姚文斌，汪億晗，蘇 蕊，連文慧，楊洪梅,2，陳長寶，劉淑瑩

(1.長春中醫(yī)藥大學，吉林省人參科學研究院，吉林 長春 130117；2.中國科學院長春應用化學研究所，吉林 長春 130022)

含二硫鍵的蛋白質/多肽的MALDI-TOF MS源內裂解研究

姚文斌1，汪億晗1，蘇 蕊1，連文慧1，楊洪梅1,2，陳長寶1，劉淑瑩1

(1.長春中醫(yī)藥大學，吉林省人參科學研究院，吉林 長春 130117；2.中國科學院長春應用化學研究所，吉林 長春 130022)

應用基質輔助激光解吸電離飛行時間質譜(MALDI-TOF MS)法研究含二硫鍵的人胰島素與甘精胰島素酶解液的源內裂解(ISD)。比較了不同基質種類及不同結晶狀態(tài)對含二硫鍵的人胰島素與甘精胰島素酶解液的源內裂解的影響。結果表明，含二硫鍵的蛋白質的ISD發(fā)生受激光點照射位置的影響，在不同基質與結晶形態(tài)的條件下，含二硫鍵的蛋白質的ISD碎片信息不同。通過分析比較，含二硫鍵的蛋白質的ISD較容易控制，并且其基質的種類及結晶狀態(tài)作用很關鍵。需要獲得大量碎片時，使激光照射在樣品和阿魏酸(FA)基質形成的大結晶處；不希望出現(xiàn)碎片時，可使用2,4,6-三羥基苯乙酮(THAP)為基質，或使激光照射在樣品和其他基質形成的細小均勻結晶處。

含二硫鍵的蛋白質/多肽；基質輔助激光解吸電離飛行時間質譜(MALDI-TOF MS)；源內裂解(ISD)；基質；結晶

基質輔助激光解吸電離(MALDI)技術是由Karas和Tanaka等[1-2]發(fā)明的，由于基質輔助激光解吸電離飛行時間質譜(MALDI-TOF MS)具有質量范圍寬、耐鹽和靈敏度高等優(yōu)點[3-5]，利用該技術分析蛋白質的應用日益廣泛。二硫鍵是一種常見的蛋白質翻譯后修飾，對穩(wěn)定蛋白質的空間結構[6-9]、維持正確的折疊構象[10-12]、保持及調節(jié)生物活性[13-15]等都有著重要作用。確定二硫鍵在蛋白質中的位置對于鑒定蛋白質一級結構有重要的意義，是研究含有二硫鍵的活性多肽/蛋白質化學結構的重要方面。目前，MALDI MS已經(jīng)成為分析二硫鍵的強有力工具[16-17]。

雖然MALDI通常被認為是一種軟離子化過程，但是在離子化過程中也會出現(xiàn)嚴重的碎裂，例如，會發(fā)生二硫鍵連接的多肽的快速裂解[18-20]現(xiàn)象。這種快速裂解受激光強度和所用基質的影響[20-22]，在以芥子酸為基質的質譜圖中，這種快速碎裂方式并不常見。通過研究二硫鍵連接的多肽的快速裂解現(xiàn)象發(fā)現(xiàn)，激光強度起著重要的作用，其他參數(shù)對碎裂程度的影響則很小。Zhao等[22]報道的二硫鍵源內裂解具有如下規(guī)律：如果多肽結構中是成對的鏈間二硫鍵，主要發(fā)生自由基重排；只要結構中有奇數(shù)個半胱氨酸就可以獲得還原型的源內裂解碎片。

本研究擬分析結晶狀態(tài)和基質種類對含二硫鍵的蛋白質源內裂解(ISD)的影響，以促進或避免ISD的發(fā)生，希望為使用MALDI-TOF MS分析含二硫鍵的蛋白質/多肽提供支持信息。

1 實驗部分

1.1 儀器和試劑

Voyager DE-STR MALDI-TOF MS儀：美國AB公司產(chǎn)品，配有N2激光器(337 nm)；α-氰基-4-羥基肉桂酸(CHCA)、芥子酸(SA)、阿魏酸(FA)、2,5-二羥基苯甲酸(DHB)、2,4,6-三羥基苯乙酮(THAP)、3-氨基喹啉(3-AQ)等基質：均為美國Sigma公司產(chǎn)品；胰蛋白酶： Promega(北京)生物技術有限公司產(chǎn)品；人胰島素和甘精胰島素：通化東寶制藥有限公司產(chǎn)品；三氟乙酸(TFA)：色譜級，美國Tedia公司產(chǎn)品；實驗用水：由美國Millipore公司Milli-Q超純水機制備。

