Simultaneous Determination of Post-Translational Racemization and

Nov 27, 2013 - Typical markers of protein aging are spontaneous post-translational modifications such as amino acid racemization (AAR) and amino acid ...
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Simultaneous Determination of Post-Translational Racemization and Isomerization of N‑Terminal Amyloid‑β in Alzheimer’s Brain Tissues by Covalent Chiral Derivatized Ultraperformance Liquid Chromatography Tandem Mass Spectrometry Koichi Inoue,† Daiju Hosaka,† Nana Mochizuki,† Hiroyasu Akatsu,‡,§ Kaname Tsutsumiuchi,∥ Yoshio Hashizume,‡ Noriyuki Matsukawa,§ Takayuki Yamamoto,‡ and Toshimasa Toyo’oka*,† †

Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Department of Neuropathology, Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan § Department of Neurology, Nagoya City University, Graduate School of Medical Sciences, Nagoya, Japan ∥ College of Bioscience and Biotechnology, Chubu University, Kasugai, Japan ‡

S Supporting Information *

ABSTRACT: Typical markers of protein aging are spontaneous post-translational modifications such as amino acid racemization (AAR) and amino acid isomerization (AAI) during the degradation of peptides. The post-translational AAR and AAI could significantly induce the density and localization of plaque deposition in brain tissues. Alzheimer’s disease (AD) is reliably related to the formation and aggregation of amyloid-β peptide (Aβ) plaques in the human brain. No current analytical methods can simultaneously determine AAR and AAI during the degradation of Aβ from AD patients. We now report a covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry (CCD-UPLC-MS/MS) method for the determination of post-translational AAR and AAI of N-terminal Aβ (N-Aβ1−5) in human brain tissues. When subjected to tryptic N-Aβ1−5 from post-translationally modified natural Aβ in focal brain tissues by the CCD procedure, it was monitored at m/z 989.6→ 637.0/678.9 during electrospray collision-induced dissociation. These N-Aβ1−5 fragments with L-aspartic acid (L-Asp), D-Asp, LisoAsp, and D-isoAsp could be separated using the UPLC system with a conventional reversed-phase column and mobile phase. The quantification of these peptides was determined using a stable isotope [15N]-labeled Aβ1−40 internal standard. The CCDUPLC-MS/MS assay of potential N-Aβ1−5 allowed for the discovery of the present and ratio levels of these N-Aβ1−5 sequences with L-Asp, D-Asp, L-isoAsp, and D-isoAsp.

T

Amyloidosis, the most widely known appearance of frequent pathology, may still dramatically impact the outcome from therapy and prevention with aging degeneration. The pathogenic aggregation of the amyloid-β (Aβ) peptides is considered a hallmark of the progression of Alzheimer’s disease (AD), the leading cause of senile dementia in the elderly and one impact of dementia as an increasing threat to global health. Efforts to extract and analyze the composition of Aβ plaques from human brains of AD patients started in the 1970s.2,3 The first successful protocol to purify and analyze the Aβ sequence of amino acids was developed using amino acid analyzer liquid chromatography (LC) techniques. 4 They reported the sequence of the N-terminal 24 amino acids in Aβ, showing

he spontaneous accumulation of the post-translational modifications within biological proteins can be regarded as an aging process. The accumulation of modified proteins may disrupt biochemical functions by affecting protein expression, clearance, turnover, cell signaling, and induction of apoptosis, suggesting that protein aging could have both physiological and pathological markers. Nearly all age-related neurodegenerative diseases involve the misfolding and accumulation of specific proteins in the brain regions. More than 50 diseases of abnormal protein deposition have been identified in the brain and systemic tissues of humans.1 Under pathogenic conditions such as amino acid substitutions, the post-translational modifications such as cleavage, phosphorylation, oxidation, protein density, and/or misfolding of structured proteins, some proteins are liable to be dysfunctional, self-aggregate, and accumulate inside or outside of neuron cells in specific brain regions. © 2013 American Chemical Society

