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A new methodology for simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 by column-switching LC/MS/MS

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Abstract

This article details the development of a novel method that overcomes the drawbacks of sandwich ELISA (sELISA) and allows reliable evaluation of simultaneous quantification of the amyloid (Aβ)-peptides, total-Aβ, Aβx-38, Aβx-40, and Aβx-42, in rat brain by optimized sample purification and column-switching liquid chromatographic-tandem mass spectrometry (LC/MS/MS). This method provides accurate analyses of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 with a linear calibration range between 0.05 and 45 ng/mL. Verification for accuracy and precision of biological samples were determined by a standard addition and recovery test, spiked with synthetic Aβ1-38, Aβ1-40, and Aβ1-42 into the rat brain homogenate. This method showed <20% relative error and relative standard deviation, indicating high reproducibility and reliability. The brain concentrations of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 after oral administration of flurbiprofen in rats were measured by this method. Aβx-42 concentrations (4.57 ± 0.69 ng/g) in rats administered flurbiprofen were lower than those in untreated rats (6.48 ± 0.93 ng/g). This was consistent with several reports demonstrating that NSAIDs reduced the generation of Aβ. We report here a method that allows not only the quantification of specific molecular species of Aβ but also simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42, thus overcoming the drawbacks of sELISA.

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References

  1. Hampel H, Shen Y, Walsh DM, Aisen P, Shaw LM, Zetterberg H, Trojanowski JQ, Blennow K (2010) Biological markers of amyloid β-related mechanisms in Alzheimer’s disease. Exp Neurol 223:334–346

    Article  CAS  Google Scholar 

  2. Gabelle A, Roche S, Gény C, Bennys K, Labauge P, Tholance Y, Quadrio I, Tiers L, Gor B, Chaulet C, Vighetto A, Croisile B, Krolak-Salmon P, Touchon J, Perret-Liaudet A, Lehmann S (2010) Correlations between soluble α/β forms of amyloid precursor protein and Aβ38, 40, and 42 in human cerebrospinal fluid. Brain Res 1357:175–183

    Article  CAS  Google Scholar 

  3. Bibl M, Lewczuk P, Esselmann H, Mollenhauer B, Klafki HW, Welge V, Wolf S, Trenkwalder C, Otto M, Kornhuber J, Wiltfang J (2008) CSF amyloid-β 1-38 and 1-42 in FTD and AD: biomarker performance critically depends on the detergent accessible fraction. Proteomics Clin Appl 2:1548–1556

    Article  CAS  Google Scholar 

  4. Bibl M, Mollenhauer B, Lewczuk P, Esselmann H, Wolf S, Otto M, Kornhuber J, Rüther E, Wiltfang J (2011) Cerebrospinal fluid tau, p-tau 181 and amyloid-β38/40/42 in frontotemporal dementias and primary progressive aphasias. Dement Geriatr Cogn Disord 31:37–44

    Article  CAS  Google Scholar 

  5. Lanz TA, Schachter JB (2006) Demonstration of a common artifact in immunosorbent assays of brain extracts: development of a solid-phase extraction protocol to enable measurement of amyloid-beta from wild-type rodent brain. J Neurosci Methods 157:71–81

    Article  CAS  Google Scholar 

  6. Best JD, Jay MT, Otu F, Ma J, Nadin A, Ellis S, Lewis HD, Pattison C, Reilly M, Harrison T, Shearman MS, Williamson TL, Atack JR (2005) Quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]. J Pharmacol Exp Ther 313:902–908

    Article  CAS  Google Scholar 

  7. Jablonowska A, Bakun M, Kupniewska-Kozak A, Dadlez M (2004) Alzheimer’s disease Abeta peptide fragment 10-30 forms a spectrum of metastable oligomers with marked preference for N to N and C to C monomer termini proximity. J Mol Biol 344:1037–1049