1.2 蛋白質酶解

取50 μL 1 g/L的人胰島素和甘精胰島素，分別溶解在pH 7.8的50 mmol/L NH4HCO3溶液中，于37 ℃用胰蛋白酶進行水解；酶/底物的質量比為1∶50，酶解8 h；最后將酶解液放入-20 ℃冰箱中終止酶解反應。

1.3 MALDI-MS分析

樣品直接在MALDI靶上制備：取0.5 μL0.2 g/L的被分析物與同體積10 g/L的基質混合，基質溶解在含0.1%TFA的水-乙腈(1∶1，V/V)溶液中。將50 mmol/L NH4HCO3-NH3·H2O緩沖溶液(pH 9.0)添加到混合溶液中，調節(jié)溶液至pH 8。

采用正離子模式采集，測試每一個樣品時均優(yōu)化了加速電壓、光柵電壓和延遲時間等參數(shù)。激光頻率為3 Hz，掃描50次。利用外標法進行校正，校正液包括CHCA(190.050 4 u)和血管緊張素Ⅰ(1 296.685 3 u)。

2 結果與討論

采用MALDI-TOF MS分析含二硫鍵的蛋白質時，注意到含二硫鍵的蛋白質ISD的發(fā)生受激光點照射位置的影響。以人胰島素和甘精胰島素為例，評價了結晶狀態(tài)對ISD碎裂的影響。酶切位點示于圖1。激光照射人胰島素與FA基質形成非常細小的均勻結晶位置的譜圖沒有產(chǎn)生碎片，示于圖2a。從圖2a可見，僅有2個離子m/z859.46和m/z4 866.23的酶解肽段。把激光照射在大結晶處獲得圖2b，比較圖2b與圖2a可看出，圖2b中含有質量差為32 u的3個特征離子(m/z2 454.24、2 486.21、2 518.18)，是通過對稱和非對稱斷裂m/z4 866.23的多肽的二硫鍵而產(chǎn)生，可能是熱力學的原因，是斷裂偶數(shù)個化學鍵后的自由基重排反應，而非還原性的ISD，這與前期的文獻[22]報道一致。

圖1 人胰島素(a)和甘精胰島素(b)的結構式和相對分子質量Fig.1 Structures and molecular weights of human insulin (a) and insulin glargine (b)

注：左圖為以FA為基質的經(jīng)胰蛋白酶酶解后人胰島素的位點照片圖2 均勻結晶位置(a)和大結晶處(b)所得的MALDI質譜圖Fig.2 MALDI mass spectrum recorded in a region where crystals are very finely distributed (a) and recorded on a large crystal (b)

分析甘精胰島素的質譜圖觀察到了相似的現(xiàn)象，結果示于圖3。從圖3可見，除了m/z4 809.20離子外，所有質譜峰與圖2中結果相同。結果證實，含二硫鍵的蛋白質ISD的發(fā)生依賴于其結晶狀態(tài)，這與采用MALDI ISD研究全甲基化糖的結果[23]一致。在其報道中，因為糖對陽離子的親和性較高，所以碎裂可以由樣品點無定形區(qū)域的高濃度鈉所避免。另一方面，由于基質晶體處沒有鈉離子，糖苷鍵被質子化而斷裂。這種機理不能解釋當前的實驗現(xiàn)象，因為多肽是被質子化的，對于此現(xiàn)象的合理解釋是：結晶越小，樣品吸收的激光強度越小，MALDI過程越溫和；反之，結晶越大，樣品吸收的激光強度越大，MALDI過程越劇烈[24]。

注：左圖為以FA為基質的經(jīng)胰蛋白酶酶解后甘精胰島素的位點照片；※號標記為通過對稱和非對稱斷裂二硫鍵而產(chǎn)生的碎片離子圖3 均勻結晶位置(a)和大結晶處(b)所得到的MALDI質譜圖Fig.3 MALDI mass spectrum recorded in a region where crystals are very finely distributed (a) and recorded on a large crystal (b)

為更加透徹地理解這種現(xiàn)象，考察了CHCA、SA、DHB、AQ和THAP基質。在這些基質中，與FA相似，CHCA和SA也可以使含二硫鍵的蛋白質斷裂，同樣具有結晶狀態(tài)依賴性。與FA不同的是，ISD并不是在所有的大結晶上都能觀察到，表明CHCA(SA)與FA形成結晶過程的差異性。使用DHB和AQ為基質時，在大結晶處可以觀察到較小的碎片，結果示于圖4，小結晶處觀察不到碎片。使用THAP為基質時，觀察不到碎片，可能由于它的結構中，三個羥基相對于每一個羥基而言都處于中間位置，無法發(fā)生氧化還原反應[23]。

注：*號標記為通過對稱斷裂二硫鍵而產(chǎn)生的碎片離子圖4 以DHB(a)與AQ(b)為基質，甘精胰島素經(jīng)胰蛋白酶酶解后所得的MALDI質譜圖Fig.4 MALDI mass spectra of insulin glargine tryptic digests using DHB (a) and AQ (b) as the matrices