Received: October 14, 2013 Accepted: November 27, 2013 Published: November 27, 2013 797

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event in the Aβ production before the β-secretase cleavage (BACE1). This means that the detection of AAR and AAI in the N-terminal sequence has two aspects used to evaluate the production and aggregation of Aβ. In any case, the consequences of these chemical modifications are site-specific and can affect the age-related accumulation according to the structural alterations produced by the site-specific incorporation of modified N-terminal residues. There have been many studies of AAR and AAI events of Aβ in vivo and/or in vitro, but its role in the natural Aβ pathogenesis in the focal brain tissues is still unclear. In the case of AAR in the Aβ sequence, Edman degradation with LC amino acid analysis was used for the determination of the chiral free-amino acids from extracted natural or artifact Aβs.9,16 The O-acyl isopeptide method was developed for evaluation of the solubility and stability of Aβ related to AAR.17 In the case of AAI in the Aβ sequence, the frequently employed methods for AAI in the Aβ sequence are MS techniques.18,19 Sargaeva et al. used electron capture dissociation (ECD) Fourier-transform ion cyclotron resonance (FTICR) MS for the identification of Asp and isoAsp in the Aβ sequence based on signature backbone cleavage ions (m/z c + 57 and z − 57) produced upon radical-mediated fragmentation.18 Ni et al. applied electron transfer dissociation (ETD) MS based on the same cleavage ions.19 However, an MS assay may be not utilized to discover signature ions of both Asp residues in the Aβ sequences from the spurious background noise of biological samples. To chromatographically separate the AAI peptides using LC techniques, sufficient results could not be acquired with model peptide sequences.19−21 On the other hand, LC separation would be needed to analyze nondifferences in the Asp-isomers with identical mass in biological samples. In this study, covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry (CCD-UPLCMS/MS) was used to simultaneously analyze the posttranslational AAR and AAI of N-terminal Aβ (N-Aβ1−5) in the human brain. When subjected to tryptic N-Aβ1−5 from posttranslationally modified natural Aβ in brain tissues monitored by CCD-electrospray ionization (ESI) with collision-induced dissociation (CID), these N-Aβ1−5 sequences with L-Asp, DAsp, L-isoAsp, and D-isoAsp could be separated using the UPLC system with a conventional reversed-phase column and mobile phase. Our approach overcomes the limitations of simultaneous AAR and AAI monitoring of the modified Aβ peptides in AD brain.

similarity between AD and Down’s syndrome. In the 1990s, various reports strongly suggested that Aβ sequences isolated from AD and Down’s syndrome brains were post-translationally modified by racemization and isomerization of the amino acids in protein.5−9 The N-terminal structure of Aβ, extracted from senile plaques such as neuritic deposits, showed cleaved Nterminal aspartic acid (1Asp) of about 8%, the formation of isoaspartate forms (iso1Asp) of about 20%, pyroglutamate-3 (p3Glu), the cyclization of the N-terminal glutamyl residue of about 51%, and the native form of only about 20% by amino acid analysis and mass spectrometric (MS) analysis with the tryptic digestion and Edman degradation.10 These pathways of the post-translational N-terminal modification of Aβ are characterized to represent the most frequent type of aging protein damage. These reactions proceed through the formation of a cyclic succinimide intermediate, which rapidly processed to spontaneous chemical modifications such as amino acid racemization (AAR) and amino acid isomerization (AAI) during the degradation of Asp-included peptides (Scheme 1).11 During the spontaneous chemical modification Scheme 1. Formation of a Cyclic Succinimide Intermediate That Occurred by Spontaneous Chemical Modifications Such As Amino Acid Racemization (AAR) and Amino Acid Isomerization (AAI) during Degradation of Asp-Included Peptides

Isomerization and racemization of L-aspartate occur spontaneously in proteins and proceed through a common transient cyclic succinimidyl intermediate. Succinimide is hydrolyzed immediately to form two more stable compounds: L-isoaspartate and L-aspartate. Alternatively, L-succinimide racemizes to D-succinimide, which is rapidly hydrolyzed to D-isoaspartate and D-aspartate.

