Please use this identifier to cite or link to this item:
標題: 基質輔助雷射脫附游離法搭配微結構濃縮晶片應用於磷酸化胜肽與黏多醣之快速檢測
Application of MALDI-TOF MS with radiate micro-structure chip in rapid screening of phosphopeptides and mucopolysaccharides
作者: 李坤頤
Li, Kun-In
關鍵字: 基質輔助;MALDI;質譜儀;黏多醣;磷酸化;晶片;MS;mucopolysaccharidois;phosphorylation
出版社: 分子生物學研究所
引用: 中華民國 台灣黏多醣症協會網站 財團法人罕見疾病基金會 Alverdi V, Di Pancrazio F, Lippe G, Pucillo C, Casetta B, Esposito G. Determination of protein phosphorylation sites by mass spectrometry: a novel electrospray-based method. Rapid Commun Mass Spectrom. (2005) 19:3343-8. Anson DS, Bielicki J, Hopwood JJ. Correction of mucopolysaccharidosis type I fibroblasts by retroviral-mediated transfer of the human alpha-L-iduronidase gene. Hum Gene Ther. (1992) 3:371-9. Armstrong DW, Zhang LK, He L, Gross ML. Ionic liquids as matrixes for matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem. (2001) 73:3679-86. Asara JM, Allison J. Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts. J Am Soc Mass Spectrom. (1999) 10:35-44. Blume-Jensen PH, Hunter T. Oncogenic kinase siganlling. Nature. (2001) 411 :355-65. Byers S, Rozaklis T, Brumfield LK, Ranieri E, Hopwood JJ. Glycosaminoglycan accumulation and excretion in the mucopolysaccharidoses: characterization and basis of a diagnostic test for MPS. Mol Genet Metab. (1998) 65:282-90. Fairbairn LJ, Lashford LS, Spooncer E, McDermott RH, Lebens G, Arrand JE,Arrand JR, Bellantuono I, Holt R, Hatton CE, Cooper A, Besley GT, Wraith JE, Anson DS, Hopwood JJ, Dexter TM. Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow. Proc Natl Acad Sci U S A. (1996) 93:2025-30. Ficarro SB, Parikh JR, Blank NC, Marto JA. Niobium(V) oxide (Nb2O5): application to phosphoproteomics. Anal Chem. (2008) 80:4606-13. Fukuyama Y, Nakaya S, Yamazaki Y, Tanaka K. Ionic liquid matrixes optimized for MALDI-TOF MS of sulfated/sialylated/Neutral oligosacchrides and glycopeptides. Anal Chem. (2008) 80:2171-9. Fuller M, Meikle PJ, Hopwood JJ. Glycosaminoglycan degradation fragments in mucopolysaccharidosis I. Glycobiology. (2004) 14:443-50. Fuller M, Rozaklis T, Ramsay SL, Hopwood JJ, Meikle PJ. Disease-specific markers for the mucopolysaccharidoses. Pediatr Res. (2004) 56:733-8. Gundry RL, Edward R, Kole TP, Sutton C, Cotter RJ. Disposable hydrophobic surface on MALDI targets for enhancing MS and MS/MS data of peptides. Anal Chem. (2005) 77:6609-17. Guo Z, Zhang Q, Zou H, Guo B, Ni J. A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry. Anal Chem. (2002) 74:1637-41. Guo Y, Conrad HE, The disaccharide compositions of heparin and heparin sulfates, Anal. Bio. (1989) 176:97-104. Hart SR, Waterfield MD, Burlingame AL, Cramer R. Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS. J Am Soc Mass Spectrom. (2002) 13:1042-51. Hopwood JJ, Harrison JR. High-resolution electrophoresis of urinary glycosaminoglycans: an improved screening test for the mucopolysaccharidoses. Anal Biochem. (1982) 119:120-7. Hopwood JJ, Elliott H, N-Acetylglucosamine 6-sulfate residues in keratin sulfate and heparin sulfate. Biochem Int. (1983) 6:141-8. Hopwood JJ, Elliott H. Urinary excretion of sulphated N-acetylhexosamines in patients with various mucopolysaccharidoses. Biochem J. (1985) 229:579-86. Kakkis ED, Matynia A, Jonas AJ, Neufeld EF. Overexpression of the human lysosomal enzyme alpha-L-iduronidase in Chinese hamster ovary cells. Protein Expr Purif. (1994) 5:225-32. Kakkis ED, McEntee MF, Bedolla C, Chien SL, Shull RM. Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysaccharidosis I. Biochem Mol Med. (1996) 58:156-67. Kim J, Camp DG 2nd, Smith RD. Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry. J Mass Spectrom. (2004) 39:208-15. Kjellström S, Jensen ON. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins. Anal Chem. (2004) 76:5109-17. Kosmulski M. The significance of the difference in the point of zero charge between rutile and anatase. Adv Colloid Interface Sci. (2002) 99:255-64. Laremore TN, Murugesan S, Park TJ, Avci FY, Zagorevski DV, Linhardt RJ. Matrix-assisted laser desorption/ionization mass apectrometric analysis of uncomplexed highly sulfated oligosaccharides using ionic liquid matrices. Anal Chem. (2006) 78:1774-9. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jørgensen TJ. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics. (2005) 4:873-86. Lee A, Yang HJ, Lim ES, Kim J, Kim Y. Enrichment of phosphopeptides using bare magnetic particles. Rapid Commun Mass Spectrom. (2008) 22:2561-4. Li Y, Wu J, Qi D, Xu X, Deng C, Yang P, Zhang X. Novel approach for the synthesis of Fe3O4@TiO2 core-shell microspheres and their application to the highly specific capture of phosphopeptides for MALDI-TOF MS analysis. Chem Commun (Camb). (2008) 7:564-6. Liu H, Stupak J, Zheng J, Keller BO, Brix BJ, Fliegel L, Li L. Open Tubular Immobilized Metal Ion Affinity Chromatography Combined with MALDI MS and MS/MS for Identification of Protein Phosphorylation Sites. Anal Chem. (2004) 76:4223-32. Mada A, Toyoda H, Imanari T, Utility of a carbon column for high-performance liquid chromatographic separation of unsaturated disaccharides produced from glycosaminoglycans, Anal. Sci. (1992) 8:793-797. Mason KE, Meikle PJ, Hopwood JJ, Fuller M. Characterization of sulfated oligosaccharides in mucopolysaccharidosis type IIIA by electrospray ionization mass spectrometry. Anal Chem. (2006) 78:4534-42. Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA (1999) 281:249-254. Oguma T, Tomatsu S, Montano AM, Okazaki O. Analytical method for the determination of disaccharides derived from keratan, heparan, and dermatan sulfates in human serum and plasma by high-performance liquid chromatography/turbo ionspray ionization tandem mass spectrometry. Anal Biochem. (2007) 368:79-86. Owen SJ, Meier FS, Brombacher S, Volmer DA. Increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates. Rapid Commun Mass Spectrom. (2003) 17:2439-49. Pan C, Xu S, Zhou H, Fu Y, Ye M, Zou H. Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS. Anal Bioanal Chem. (2007) 387:193-204. Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem. (2004) 76:3935-43. Porath J, Carlsson J, Olsson I, Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975 Dec 18;258(5536):598-9. Posewitz MC, Tempst P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal Chem. (1999) 71:2883-92. Qiu G, Toyoda H, Toida T, Koshiishi I, Imanari T. Compositional analysis of hyaluronan, chondroitin sulfate and dermatan sulfate: HPLC of disaccharides produced from the glycosaminoglycans by solvolysis. Chem Pharm Bull (Tokyo). (1996) 44:1017-20. Richal L. VS, David MH. 9-aminoacridine as a matrix for negative mode matrix-assisted laser desorption/ionization. Rapid Commun Mass Spectrom. (2002) 16: 1575-1581. Schuerenberg M, Luebbert C, Eickhoff H, Kalkum M, Lehrach H, Nordhoff E. Prestructured MALDI-TOF MS sample supports. Anal Chem. (2000) 72:3436-42. Scriver CR, Stanbury JB, Wyngaarden JB. The mucopolysaccharide storage diseases. The Metabolic Basis of Inherited Disease 5th ed. New York (1983) 751-761. Shull RM, Kakkis ED, McEntee MF, Kania SA, Jonas AJ, Neufeld EF. Enzyme replacement in a canine model of Hurler syndrome. Proc Natl Acad Sci U S A. (1994) 91:12937-41. Tan F, Zhang Y, Mi W, Wang J, Wei J, Cai Y, Qian X. Enrichment of phosphopeptides by Fe3+-immobilized magnetic nanoparticles for phosphoproteome analysis of the plasma membrane of mouse liver. J Proteome Res. (2008) 3:1078-87. Tsai CF, Wang YT, Chen YR, Lai CY, Lin PY, Pan KT, Chen JY, Khoo KH, Chen YJ. Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics. J Proteome Res. (2008) 7:4058-69. vander Verken P, Dirksen EH, Ruijter E, Elgersma RC, Heck AJ, Rijkers DT, Slijper M, Liskamp RM. Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides. Chembiochem. (2005) 6:2271-80. Vivès RR, Goodger S, Pye DA. Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides. Biochem J. (2001) 354:141-7. Zhao L, Wu RA, Han GH, Zhou HJ, Ren LB, Tian RJ, Zou HF. The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis. J. Am. Soc. Mass Spectrom. (2008) 19:1176-86. Zhou W, Merrick BA, Khaledi MG, Tomer KB. Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom. (2000) 11:273-82. Zolnierowica S, Bollen M. Protein phosphorylation and protein phosphatases EMBO J. (2000) 19:483–488.
