胃蛋白酶结合柱后在线还原法分析重组人粒细胞刺激因子的二硫键
|
摘要
建立了一种利用胃蛋白酶在酸性条件下酶切,结合柱后在线还原法和液相色谱-质谱联用系统分析蛋白质二硫键的方法。在酸性体系中,采用胃蛋白酶水解蛋白质,能够最大限度地维持蛋白质二硫键的原有构象;柱后在线还原法的应用能够弥补胃蛋白酶酶切位点专一性差、数据解析困难等不足。本研究将二者相结合并成功用于分析重组人粒细胞刺激因子(rhG-CSF)的二硫键和未配对半胱氨酸。实验结果表明,采用本方法测得经N-乙酰马来酰亚胺烷基化处理的rhG-CSF中二硫键配对方式为Cys36-Cys42和Cys64-Cys74,Cys17全部被烷基化试剂结合,且未检测到二硫键错配,实现了二硫键的完全定位。不经过烷基化处理,采用本方法测得rhG-CSF中Cys36-Cys42、Cys64-Cys74和Cys17的错配比例分别为9.1%、0%和12.6%;基于胰凝乳蛋白酶的常规方法检测到的二硫键错配比例依次为73.4%、58.1%和97.5%;采用Glu-C和胰蛋白酶联合酶解的常规方法检测到的错配比例依次为40.3%、5.2%和22.3%。与常规方法相比,本方法检测到的二硫键错配比例更低,测定结果能够更准确地反映二硫键在蛋白分子内的实际存在状态。
A disulfide bond analysis method was presented in this study, which digested protein with pepsin, and combined post-column reduction with liquid chromatography-tandem mass spectrometry for peptide analysis. When protein was digested in an acidic environment, the original conformation of the disulfide bond could be maintained as much as possible; the method of post-column online reduction could make up for the poor specificity of pepsin and difficulty in data analysis. This method was used to analyze the disulfide bonds of recombinant human granulocyte-colony stimulating factor(rhG-CSF), and the results showed that disulfide bonds in NEM treated rhG-CSF were Cys36-Cys42 and Cys64-Cys74, leaving Cys17 alkylated by NEM, and no scrambling signal was detected. When analyzing the disulfide bond of rhG-CSF by this method without alkylating, the scrambling ratios were 9.1%(Cys36-Cys42), 0%( Cys64-Cys74) and 12.6%(Cys17), the results obtained from traditional method with chymotrypsin and Glu-C + trypsin were 73.4%, 58.1%, 97.5% and 40.3%, 5.2%, 22.3% correspondingly. Compared with the conventional method, this method showed a lower disulfide bond scrambling ratio, the measurement could more accurately represent the actual existence state of the disulfide bond in the protein.
A disulfide bond analysis method was presented in this study, which digested protein with pepsin, and combined post-column reduction with liquid chromatography-tandem mass spectrometry for peptide analysis. When protein was digested in an acidic environment, the original conformation of the disulfide bond could be maintained as much as possible; the method of post-column online reduction could make up for the poor specificity of pepsin and difficulty in data analysis. This method was used to analyze the disulfide bonds of recombinant human granulocyte-colony stimulating factor(rhG-CSF), and the results showed that disulfide bonds in NEM treated rhG-CSF were Cys36-Cys42 and Cys64-Cys74, leaving Cys17 alkylated by NEM, and no scrambling signal was detected. When analyzing the disulfide bond of rhG-CSF by this method without alkylating, the scrambling ratios were 9.1%(Cys36-Cys42), 0%( Cys64-Cys74) and 12.6%(Cys17), the results obtained from traditional method with chymotrypsin and Glu-C + trypsin were 73.4%, 58.1%, 97.5% and 40.3%, 5.2%, 22.3% correspondingly. Compared with the conventional method, this method showed a lower disulfide bond scrambling ratio, the measurement could more accurately represent the actual existence state of the disulfide bond in the protein.