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EXPERIMENTAL SECTION Materials. Synthetic N-Aβ1−5 peptides with L-Asp, D-Asp, LisoAsp, and D-isoAsp (peptide purity: >95%, molecular weight: 636.65) were obtained from Eurofins Operon (Wien, Austria). Aβ1−40, 1−42, 1−43 and stable isotope [15N]-labeled Aβ1−40 were obtained from the rPeptide Co. (Athens, GA). The covalent chiral derivatized reagent of (R)-(-)-4-(N, N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole (R-DBD-Py-NCS) for HPLC labeling was obtained from the Tokyo Chemical Industry (Tokyo, Japan). All other chemicals were of analytical grade and were used without further purification. UPLC-MS/MS Equipment and Conditions. The UPLCMS/MS was performed using a Waters ACQUITY UPLC/ Xevo TQ-S system (Waters, Milford, MA) that was coupled to a triple quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source. LC separation was performed

of Aβ, the AAR and AAI of Asp for protein functionality is the existence of a specific repair system based on protein Lisoaspartyl O-methyltransferase (PIMT).12 However, the PIMT repair system is not completely efficient in that the D-Asp residue is not recognized and equally functional in all tissues.13 It has been reported that the modified Asp sequence dramatically increased with aging in the brain.14 The AAR and AAI represent the major nonenzymatic and chemical modifications affecting the Aβ folding and degradation in the pathology of aging dementia. Recently, an interesting study was reported that when isoAsp is present at position 672 in the amyloid precursor protein (APP), cathepsin B can catalyze the cleavage between methionine (Met) at 671 and isoAsp at 672 with a high efficiency.15 Since spontaneous AAI cannot readily take place in the post-translational N-terminal modification, iso1Asp formation in the native Aβ can only occur as an early 798

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using an Acquity UPLC BEH C18 (2.1 × 100 mm, 1.7 μm; Waters, Milford, MA). The mobile phase consisted of 50 mM aqueous ammonium formate with 0.01% formic acid (Solvent A) and 0.01% formic acid in methanol (Solvent B). Other conditions are shown in the Supporting Information. Preparation and Covalent Chiral Derivatization of Aβ Peptides. The Aβ peptides were dissolved in their original vial with water and acetonitrile (50/50, v/v) by sonication for 30 s to produce 1 mM solutions. This water/acetonitrile solution was already applied to prepare the Aβ1−16 and Aβ1−40 peptides for the LC/MS experiment.22,23 For the optimal derivatized conditions, this protocol was used for measuring the AAR and AAI of N-Aβ1−5 as derivatives in the brain tissues (Supporting Information). Extraction, SPE, and Tryptic Procedure. In the first step, the brain tissues and zirconia beads (5.0 mm) in the tubes were placed in the holes of an aluminum block and immediately homogenized for 3 min by a Shake Master (Bio Medical Sciences, Tokyo, Japan). These supernatant solutions were applied to the solid phase extraction (SPE) using InterSep MC1 (30 mg/1 mL, GL Science Co., Tokyo, Japan). These eluants were dried under a stream of nitrogen at 30 °C. These samples were dissolved in 90 μL of 50 mM ammonium hydrogen carbonate in water, 10 μL of trypsin, and were then incubated at 37 °C for 6 h. These solutions were then subjected to derivatization. For the optimal extraction and tryptic procedures of Aβ, we utilized the UPLC-MS/MS analysis of the stable tryptic part of LVFFAEDVGSNK (m/z 663.6 → m/z 185.2) from the AD brain tissues. Sample Information. The utilized tissues from the Fukushimura Brain Bank were used for the accurate, reliable, and detailed pathological evaluation of AD.24 All of our cases were reviewed and discussed with several doctors at a clinicopathological conference regarding neuropathological staining.25