    Article  CAS  Google Scholar 

  8. Kuo YM, Kokjohn TA, Kalback W, Luehrs D, Galasko DR, Chevallier N, Koo EH, Emmerling MR, Roher AE (2000) Amyloid-beta peptides interact with plasma proteins and erythrocytes: implications for their quantitation in plasma. Biochem Biophys Res Commun 268:750–756

    Article  CAS  Google Scholar 

  9. Slemmon JR, Painter CL, Nadanaciva S, Catana F, Cook A, Motter R, Seubert P (2007) Distribution of Abeta peptide in whole blood. J Chromatogr B Anal Technol Biomed Life Sci 846:24–31

    Article  CAS  Google Scholar 

  10. Du Y, Dodel R, Hampel H, Buerger K, Lin S, Eastwood B, Bales K, Gao F, Moeller HJ, Oertel W, Farlow M, Paul S (2001) Reduced levels of amyloid beta-peptide antibody in Alzheimer disease. Neurology 57:801–805

    CAS  Google Scholar 

  11. Mruthinti S, Buccafusco JJ, Hill WD, Waller JL, Jackson TW, Zamrini EY, Schade RF (2004) Autoimmunity in Alzheimer’s disease: increased levels of circulating IgGs binding Abeta and RAGE peptides. Neurobiol Aging 25:1023–1032

    Article  CAS  Google Scholar 

  12. Lanz TA, Karmilowicz MJ, Wood KM, Pozdnyakov N, Du P, Piotrowski MA, Brown TM, Nolan CE, Richter KE, Finley JE, Fei Q, Ebbinghaus CF, Chen YL, Spracklin DK, Tate B, Geoghegan KF, Lau LF, Auperin DD, Schachter JB (2006) Concentration-dependent modulation of amyloid-beta in vivo and in vitro using the gamma-secretase inhibitor LY-450139. J Pharmacol Exp Ther 319:924–933

    Article  CAS  Google Scholar 

  13. Oe T, Ackermann BL, Inoue K, Berna MJ, Garner CO, Gelfanova V, Dean RA, Siemers ER, Holtzman DM, Farlow MR, Blair IA (2006) Quantitative analysis of Amyloid beta peptides in cerebrospinal fluid of Alzheimer’s disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 20:23–35

    Article  Google Scholar 

  14. Bateman RJ, Siemers ER, Mawuenyega KG, Wen G, Browning KR, Sigurdson WC, Yarasheski KE, Friedrich SW, Demattos RB, May PC, Paul SM, Holtzman DM (2009) A gamma-secretase inhibitor decreases amyloid-beta production in the central nervous system. Ann Neurol 66:48–54

    Article  CAS  Google Scholar 

  15. Lame ME, Chambers EE, Blatnik M (2011) Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry. Anal Biochem 419:133–139

    Article  CAS  Google Scholar 

  16. Kamiie J, Ohtsuki S, Iwase R, Ohmine K, Katsukura Y, Yanai K, Sekine Y, Uchida Y, Ito S, Terasaki T (2008) Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res 25:1469–1483

    Article  CAS  Google Scholar 

  17. Kawakami H, Ohtsuki S, Kamiie J, Suzuki T, Abe T, Terasaki T (2011) Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection. J Pharm Sci 100:341–352

    Article  CAS  Google Scholar 

  18. Stine WB Jr, Dahlgren KN, Krafft GA, LaDu MJ (2003) In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J Biol Chem 278:11612–11622

    Article  CAS  Google Scholar 

  19. Jemal M, Schuster A, Whigan DB (2003) Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard. Rapid Commun Mass Spectrom 17:1723–1734

    Article  CAS  Google Scholar 

  20. Li W, Cohen LH (2003) Quantitation of endogenous analytes in biofluid without a true blank matrix. Anal Chem 75:5854–5859

    Article  CAS  Google Scholar 

  21. Sergeant N, Bombois S, Ghestem A, Drobecq H, Kostanjevecki V, Missiaen C, Wattez A, David JP, Vanmechelen E, Sergheraert C, Delacourte A (2003) Truncated beta-amyloid peptide species in pre-clinical Alzheimer’s disease as new targets for the vaccination approach. J Neurochem 85:1581–1591