黏多醣儲積症 (mucopolysaccharidosis, MPS) 的起因為人體負責降解黏多醣之基因發生缺陷所致,造成無法代謝的黏多醣持續在人體內如關節、組織、器官等各處大量累積。隨著酵素缺乏的程度差異, MPS 病患的病症也有所差異,但多伴隨如器官肥大、臉部畸形、多發性成骨不全 (dysostosis multiplex),以及聽力、視力,關節移動能力的受損,或是呼吸道與心血管受損等問題。於此希望建立一套搭配 MALDI-TOF MS 快速純化鑑定尿液中黏多醣的技術,使可能的病患可被提早檢測之目的。
樣品前濃縮與純化處理是最經常應用於增加MALDI-TOF MS 靈敏度的兩項方法,於此我們使用微結構晶片對樣品進行濃縮,增加 MALDI-TOF MS 偵測之靈敏度。比較相同體積的樣品與基質混合液點於傳統樣品盤與微結構晶片之分析結果,後者所形成之面積較前者為小,且訊號強度可放大 124% ~ 294% 不等。藉由衍生化步驟並使用陰離子交換樹脂、HLB 固相萃取可成功純化出尿液中之黏多醣分子,預期這些醣類分子可作為偵測不同黏多醣儲積症之生物標誌 (biomarker)。
可逆性的蛋白質磷酸化修飾作用可調控細胞中如增生、分化、轉錄等現象,而質譜儀是目前最常被廣泛用於鑑定磷酸化蛋白質的工具,藉由質譜儀不僅可找出磷酸化蛋白質,甚至其磷酸化位置也可被鑑定。由於磷酸化蛋白於生物體內的含量極低,加上直接以酵素水解後的胜肽混合物作為樣品分析,會發生離子抑制效應現象 (ion suppression) ,因此在進入質譜儀分析前,樣品之濃縮以及磷酸化胜肽之純化倍顯重要。因此希望藉由 Fe3O4 奈米磁性粒子搭配微結構晶片建立一快速且簡單的純化步驟,藉由與其他實驗步驟相較,本方法整體實驗時間只需耗費五分鐘;而利用相同濃度之樣品所獲得之實驗結果,on-chip 步驟之磷酸化胜肽訊號強度較 in-tube 步驟之訊雜比 (S/N ratio) 可高出27倍。

The lysosomal storage disorder mucopolysaccharidoses (MPS) caused by a deficiency in the activity of the lysosomal exohydrolases. Undegraed mucopolysacchrides accumulated in joints, tissues, and organs. The MPS share many clinical manifestations, including organomegaly, abnormal facial features, and dysostosis multiplex. Impaired hearing, vision, and joint mobility, as well as abnormal airway and cardiovascular function, are common, although there is wide clinical heterogeneity within each enzyme deficiency. However experiment diagnostic methods, have not be well established through MALDI-TOF mass spectrometry, allow patients to be treated earlier in the course of their disease.
Sample preconcentration and purification are most often used procedure to improve MALDI sensitivity. Herein, we used micro-structure chip for concentrating samples for MALDI-TOF MS analysis. Sample spots formed on our chip were much smaller than those on an unmodified plate with the same total volume, and significant enhanced results were obtained by MALDI-TOF MS. And signal intensity analyzed by chip was enhanced to 124% ~ 294% when compared to MALDI-TOF MS target plate. Oligosaccharides isolated from the urine of normal people using PMP derivatization, anion exchange and HLB extraction chromatography were identified using MALDI-TOF MS. We suggest that these glycosaminoglycan-derived oligosaccharides may be useful biochemical markers for the identification and the clinical management of mucopolysaccharidosis patients.
Reversible protein phosphorylation is a critical mediator of many cellular processes including proliferation, differentiation, and transcription. Mass spectrometry (MS) has recently become an important tool for characterization of phosphorylation proteins. Not only can phosphorylation be identified, but phosphorylation sites can also be assigned. However, direct analysis of phosphopeptides in crude enzyme-digested complex mixtures is problematic due to “ion suppression” effects and low abundance of phosphopeptides, so it is important to preconcentrate low concentration of samples and low abundance of phosphopeptides before MS analysis. Here we demonstrated another simple phosphopeptides purification procedures for MALDI-TOF MS analysis by using Fe3O4 as purification beads with micro-scale structure chip. Phosphopeptide enrichment was extremely rapid, taking approximately 5 min per sample when compared to other enrichment methods. Signal-to-noise ratio of phosphopeptides after on-chip procedure was twenty-seven times higher than in-tube procedure.
其他識別: U0005-1708200913574600
Appears in Collections:分子生物學研究所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.