Scheme 2. Tryptic Digestion and Experimental Approach for the Post-Translational AAR and AAI Formation of NTerminal Aβ Sequences

Natural Aβ sequences are termed from the N-terminal Asp to the Cterminal 38Gly, 40Val, 42Ala, or other types. Moreover, 13,14His and/or 35 Met are oxidized. These Aβ sequences were cleavage by trypsin and produced to four fragments such as DAEFR, HDSGYEVHHQ, and others. The N-terminal fragment (DAEFR) is focused for determining AAR and AAI formation of N-terminal 1Asp in the natural Aβ sequence. This N-terminal fragment is deviated by R-DBD-Py-NCS for the UPLC separation of AAR and AAI formation of N-terminal 1 Asp in natural Aβ sequences.

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RESULTS AND DISCUSSION CCD-UPLC-MS/MS Analysis of N-Terminal Aβ Sequence. The Aβ sequences in the plaque, aggregation, fibrils, insoluble polymers, and soluble oligomers were built from the N-terminal position starting with Asp to the C-terminal position at 40, 42, 43, or others in the AD brain. After tryptic digestion, four main parts of the fragments were dissected, as shown in Scheme 2. In this study, the N-terminal part of Aspalanine (Ala)-glutamic acid (Glu)-phenylalanine (Phe)-arginine (Arg) (1DAEFR5; N-Aβ1−5) was the focus based on three reasons from previous reports. First, cathepsin B is more effective than BACE1 in processing the 672Asp-containing peptide derivatives and can cleave the 672isoAsp-containing peptides such as APP, which occurs with a high catalytic efficiency.15 BACE1 is a type I transmembrane aspartyl protease that is involved in the generation of the Aβ component in brain tissue. This enzyme has been found to be especially efficacious to hydrolyze the variant of APP and was in fact discovered using this APP sequence as molecular bait. BACE1 was able to cleave readily only the sample peptide representing the APP Swedish mutated sequence at the prior position 1Asp, while cathepsin B hydrolyzed the structures of the isoAsp residue at position-1 in APP.15 BACE1 and cathepsin B could jointly participate in cleaving the N-terminal part, including 672Asp or 672isoAsp. The majority of AD dementia patients may be altered in the extent of cleavage and clearance of APP, and therefore, specific inhibitors of cathepsin

B represent candidate drugs for AD dementia.26,27 Second, the formation of pyroglutamate-modified Aβ is a multistep process requiring the removal of the two N-terminal amino acids, such as 1Asp and 2Ala, by elimination to expose the 3Glu site at the third position of the Aβ peptides.28 Third, the AAR of the Nterminal 1Asp residue could suppress the Aβ fibril formation regarding Asp at position 7 or 23.29 Thus, a valuable idea was conceived that the AAR of the N-terminal 1Asp is accelerated for the inhibition of the Aβ fibril and aggregation. Accordingly, the analysis of the N-terminal Aβ sequence in the AD brain would be needed for discussing these queries related to the Nterminal 1Asp. In this study, based on a novel CCD-UPLC-MS/ MS assay of N-Aβ1−5, an initial experiment was performed to determine the CCD reaction and MS ionizations of the Aβ model peptides. The full scan and daughter mass spectra were evaluated to determine the relative intensities of ions in a given mass range in the positive ESI mode. These N-Aβ1−5 sequences with the L-Asp, D-Asp, L-isoAsp, and D-isoAsp structures could be reacted with R-DBD-Py-NCS in a solution of triethylamine. It was clear that the CCD reaction of the N-Aβ1−5 model peptides occurs at higher temperatures from 20 to 70 °C. In this study, the reaction condition was the highest at 70 °C and achieved a plateau after 4 h in the solution of 2% triethyamine 799

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(Figure 1). The mass spectrum of the typical Aβ1−5 peptide (NAβ1−5 sequence with L-Asp) showed a signal at m/z 989.6 [M

The derivatized condition was used for measuring the AAR and AAI of N-Aβ1−5 by UPLC-MS/MS assay. A 100 μL solution of the standard was added to 50 μL of 5 mM R-DBD-Py-NCS in acetonitrile and 50 μL of 2% triethyamine at 70 °C for 0−6 h. The mixed solution was dried and added to 50 μL of the mobile phase solution.