    Article  CAS  Google Scholar 

  22. Watanabe K, Segawa T, Nakamura K, Kodaka M, Konakahara T, Okuno H (2001) Identification of the molecular interaction site of amyloid beta peptide by using a fluorescence assay. J Pept Res 58:342–346

    Article  CAS  Google Scholar 

  23. Tjernberg LO, Tjernberg A, Bark N, Shi Y, Ruzsicska BP, Bu Z, Thyberg J, Callaway DJ (2002) Assembling amyloid fibrils from designed structures containing a significant amyloid β-peptide. Biochem J 366:343–351

    CAS  Google Scholar 

  24. Heinig K, Wirz T, Yuan M, Tingler M, Mylott W (2011) An improved LC-MS/MS method for the determination of taspoglutide in plasma and urine using orthogonal HILIC-RP column switching, ultra-performance LC separation and 'wrong-way-round' electrospray ionization. Biomed Chromatogr. doi:10.1002/bmc.1593

  25. Kita Y, Takahashi T, Uozumi N, Shimizu T (2005) A multiplex quantitation method for eicosanoids and platelet-activating factor using column-switching reversed-phase liquid chromatography-tandem mass spectrometry. Anal Biochem 342:134–143

    Article  CAS  Google Scholar 

  26. Mitulovic G, Stingl C, Steinmacher I, Hudecz O, Hutchins JR, Peters JM, Mechtler K (2009) Preventing carryover of peptides and proteins in nano LC-MS separations. Anal Chem 81:5955–5960

    Article  CAS  Google Scholar 

  27. Leuchtenberger S, Beher D, Weggen S (2006) Selective modulation of Abeta42 production in Alzheimer's disease: non-steroidal anti-inflammatory drugs and beyond. Curr Pharm Des 12:4337–4355

    Article  CAS  Google Scholar 

  28. Eriksen JL, Sagi SA, Smith TE, Weggen S, Das P, McLendon DC, Ozols VV, Jessing KW, Zavitz KH, Koo EH, Golde TE (2003) NSAIDs and enantiomers of flurbiprofen target γ-secretase and lower Aβ42 in vivo. J Clin Invest 112:440–449

    CAS  Google Scholar 

  29. Kounnas MZ, Danks AM, Cheng S, Tyree C, Ackerman E, Zhang X, Ahn K, Nguyen P, Comer D, Mao L, Yu C, Pleynet D, Digregorio PJ, Velicelebi G, Stauderman KA, Comer WT, Mobley WC, Li YM, Sisodia SS, Tanzi RE, Wagner SL (2010) Modulation of g-secretase reduces β-amyloid deposition in a transgenic mouse model of Alzheimer’s disease. Neuron 67:769–780

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Pharmacokinetics Research Laboratory, Dainippon Sumitomo Pharmaceutical Co., Ltd., Enoki 33-94, Suita-shi, Osaka, 564-0053, Japan

    Ken-ichi Watanabe, Chihiro Ishikawa, Kimihiko Sato, Setsuko Komuro, Tetsuya Nakagawa, Naruaki Nomura & Masashi Yabuki

  2. Genomic Science Laboratories, Dainippon Sumitomo Pharmaceutical Co., Ltd., 3-1-98 Kasugade Naka, Konohana-ku, Osaka, 554-0022, Japan

    Hiroshi Kuwahara

  3. Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama, 930-0194, Japan

    Shiro Watanabe

Authors
  1. Ken-ichi Watanabe
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  2. Chihiro Ishikawa
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  3. Hiroshi Kuwahara
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  4. Kimihiko Sato
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Correspondence to Ken-ichi Watanabe.

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Watanabe, Ki., Ishikawa, C., Kuwahara, H. et al. A new methodology for simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 by column-switching LC/MS/MS. Anal Bioanal Chem 402, 2033–2042 (2012). https://doi.org/10.1007/s00216-011-5648-1

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  • DOI: https://doi.org/10.1007/s00216-011-5648-1

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