Human exposure to AAs, for example through the use of Chinese medicine, is known to induce a unique type of rapid progressive kidney fibrosis named aristolochic acid nephropathy (AAN).9,10 4


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Because of the traditional practice of herbal medicine, particularly in Asian countries, it is estimated that over 100 million people may be at risk of AA-associated toxicity.11

AAs have been classified as one of the known rodent and human carcinogens (Group I) by IARC,12 resulting in tumour formation in the forestomach, renal cortex, renal pelvis, and urinary bladder in rats13-15 and urothelial tumour formation in humans16,17. Recently, chronic food poisoning by AAs ingested through AA-tainted food grains and baking flour was also discovered to be one of the main etiological pathways in the development of Balkan endemic nephropathy (BEN),18-20 a renal fibrotic disease that has affected numerous farmers living alongside tributaries of the Danube River in the Balkan Peninsula for over 60 years.21,22 In addition to its similar clinical and morphological properties to AAN, BEN is also characterized by its coexistence with cancer in the upper urothelial tract.23,24

While the genotoxicity and nephrotoxicity of AAs have long been recognized, the pathophysiological mechanism underlying the destructive renal fibrotic process has remained unclear.25 It was suggested that a reactive aristolactam nitronium intermediate is formed during metabolism of AAs, which could react with DNA to generate adducts linked to the development of upper urothelial cancers and renal fibrosis.21, 26-27 Emerging evidence also points to a potential causative role of oxidative stress in the pathophysiology of AAs.28 For example, elevated levels of methylglyoxal (generated in lipid peroxidation) and depletion of glutathione (cellular reducing agent) were observed in the kidneys of AA-exposed mice.29,30 Under such conditions of oxidative stress, which is comparable to oxidative and nitrosative stresses in response to inflammation, 5



which is also one of the seven hallmarks of cancer31,32, it is common to observe the formation of FLys in tissues. Results from previous studies suggested FLys could play an important role in epigenetic regulation affecting cell signaling and gene expression,6,33 therefore we hypothesized that the formation of FLys may also play a role in the pathophysiology of AAs in humans (Figure 1).

The objective of this study was to define the potential causative role of lysine formylation in the pathophysiological processes induced by AAs in humans. We initiated the study by developing a liquid chromatography-tandem mass spectrometry (LC−MS/MS) method to quantitate the amount of FLys modifications in proteins extracted from aristolochic acid I (AA-I, a predominant and potent nephrotoxic species in the AA family, Figure 1) exposed cells and rats. We then validated this analytical method and determined the overall efficiency of the enzymatic hydrolysis and extraction steps using a synthetic peptide containing a single FLys residue. Using deuterium-labeled FLys (4,4,5,5-[2H]-FLys) as an internal standard, the validated method was then applied to quantitate FLys in proteins isolated from AA-I-exposed Escherichia coli (E. coli) cells and rat tissues. An initial study in E. coli cells exposed to 3 - 24 µM of AA-I clearly showed that the formation of FLys adducts in the isolated bacterial proteins were exposure timeand concentrationdependent. A subsequent in vivo study revealed organotrophic and dosagedependent formation of FLys in protein samples isolated from kidney of AA-I dosed rats. The FLys, representing a well-studied example of pathophysiology, may in part explain the nephrotoxic and genotoxic properties of AAs.

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EXPERIMENTAL SECTION

Chemicals and Reagents. All chemicals and reagents were of the highest purity available and were used without further purification unless otherwise stated. L-Lysine, 9Fluorenylmethoxycarbonyl chloride (Fmoc-Cl), and Streptomyces griseus protease were obtained from Sigma (St. Louis, MO). N6-formyl-lysine was obtained from Santa Cruz Biotechnology (Dallas, TX). Bovine serum albumin (BSA) was purchased from Bio-Rad Laboratories (Hercules, CA). 4,4,5,5-[2H]Lysine was purchased from Cambridge Isotope Laboratories (Andover, MA). Isotope labeled N6-formyl-lysine (4,4,5,5-[2H]-N6-formyl-lysine) was synthesized before by reacting 4,4,5,5-[2H]lysine with acetic anhydride in 98% formic acid.34 A N6-formyl-lysine-containing peptide (Asp-Gly-Ala-Leu-Phe-FLys-Tyr-Glu-Val-Thr) were acquired from GL Biochem (Shanghai, China). Acetonitrile and methanol were purchased from J.T. Baker (Philisburg, NJ).