Investigation of optimal reaction time at 70 °C for the covalent chiral derivatized N-Aβ1−5 model peptides. 1

+H]+ by the CCD reaction (Figure 2A). The results of the daughter scan from the precursor ions of m/z 678.9, 637.0, and 522.6 are shown in Figure 2B. These fragment patterns of m/z 678.9, 637.0, and 522.6 from the typical Aβ1−5 peptide are shown in Figure 2C. When the collision energy (40 V) was used in the product ions of [M + H]+, the major fragment ions at m/z 989.6 → m/z 637.0/678.9 were observed using a collision energy of 20 eV. Moreover, other Aβ model peptides showed comparable MS spectral patterns in the ESI positive mode. Based on this result, these ions in the SRM mode were used for the analysis of N-terminal Aβ sequences with AAR and AAI in biological samples. To utilize an MS assay of nondifferences of peptide sequences with an identical mass in biological samples, the chromatographic techniques should be needed for the development of an accurate, reliable, and sensitive method for biological samples. In the effort of chromatographically separating the AAR and AAI peptides, we think that this sufficient result could not be acquired using traditional separation techniques. In our laboratory, an indirect derivatization-based assay could be used to identify potential low molecules such as drug, pesticide, and biomarkers in various complicated samples for the analysis of chiral compounds based on the reversed-phase LC system.30−32 The CCD-UPLC-MS/ MS assay to simultaneously screen the AAR and AAI of peptides as an effective method in biological samples, however, has not been reported. In this study, several reversed-phase columns and mobile phases (added formic acid, acetic acid, and TFA: concentration of 0.1% in water/acetonitrile or methanol) were evaluated for the separation of these N-Aβ1−5 sequences with L-Asp, D-Asp, L-isoAsp, and D-isoAsp (Figure 3B). On the basis of this result, a separation of L-Asp (retention time (RT): 28.5 min), D-Asp (RT: 24.3 min), L-isoAsp (RT: 25.0 min), and D-isoAsp (RT: 21.3 min) could be achieved using the UPLC BEH C18 column with a mobile phase consisting of 0.1% formic acid in water/methanol. The RT of [15N]-labeled N-terminal Asp residue internal standard from a stable isotope Aβ1−40 was

(A) MS spectrum of N-Aβ1−5 in ESI-positive mode. (B) MS/MS spectrum (m/z 989.56→) of N-Aβ1−5 in ESI-positive mode. (C) MS/ MS fragment pattern of covalent chiral derivatized N-Aβ1−5. The UPLC-MS/MS was performed using a Waters ACQUITY UPLC/ Xevo TQ-S system that was coupled to a Quadrupole mass spectrometer fitted with an ESI source in the positive ionization mode. The mobile phase consisted of 50 mM aqueous ammonium formate with 0.01% formic acid and 0.01% formic acid in methanol with the flow rate of 0.4 mL/min. 2