引文
1 Zhao L, Almaraz R T, Xiang F, Hedrick J L, Franz A H. J. Am. Soc. Mass Spectrom., 2009, 20(9): 1603-1616
2 Wypych J, Li M, Guo A, Zhang Z, Martinez T, Allen M J, Fodor S, Kelner D N, Flynn G C, Liu Y D, Bondarenko P V, Ricci M S, Dillon T M, Balland A. J. Biol. Chem., 2008, 283(23): 16194-16205
3 Wu J, Watson J T. Protein Sci., 1997, 6(2): 391-398
4 Yano H, Kuroda S, Buchanan B B. Proteomics, 2002, 2(9): 1090-1096
5 Gorman J J, Wallis T P, Pitt J J. Mass Spectrom. Rev., 2002, 21(3): 183-216
6 Pace C N, Grimsley G R, Thomson J A, Barnett B J. J. Biol. Chem., 1988, 263(24): 11820-11825
7 Klúver E, Schulz-Maronde S, Scheid S, Meyer B, Forssmann W G, Adermann K. Biochemistry, 2005, 44(28): 9804-9816
8 Resemann A, Liu-Shin L, Tremintind G, Malhotra A, Fung A, Wang F, Ratnaswamy G, Suckau D. MABS, 2018, 10(8): 1200-1213
9 LIU Zhong-Hua, LIANG Song-Ping. Chem. J. Chinese Universities, 2003, 24(10): 1815-1819刘中华, 梁宋平. 高等学校化学学报, 2003, 24(10): 1815-1819
10 Roy D, Lakshminarayanan M. Biopolymers, 2016, 106(2): 196-209
11 Marques J R F, Da Fonseca R R, Drury B, Melo A. J. Theor. Biol., 2010, 267(3): 388-395
12 Gallegos-Perez J L, Rangel-Ordonez L, Bowman S R, Ngowe C O, Watson J T. Anal. Biochem., 2005, 346(2): 311-319
13 Wefing S, Schnaible V, Hoffmann D. Anal. Chem., 2006, 78(4): 1235-1241
14 Mo J, Tymiak A A, Chen G. Rapid Commun. Mass Spectrom., 2013, 27(9): 940-946
15 Dangoria N S, Delay M L, Kingsbury D J, Mear J P, Uchanskaziegler B, Ziegler A, Colbert R A. J. Biol. Chem., 2002, 277(26): 23459-23468
16 Niwa J, Yamada S, Ishigaki S, Sone J, Takahashi M, Katsuno M, Tanaka F, Doyu M, Sobue G. J. Biol. Chem., 2007, 282(38): 28087-28095
17 Wang L, Deng H X, Grisotti G, Zhai H, Siddique T, Roos R P. Hum. Mol. Genet., 2016, 18(9): 1642-1651
18 Morris H R, Pucci P. Biochem. Biophys. Res. Commun., 1985, 126(3): 1122-1128
19 Robb R J, Kutny R M, Panico M, Morris H R. Chowdhry V. Proc. Natl. Acad. Sci. USA, 1984, 81(20): 6486-6490
20 Lu S, Fan S B, Yang B, Li Y X, Meng J M, Wu L, Li P, Zhang K, Zhang M J, Fu Y. Nat. Methods, 2015, 12(4): 329-336
21 Sung W C, Chang C W, Huang S Y, Wei T Y, Huang Y L, Lin Y H, Chen H M, Chen S F. Biochim. Biophys. Acta, 2016, 1864(9): 1188-1194
22 Liu-Shin L P, Fung A,Malhotra A, Ratnaswamy G. MABS, 2018, 10(8): 1190-1199
23 Ishikawa M, Iijima H, Satake-Ishikawa R, Tsumura H, Iwamatsu A, Kadoya T, Shimada Y, Fukamachi H, Kobayashi K, Matsuki S, Asano K. Cell Struct. Funct., 1992, 17(1): 61-65
24 Schmelzer C E, Sch?ps R, Ulbrich-Hofmann R, Neubert R H, Raith K. J. Chromatogr. A , 2004, 1055(1-2): 87-92
25 Liu F, Van Breukelen B, Heck A J. Mol. Cell. Proteomics, 2014, 13(10): 2776-2786
26 Li X, Xu W, Paporello B, Richardson D, Liu H. Anal. Biochem., 2013, 439(2): 184-186
27 Liu H, Lei Q P, Washabaugh M. Anal. Chem., 2016, 88(10): 5080-5087
28 Li X, Wang F, Xu W, May K, Richardson D, Liu H. Anal. Biochem., 2013, 436(2): 93-100
29 Schneider A, Kuhn H G, Sch?bitz W R. Cell Cycle, 2005, 4(12): 1753-1757