E. coli Experiment. E. coli (DH5α; ATCC, Manassas, VA) after growing to mid-log phase was harvested by centrifugation. The cells, after being washed twice with phosphate buffered saline (PBS, 11.8 mM, pH 7.4), were resuspended in PBS for AA-I exposure. Around 0.5 g of E. coli cells in 5 mL of PBS was added with formaldehyde, Fe2+-EDTA, or AA-I at final concentrations ranged from 0.5 - 2 mM, 0.5 - 2 mM, and 3 - 24 µM, respectively. After 1 h of exposure, cells were collected by centrifugation, washed thrice with PBS, and then resupended in cell-lysis buffer (50 mM Tris buffer containing 20% glycerol, pH 8.0). The samples were then subjected to ultrasonication at 4 °C, centrifuged (18,000 rpm, 4 °C for 50 min), and the supernatant 7



containing soluble cellular proteins was collected. Saturated ammonium sulfate was then added to the supernatant to precipitate the proteins. The collected protein samples were dissolved in water, followed by addition of the internal standard, then the samples were hydrolyzed using protease as described previously,6, 35 and the concentration of FLys was determined by the LC−MS/MS method described below. Using a similar approach, the time-dependent formation of FLys in E. coli cells were also studied by exposing cells to 3 or 15 µM of AA-I for different durations (0, 0.5, 1, 3, and 5 h).

Animal Experiment. All animal experiments were conducted in accordance with the Animal Ordinance established by the Department of Health, HKSAR. The protocol for animal experiments was approved by the Animal Ethics Committee, HKUST. Sprague−Dawley rats (male, ~200 g) were obtained from the Animal and Plant Care Facility, HKUST. Fifteen rats were randomly divided into three groups. The high-dosage (n = 5) and low-dosage (n = 5) groups received a single oral gavage of 30 and 10 mg/kg of AA-I in 1 mL of 1% sodium hydrogen carbonate solution; the control group (n = 5) received an equal volume of the dosing vehicle. Twenty-four hours after the dosage, the rats were sacrificed by decapitation, and the kidney and liver were harvested. ~100 mg of tissue samples was homogenized in an ice bath, the tissue protein was isolated and digested using the method described above, and the FLys adduct concentration was determined using an isotope dilution LC−MS/MS method described below.

To investigate the kinetics of FLys formation and persistence of the adduct, Sprague−Dawley rats were given a single oral dose (30 mg/kg) of AA-I as the sodium salt in water; control 8


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animals received only sodium hydrogen carbonate solution (1 %, w/w). Rats (3 per group) were killed on 1, 2, 3, and 8 days post-treatment. The kidney and liver were collected, rinsed with 1.15% KCl, and stored at –80°C until protein isolation. The tissue protein was isolated, digested, and analyzed by LC−MS/MS method.

LC−MS/MS Analysis of N6-Formyl-Lysine. FLys was quantified using LC−MS/MS method as described previously,35, 36 with modifications. Amino acids in the protein hydrolysates were first separated on a Waters Acquity UPLC system equipped with a 100 × 2.1 mm inner diameter, 1.7 μm, BEH Amide column (Waters Corporation, Milford, MA). Ten microliters of the sample was injected onto the column, which was eluted at a constant flow rate of 0.3 mL/min at room temperature. The binary solvent system was 0.1% acetic acid in water (A) and acetonitrile (B), with the gradient elution starting at 90% B, then decreased linearly to 25% B over 5 min, and kept at 25% B for 4 min before the column re-conditioning.