3 結論

本實驗應用MALDI-TOF MS研究了結晶狀態(tài)和基質對人胰島素與甘精胰島素兩個含二硫鍵的蛋白質的ISD影響。通過分析比較，含二硫鍵的蛋白質的ISD較容易控制，其基質的種類及結晶狀態(tài)起著關鍵的作用。在基質考察中，F(xiàn)A、CHCA和SA均可以使含二硫鍵的蛋白質斷裂，同樣具有結晶狀態(tài)依賴性。與FA不同的是，使用CHCA和SA為基質時，并不能在所有的大結晶上都觀察到ISD，表明CHCA(SA)與FA形成結晶過程具有差異性。使用DHB和AQ為基質時，可以觀察到較小的碎片。使用THAP為基質時，觀察不到碎片。因此，當需要大量碎片時，可以使用FA為基質，不希望出現(xiàn)碎片時可以使用THAP為基質。這為使用MALDI-TOF MS分析含二硫鍵的蛋白質/多肽提供理論指導。

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In-Source Decay of Disulfide Bond-Containing Proteins/Peptides by MALDI-TOF MS

YAO Wen-bin1, WANG Yi-han1, SU Rui1, LIAN Wen-hui1, YANG Hong-mei1,2, CHEN Chang-bao1, LIU Shu-ying1

(1.JilinGinsengAcademy,ChangchunUniversityofChineseMedicine,Changchun130117,China;2.ChangchunInstituteofAppliedChemistryChineseAcademyofSciences,Changchun130022,China)

The disulfide bond is one of the most common post-translational modifications in proteins, of which determination is essential to the comprehensive understanding of protein structures. Disulfide bond analysis has gone through great improvement due to the development of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), especially in terms of speed and sensitivity. In general, the characterization of disulfide-containing peptides is achieved by the reduction of disulfide bonds followed by alkylation. The identification of disulfide/cysteine-containing peptides in digests of proteins is essential to structure elucidation of a protein. In order to present a deep understanding of some phenomena occurring in MALDI MS of disulfide/cysteine-containing proteins, the current work systematically investigated effects of co-crystal size and matrix on MALDI-In Source Decay (ISD) fragmentation of disulfide-containing proteins. Imaging experiments were performed to evaluate the influence of laser shot location on the fragmentation of human insulin and insulin glargine, which were selected as model compounds. The spectrum that was recorded on very finely distributed crystals of FA spot does not exhibit fragments. While a characteristic ‘triplet’ ions with a mass separation of 32 u generated by both symmetric and nonsymmetric cleavages of the disulfide bonds was observed on large crystals. Probably for thermodynamic reasons, the tryptic peptide was subjected to the cleavage of even number of chemical bonds gave rise to radical recombination without reductive ISD. Among several matrices tested including ferulic acid (FA),α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), 3-aminoquinolin (AQ), and 2,4,6-trihydroxy acetophenone (THAP), FA was shown to be a versatile matrix allowing one to induce or prevent ISD according to the location of laser shots. CHCA and SA were found to promote ISD of disulfide-containing proteins, in a location dependent manner. However, unlike in CHCA (or SA), ISD was not systematically observed on all crystals for FA, suggesting differences between the crystallization processes of CHCA (or SA) and FA. Minor fragments were observed when using DHB and AQ as matrices. As for THAP, no fragmentation was observed probably because its three OH-groups in meta-position relative to each other resulted in the nonoccurrence of redox reaction. The studies provide insights into the experimental conditions required for determination of disulfide-containing protein by MALDI MS and are helpful for mass spectrum interpretation, opening the way to more rational studies of disulfide/cysteine-containing proteins by MALDI mass spectrometry.

disulfide-containing protein/peptide; MALDI-TOF MS; in-source decay; matrix; crystal

2016-05-06;

2016-09-21

吉林省科技廳項目(20170623026TC，20160101220JC，20160204027YY)；吉林省衛(wèi)生技術創(chuàng)新項目(2016J098)資助

姚文斌(1991—)，男(漢族)，山西人，碩士研究生，中藥化學專業(yè)。E-mail: 2390697021@qq.com

陳長寶(1967—)，男(漢族)，吉林人，研究員，從事中藥及生物制藥研究。E-mail: ccb2021@126.com
劉淑瑩(1943—)，女(漢族)，黑龍江人，研究員，從事中藥化學和有機質譜學研究。E-mail: liusy01@ccucm.edu.cn

時間：2017-04-13；網(wǎng)絡出版地址：http:∥www.cnki.net/kcms/detail/11.2979.TH.20170413.0929.012.html

O657.63

A

1004-2997(2017)03-0302-06

10.7538/zpxb.2016.0075