30 Basu S, Dunn A, Ward A. Int. J. Mol. Med., 2002, 10(1): 3-10
2 Wypych J, Li M, Guo A, Zhang Z, Martinez T, Allen M J, Fodor S, Kelner D N, Flynn G C, Liu Y D, Bondarenko P V, Ricci M S, Dillon T M, Balland A. J. Biol. Chem., 2008, 283(23): 16194-16205
3 Wu J, Watson J T. Protein Sci., 1997, 6(2): 391-398
4 Yano H, Kuroda S, Buchanan B B. Proteomics, 2002, 2(9): 1090-1096
5 Gorman J J, Wallis T P, Pitt J J. Mass Spectrom. Rev., 2002, 21(3): 183-216
6 Pace C N, Grimsley G R, Thomson J A, Barnett B J. J. Biol. Chem., 1988, 263(24): 11820-11825
7 Klúver E, Schulz-Maronde S, Scheid S, Meyer B, Forssmann W G, Adermann K. Biochemistry, 2005, 44(28): 9804-9816
8 Resemann A, Liu-Shin L, Tremintind G, Malhotra A, Fung A, Wang F, Ratnaswamy G, Suckau D. MABS, 2018, 10(8): 1200-1213
9 LIU Zhong-Hua, LIANG Song-Ping. Chem. J. Chinese Universities, 2003, 24(10): 1815-1819刘中华, 梁宋平. 高等学校化学学报, 2003, 24(10): 1815-1819
10 Roy D, Lakshminarayanan M. Biopolymers, 2016, 106(2): 196-209
11 Marques J R F, Da Fonseca R R, Drury B, Melo A. J. Theor. Biol., 2010, 267(3): 388-395
12 Gallegos-Perez J L, Rangel-Ordonez L, Bowman S R, Ngowe C O, Watson J T. Anal. Biochem., 2005, 346(2): 311-319
13 Wefing S, Schnaible V, Hoffmann D. Anal. Chem., 2006, 78(4): 1235-1241
14 Mo J, Tymiak A A, Chen G. Rapid Commun. Mass Spectrom., 2013, 27(9): 940-946
15 Dangoria N S, Delay M L, Kingsbury D J, Mear J P, Uchanskaziegler B, Ziegler A, Colbert R A. J. Biol. Chem., 2002, 277(26): 23459-23468
16 Niwa J, Yamada S, Ishigaki S, Sone J, Takahashi M, Katsuno M, Tanaka F, Doyu M, Sobue G. J. Biol. Chem., 2007, 282(38): 28087-28095
17 Wang L, Deng H X, Grisotti G, Zhai H, Siddique T, Roos R P. Hum. Mol. Genet., 2016, 18(9): 1642-1651
18 Morris H R, Pucci P. Biochem. Biophys. Res. Commun., 1985, 126(3): 1122-1128
19 Robb R J, Kutny R M, Panico M, Morris H R. Chowdhry V. Proc. Natl. Acad. Sci. USA, 1984, 81(20): 6486-6490
20 Lu S, Fan S B, Yang B, Li Y X, Meng J M, Wu L, Li P, Zhang K, Zhang M J, Fu Y. Nat. Methods, 2015, 12(4): 329-336
21 Sung W C, Chang C W, Huang S Y, Wei T Y, Huang Y L, Lin Y H, Chen H M, Chen S F. Biochim. Biophys. Acta, 2016, 1864(9): 1188-1194
22 Liu-Shin L P, Fung A,Malhotra A, Ratnaswamy G. MABS, 2018, 10(8): 1190-1199
23 Ishikawa M, Iijima H, Satake-Ishikawa R, Tsumura H, Iwamatsu A, Kadoya T, Shimada Y, Fukamachi H, Kobayashi K, Matsuki S, Asano K. Cell Struct. Funct., 1992, 17(1): 61-65
24 Schmelzer C E, Sch?ps R, Ulbrich-Hofmann R, Neubert R H, Raith K. J. Chromatogr. A , 2004, 1055(1-2): 87-92
25 Liu F, Van Breukelen B, Heck A J. Mol. Cell. Proteomics, 2014, 13(10): 2776-2786
26 Li X, Xu W, Paporello B, Richardson D, Liu H. Anal. Biochem., 2013, 439(2): 184-186
27 Liu H, Lei Q P, Washabaugh M. Anal. Chem., 2016, 88(10): 5080-5087
28 Li X, Wang F, Xu W, May K, Richardson D, Liu H. Anal. Biochem., 2013, 436(2): 93-100
29 Schneider A, Kuhn H G, Sch?bitz W R. Cell Cycle, 2005, 4(12): 1753-1757
30 Basu S, Dunn A, Ward A. Int. J. Mol. Med., 2002, 10(1): 3-10