Mass spectra and fragment pattern of the typical Aβ1−5 peptide (N-Aβ1−5 sequence with L-Asp). 28.4 min in the SRM mode (m/z 989.6 → m/z 637.0/678.9) (Figure 3A). Sample Preparation and Tryptic Digestion of FullLength Aβ Peptides in Human Brain Tissues. For the analysis of the natural Aβ peptides from brain tissues using our developed method, extraction, cleanup and tryptic digestion would be needed. Thus, we investigated each condition and operation of the full-length natural Aβ peptides. For the extractive process of natural Aβ peptides, we investigated various kinds of solutions using the tryptic part of LVFFAEDVGSNK (m/z 663.6 → m/z 185.2) from AD brain tissues. This result is shown in Figure 4A. In addition, previous studies showed that the compact of natural Aβ plaques in the human brain often used the FA solution.33−36 In this study, we used 50% FA in water for the extraction of the natural Aβ peptides in the brain tissues. For the cleanup procedure of the full-length natural Aβ peptides, we then employed the SPE method as a reference.37 It suggested that recovery was approximately 90% for these Aβ species, and the key to obtaining a high recovery was in meticulously altering the elutropic composition and ion pair strength of the SPE solvents with the acetonitrile and ammonium combination as the best 800

dx.doi.org/10.1021/ac403315h | Anal. Chem. 2014, 86, 797−804

Analytical Chemistry

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(A) SRM chromatogram (m/z 989.6 → m/z 637.0/678.9) for the isotope stable [ 15 N]-labeled N-terminal peptide from Aβ 1−40 (retention time (RT): 28.4 min). (B) SRM chromatogram (m/z 989.6 → m/z 637.0/678.9) for the N-Aβ1−5 with L-Asp (RT: 28.5 min), D-Asp (RT: 24.3 min), L-isoAsp (RT: 25.0 min), and D-isoAsp (RT: 21.3 min). This separation of the derivatized N-Aβ1−5 fragment included with L-Asp, D-Asp, L-isoAsp, and D-isoAsp was achieved using the UPLC BEH C18 column with a mobile phase consisting of 0.1% formic acid in water/methanol.

(A) Investigation of extraction solution for the natural Aβ peptides in AD brain tissue. (B) Investigation of tryptic digestion time for the natural Aβ peptides in AD brain tissue. In the extractive process and tryptic digestion time of natural Aβ peptides, various kinds of solutions and tryptic digestion time were investigated using the stable tryptic part of LVFFAEDVGSNK (m/z 663.6 → m/z 185.2) from AD brain tissues.

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SRM chromatograms of N-Aβ1−5 standard solution.

recovery solvent.37 We then targeted the N-terminal Aβ sequence in the brain tissues regarding the tryptic digestion procedure. In previous reports, we investigated the tryptic digestion of the full-length Aβ1−40 and Aβ1−42 peptides under the buffer’s condition.22,23 The reaction time was investigated using the tryptic part of LVFFAEDVGSNK from the full-length Aβ1−40, Aβ1−42 and Aβ1−43 standards, as shown in Figure 4B. The reaction time of about 6 h was used in this study. Ultimately, in this study, we used the stable isotope full-length Aβ1−40 for evaluating the recovery of the natural Aβ peptides using the extraction, SPE, and tryptic procedures. The recovery of the full-length Aβ1−40 in the control brain tissues was analyzed using the dilution stable isotope tryptic [15N]-labeled N-Aβ1−5, which ranged from 96.1 to 102.3% (Average 99.2%, RSD: 2.2%, n = 5). Examination of AAR and AAI of N-Terminal Aβ Sequence in AD Brain Tissues. Shimizu et al. reviewed the AAR and AAI formation; although seizure is not a common symptom of AD patients, rapid accumulation of isomerized proteins may cause an epileptic seizure, while chronic accumulation may cause neurodegeneration.11 Alternatively, it is speculated that most proteins isomerized in matured or aged brains are associated with the progression of neurodegeneration. Moreover, the N-terminal Aβ sequence in senile plaque from AD patients indicated that DL-Asp is only 10%, and the predicted L-Asp is about 2% based on the D/L-iso7Asp ratio (55.7/19.2%) in this review.11 Successively, most of them wonder about this influence of the AAR and AAI formations on the AD brain. Thus, we examined the AAR and AAI formations of the N-terminal Aβ sequence in AD and control brain tissues (frontal lobe region) using our developed CCD-UPLC-MS/MS assay. Table 1 shows that the N-terminal Aβ sequence in the AD and control brain tissues was calculated using the unit of