The LC eluate was monitored by a Waters TQ-XS triple quadrupole mass spectrometer operated in positive electrospray ionization (ESI) mode, with the instrument parameters optimized for the highest sensitivity for FLys analysis. Multiple reaction monitoring (MRM) was performed with the MRM transition set at m/z 175→112 (quantitative transition), m/z 175→84 (qualitative transition) for FLys; and m/z 179→116 for FLys internal standard (4,4,5,5-[2H]-FLys).

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Calibration and Method Validation. The isotope-dilution method was used for the quantitation of FLys. Working standards of FLys at five different concentrations (0, 6.9, 14.8, 34.5, 69.0 nM, ) were prepared, added with equal amount of internal standard (28.7 nM), and analyzed using the developed LC−MS/MS method. A calibration curve for quantitation was established by plotting the peak area ratio of unlabeled FLys over the internal standard versus the concentration of FLys in the working standards. The minimum detection limit (MDL) was determined as the minimum amount of FLys that generates an analytical signal equal to 3 times the standard deviation from replicated analysis (n = 7) of proteins isolated from kidney/liver tissue of control rats, as reported previously.37

The method performance, which accounts for artifact formation or loss of the adduct during the enzymatic hydrolysis and sample preparation, was determined using a FLys-containing peptide. The validation entailed spiking rat tissue-isolate (mixture of kidney and liver protein, 200 𝜇g) with 0.48, 2.4, 12 per 104 lysine of the FLys-containing peptide, followed by addition of internal standard, enzymatic digestion, and LC−MS/MS analysis of the sample extracts, as described above. Parallel studies using the same amount of protein, but with no peptide added, was used as the control. The overall efficiency of the analytical method was calculated as the measured quantities of FLys recovered divided by the quantity of FLys-containing peptide added.

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RESULTS

Development of an LC−MS/MS Method to Quantify N6-Formyl-Lysine. LC−MS/MS based analytical methods had previously been developed and employed for the selective detection of FLys in proteins.38 However, these methods involve either prolonged LC runs or tedious and time-consuming sample preparation methods. For example, a 90-min chromatographic run was needed for the separation of FLys from co-eluting contaminants in the protein hydrolysates.38 The long retention time of FLys (55 min) further resulted in severe band broadening, widened chromatographic peaks (> 1.5 min) and lowering the analytical sensitivity. Another method based on phenylisothiocyanate derivatization, followed by HPLC pre-purification of the derivative and LC−MS/MS analysis of the FLys derivative-containing fractions has also been developed.6 However, this method is relatively tedious and time-consuming. Therefore, a more sensitive and user-friendly method is needed to analyze FLys in low abundance within proteins isolated from oxidative stress-exoposed cells or rat tissues.

Herein, we have developed and validated an LC−MS/MS coupled with stable isotope-dilution method for the rigorous quantification of FLys in proteins. Because FLys is highly polar and does not retain well in reverse-phase chromatography, a hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS/MS) coupled with stable isotope-dilution method was adopted to quantify the low levels of FLys in protein hydrolysates. To achieve unambiguous identification and accurate quantification of FLys in the complicated sample matrix of protein digest, we employed a qualitative MRM transition for FLys analysis (m/z 175→84), in addition 11



to the quantitative transition (m/z 175→112, Figure 2). Identification of FLys in the samples was achieved by comparing the peak area ratio of the quantitative to qualitative MRM transitions and the chromatographic migration time, as reported previously.39

Using the developed LC−MS/MS method, we conducted control experiments for various facets of the study. First, we evaluated the potential adventitious formation of FLys during the sample processing. To this end, using the LC-MS/MS method described above, we performed a parallel analysis of E. coli cell (n = 3) and rat liver tissue (n = 3) by adding anti-oxidative butylated hydroxytoluene (BHT, 100 mM) to the samples prior to the protein isolation and compared the FLys concentration to samples with no BHT added. BHT is commonly used as an anti-oxidant to reduce oxidative damage to biomacromolecules during the sample preparation process.40 The analysis showed no significant difference in the FLys concentration in protein samples isolated from cells/tissue samples with and without BHT added during the sample processing. (Figure S1).