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Investigation of extraction and tryptic procedures for the natural Aβ peptides using the stable tryptic part of LVFFAEDVGSNK from AD brain tissues.

picomoles per milligram of tissue. In addition, the typical SRM chromatograms of N-Aβ1−5 and the internal standard in AD brain tissues are shown in Figure 5. The concentration levels of the post-translational AAR and AAI formation of the Nterminal Aβ sequence were detected in the AD brain tissues. Thus, the specificity is very important, and it is defined as the noninterference at the retention times of N-Aβ1−5 and internal standard from the endogenous brain components in the SRM chromatograms. From this result for analysis of the control, no significant peaks from the Aβ presence (sample nos. 11−13, 15, 16, and 18−20 in Table 1) were found at the retention time corresponding to N-Aβ1−5 in almost all cases. Moreover, the relationship between the Braak stage and Aβ levels is not a possibility definitive correlation and will be discussed using large scale samples. On the other hand, based on this result for the AD brain, the post-translational AAR and AAI formation of the N-terminal Aβ sequence was detected in all patients. These patterns for the aging and gender difference in AD patients are shown in Figure 6. The detection levels of the N-terminal Aβ sequence from AD patients of the predictable L-Asp of about 59.0 ± 26.0% based on the D/L-iso/D-isoAsp ratio (4.8 ± 5.7/ 25.4 ± 15.0/10.8 ± 11.2%, AD patients, n = 10) were observed. This pattern of aging and gender difference allowed for the discovery of significantly interesting results in the AD patients (Figure 6). Thus, the possible AAR and AAI and the effects of the post-translational modification in AD patients would be investigated using a greater number of subjects. On the basis of the antibody’s study of the Aβ in AAI, Szendrei et al. reported 801

dx.doi.org/10.1021/ac403315h | Anal. Chem. 2014, 86, 797−804

Analytical Chemistry

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Table 1. Post-Translational AAR and AAI Formation of N-Terminal Aβ Sequence for AD and Control Brain Tissues (n = 20)a N-terminal AP sequence (pmol/mg)

a

no.

state

age

Braak

sex

L-Asp

D-Asp

L-isoAsp

D-isoAsp

total

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

AD AD AD AD AD AD AD AD AD AD control control control control control control control control control control

86 87 96 86 85 77 83 92 76 88 82 87 95 89 85 76 87 96 73 89

6 4 4 4 6 4 6 5 4 4 1 1 1 2 2 1 2 2 1 2

male female female male male male female female male female male female female male male male female female male female

3.2 0.3 0.2 1.8 1.8 2.3 7.1 1.7 0.3 0.1 ND ND ND 0.7 ND ND 0.2 ND ND ND

ND ND 0.1 0.1 0.1 0.1 ND 0.2 0.1 ND ND ND ND 0.1 ND ND ND ND ND ND

0.6 0.4 0.2 0.3 0.2 0.4 0.6 2.2 0.3 0.1 ND ND ND 0.7 ND ND ND ND ND ND

ND 0.2 0.1 0.1 ND ND 0.2 0.7 0.1 0.1 ND ND ND 0.3 ND ND ND ND ND ND

3.8 0.9 0.6 2.3 2.1 2.8 7.9 4.8 0.8 0.3 ND ND ND 1.8 ND ND 0.2 ND ND ND

ND < 0.1 pmol/mg.

linked immunoassay procedure.38 They suggested a possible mechanism of the retention of the isomerized Aβ peptide in the affected brains.38 In addition, Iwatubo et al. used the six different antibodies that specifically recognize distinct Nterminal structures of Aβ for suggesting that initial deposits in diffuse plaques begin with N-terminal Aβ with or without structural modifications such as isomerization and racemization.8 Future researchers would investigate the questions by examining the influence of different antibody epitopes and isotypes on plaque clearance and neuronal protection. In addition, the isotype of an antibody is important for either Fcor complement-mediated phagocytosis of N-terminal Aβ by microglial cells because antibody isotype defines its affinity for Fc receptors as well as its ability to activate complement for the design of antibodies with therapeutic potential.39 Thus, the accurate analytical method should be used for the evaluation of antibody and binding effects of N-terminal Aβ modifications in brain tissues. At the present stage, we cannot determine what is behind the emergence, pathological effects, elimination, and useful biomarkers of the post-translational AAR and AAI formation of the N-terminal Aβ sequence for AD. On the basis of these data, we show for the first time that the N-Aβ1−5 levels in AD brain tissues were compared by a reliable CCD-UPLCMS/MS method.

(A) SRM chromatogram (m/z 989.6 → m/z 637.0/678.9) for the isotope stable [ 15 N]-labeled N-terminal peptide from Aβ 1−40 (retention time (RT): 27.8 min). (B) SRM chromatogram (m/z 989.6 → m/z 637.0/678.9) for the N-Aβ1−5 with L-Asp (RT: 27.8 min), D-Asp (RT: 23.6 min), L-isoAsp (RT: 24.3 min), and D-isoAsp (RT: 20.7 min). Concentration levels showed (Table 1) that the Nterminal Aβ sequence in the AD and control brain tissues was calculated using the unit of picomoles per milligram of tissue.

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CONCLUSIONS

In this study, the analytical assay to simultaneously analyze the AAR and AAI of N-terminal Aβ sequence was developed and applied to monitoring the post-translational modified Aβ in brain tissues from AD patients. Our CCD-UPLC-MS/MS assay of potential N-Aβ1−5 allowed for the discovery of the presence and ratio levels of these N-Aβ1−5 sequences with L-Asp, D-Asp, L-isoAsp, and D-isoAsp. Future work is needed so that the cause-and-effect sequence of the Aβ aggregated events with AAR and AAI will be investigated using this assay.

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SRM chromatograms of N-Aβ1−5 in brain tissue from AD patients. that the diisomerized decapeptide (Asp at position 1 and 7) was used as an immunogen to generate polyclonal antibody 14 943 that recognizes the isomerized peptides preferentially when the peptide antigen structures are conserved during the enzyme802

dx.doi.org/10.1021/ac403315h | Anal. Chem. 2014, 86, 797−804

Analytical Chemistry

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(A) Pattern of aging difference for each AD patient (n = 10). (B) Pattern of gender difference of average AD patient (male, n = 5, and female, n = 5) for the decreased L-Asp.The detection levels of the N-terminal Aβ sequence from AD patients of the predictable L-Asp of about 59.0 ± 26.0% based on the D/L-iso/D-isoAsp ratio (4.8 ± 5.7/25.4 ± 15.0/10.8 ± 11.2%, AD patients, n = 10) were observed. 6

Pattern of the post-translational AAR and AAI formation of N-terminal Aβ sequence for AD brain (n = 10).

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81 54 264 5593. Tel: +81 54 264 5656. Author Contributions

K.I., D.H., and N.M. carried out most of the analytical experiments. H.A. and Y.H. performed the pathological examination, and H.A. designed and supervised the clinical work for the AD patients. N.M. conducted biological discussion. K.T. prepared and suggested the peptide’s synthesis. K.I., H.A., N.M., T.Y., and T.T. contributed to preparation of materials and provided advice on project planning and data interpretation. K.I. designed and supervised the project and wrote the manuscript. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This study was supported by grants from the Japanese Brain Bank Network for Neuroscience Research and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Norihiro Ogawa, Takeshi Kanesaka, and Chie Taniguchi for technical assistance in the clinical and pathological study, and we thank all patients and relatives of patients for organ donation.

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