Besides oxidative damage, we also determined the potential for FLys formation caused by formaldehyde present in the reagents. To this end, sodium bisulfite (79 mM) 41,42 and pentafluorophenyl hydrazine (1.5 mM) 43,44 as formaldehyde scavengers, were applied to the cell (n = 3) and liver tissue (n = 3) samples to remove free formaldehyde from the reagents and the surrounding environment before protein isolation and before protein digestion. The test revealed similar levels of FLys between cells/tissue samples treated with and without formaldehyde

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scavengers during the protein isolation (Figure S2) and protein digestion (Figure S3), further confirming the absence of artifact formation of FLys during the sample processing.

Control studies were also performed to determine the chemical stability of FLys during sample processing. Fifty picograms of FLys was added to the cell/rat tissue which was homogenized, and centrifuged. After the removal of protein by acetonitrile precipitation, FLys concentration in the supernatant was monitored by LC−MS/MS analysis. A counter experiment was performed by spiking the same amount of the standard to blank cell/tissue extracts. The chemical stability of FLys was determined by comparing the peak area of the processed and control samples. Data revealed FLys is chemically stable throughout the protein isolation process (Figure S4).

Method Accuracy, Precision, and Detection Limits. After selecting a suitable chromatographic method, optimizing the MS/MS condition, and testing for potential artifact formation during the sample processing, we evaluated the performance of the LC−MS/MS method for the analysis of FLys in cellular environment. To this end, we digested and analyzed cell-isolated protein solutions that were added with different concentrations of the FLys-bearing peptide (Asp-Gly-Ala-Leu-Phe-FLys-Tyr-Glu-Val-Thr, Figure S5). The overall efficiency was estimated by dividing the concentration of FLys by the amount of FLys-containing peptide spiked to the cell-lysates, with the background contribution of FLys from the cellular protein subtracted by analysis of the control protein sample. The yield of FLys was found to be ~99.6 ± 2.4% of the theoretical value (Table 1), which is indicative of high accuracy of the developed method. 13



Using a similar strategy, we then determined the intraday and interday reproducibility of the method by replicate measurements of protein (200 𝜇g) extracted from tissue mixed with various concentrations of the FLys-containing peptide on the same day (n = 3) and on seven different days of a week (n = 7). Our results (relative standard deviation: 8.6−10.9%, Table 1) demonstrated that the developed LC−MS/MS coupled with isotope-dilution method is highly reproducible for the captioned analysis.

We then evaluated the MDL of the developed method. The MDL for FLys, defined as the amount of FLys that generates an analytical signal equal to 3 times the standard deviation of the signals from replicated analysis of protein isolated from control rat tissues,37 were found to be 0.3 fmol for FLys. In comparison with previous studies, e.g. 1 fmol for FLys on column using an aqueous normal phase chromatography45 and 50 fmol using the PITC derivatization method,6 slightly higher sensitivity was achieved in the present study. The enhancement of the sensitivity could be attributed to the higher chromatographic efficiency of the HILIC column for FLys analysis (0.3 min vs 1.8 min peak width) and lower interference than that was done in reversedphase chromatography after PITC derivatization.

Quantification of FLys in Purified Proteins and in Protein Isolated from Cells. The validated method was then applied to quantitate the levels of FLys in purified proteins and in protein isolated from toxicant-exposed E. coli cells. To this end, we first established a calibration curve using the isotope-dilution method by LC−MS/MS analysis of working standard solutions spiked with a fixed amount of the internal standard. The peak area ratio between FLys and 14


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4,4,5,5-[2H]-FLys versus the concentration of FLys on the working standard solutions was plotted. Linear regression analysis of the plot showed excellent linearity (r2 = 0.9997) with a slope of 0.035 nM-1 (Figure S6), indicating the suitability of the developed method for the study.

Having established the calibration curve for the quantitative analysis, we initiated the study by analyzing FLys in purified BSA and proteinase K. After normalizing to the quantity of lysine, which was determined by a HPLC with fluorescence detection method as reported previously,46 the concentrations of FLys in both purified BSA and proteinase K were determined to be ~0.15 adducts per 104 lysine. The result is in reasonable agreement with the concentration (

Quantitation of N6-Formyl-Lysine Adduct Following

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