宋 妮，張秀麗，王 聰，呂志華，任素梅

(中國海洋大學醫(yī)藥學院，海洋藥物教育部重點實驗室，山東 青島 266003)

碰撞誘導解離和高能碰撞解離方式在褐藻膠寡糖結(jié)構(gòu)分析中的比較和應用

宋 妮，張秀麗，王 聰，呂志華，任素梅

(中國海洋大學醫(yī)藥學院，海洋藥物教育部重點實驗室，山東 青島 266003)

利用組合式高分辨質(zhì)譜儀(LTQ Orbitrap XL)對均一褐藻膠寡糖，包括甘露糖醛酸寡糖和古洛糖醛酸寡糖進行二級質(zhì)譜分析，比較了常規(guī)的碰撞誘導解離(CID)和高能碰撞解離(HCD)在寡糖結(jié)構(gòu)分析中的差異，并應用HCD技術分析甘露糖醛酸寡糖和古洛糖醛酸寡糖的結(jié)構(gòu)差異。研究發(fā)現(xiàn)，HCD可避免CID中的1/3效應，而且可以提供更多的糖環(huán)斷裂碎片信息，如2,5A2(m/z291)、2,4A(m/z235、411)、2,5A脫水(m/z101、277、453)以及Z2脫羧碎片(m/z307、483、659)等，進一步驗證了Z2脫羧碎片是區(qū)別甘露糖醛酸與古洛糖醛酸的特征碎片。同時，HCD能夠提供豐富的裂解碎片信息，這為褐藻膠寡糖的結(jié)構(gòu)解析提供了依據(jù)。

組合式高分辨質(zhì)譜儀(LTQ Orbitrap XL)；褐藻膠寡糖；碰撞誘導解離(CID)；高能碰撞解離(HCD)

Abstract: Alginate homooligosaccharides including oligomannuronate (M) and oligoguluronate (G) with degree of polymerization from 2 to 7 were analyzed by linear ion trap-Orbitrap mass spectrometer (LTQ Orbitrap XL) using collision induced dissociation (CID) and high energy collision induced dissociation (HCD). First of all, comparison of CID MS/MS and HCD MS/MS of either M or G was conducted. Taking the trimannuronate (M3) as an example, CID MS/MS showed them/z369, 193 and 175 ions were assigned to C2/Y2, C1/Y1and B1/Z1, respectively, which were formed due to single glycosidic bond cleavage, while HCD MS/MS caused cleavage of all glycosidic bonds and showed weak A-type ions, such as0,2A (m/z309, 485),2,5A (m/z291, 467) and0,4A3ions (m/z471). Interestingly, an internal M produced a Zint-CO2ions atm/z307, which was unique and found only in HCD MS/MS. Furthermore, CID MS/MS and HCD MS/MS of M3 and triguluronate (G3) were executed. For CID, M3 and G3 did not show significant difference, and they gave identical glycosidic bond cleavage ions C1(m/z193), B1(m/z175) together with the dehydrated and decarboxylated ions (m/z157 andm/z131). These results suggest that it is impossible to differentiate the two residues at a nonreducing terminal of M3 and G3 by CID. However, a significant difference was observed in HCD spectra for M3 and G3. It was found that besides decarboxylation fragmentm/z307, debris2,4A3(m/z411),2,5A3(m/z467) and0,4A3(m/z471) were found in M3, whereas these ions were not found in HCD MS/MS of G3, thus demonstrating that HCD MS/MS can be used to differentiate M3 and G3. In the CID MS/MS of pentamannuronate (M5), fragment ions0,2A3(m/z837),0,4A3(m/z823) and2,5A3(m/z819) was found apparently, however, HCD displayed rich fragment ions in the low mass region, which should be ascribed to the characteristics of high-energy dissociation of HCD. For heptamannuronate (M7) and heptaguluronate (G7), doubly charged protonated species were collected as parent ions for CID and HCD. HCD MS/MS provided more fragment ions than CID MS/MS, especially, Zint-CO2ion (m/z307, 483, 659, 835 and 1 011) signal was apparently showed in M7 HCD spectra. Taken together, this work demonstrates that HCD MS/MS provides rich product ions and has no low mass cut off. The fragment ions in HCD spectra have high mass accuracy and resolution. These characteristics of HCD spectra complement the power of CID and allow easy spectrametric interpretation and high confidence in structural elucidation for alginate oligosaccharides.

Keywords： linear ion trap-Orbitrap mass spectrometer (LTQ Orbitrap XL); alginate homooligosaccharides; collision induced dissociation (CID); high energy collision induced dissociation (HCD)

褐藻膠是一種直鏈的多糖類化合物，結(jié)構(gòu)單元是β-(1→4)連接的D-甘露糖醛酸(M)和α-(1→4)連接的L-古洛糖醛酸(G)，其在人類疾病、植物生長和抑菌等方面具有生物活性。褐藻酸及其寡糖的活性和應用與褐藻酸中甘露糖醛酸和古洛糖醛酸的比例以及位置有著密切的關系[1-2]。近年來，電噴霧離子化-碰撞誘導解離-串聯(lián)質(zhì)譜(ESI-CID-MS/MS)技術促進了寡糖結(jié)構(gòu)及其序列分析的發(fā)展[3-8]。如，張真慶等[9]利用Q-TOF質(zhì)譜儀的CID技術對褐藻酸中甘露糖醛酸和古洛糖醛酸的異構(gòu)體進行了MS/MS序列分析。

高能碰撞解離(HCD)是一項新型的質(zhì)譜裂解技術，與傳統(tǒng)的碰撞誘導解離(CID)相比，HCD能夠提供穩(wěn)定的高能裂解模式，可以改善CID裂解中產(chǎn)生的低質(zhì)量碎片丟失(1/3 cut-off)效應。因此，將HCD應用到寡糖序列分析中，利用其高能特點可以提供不同斷裂位點的糖鏈碎片；另外，因其與Orbitrap相連能夠提供高精度的質(zhì)量數(shù)(<3×10-6)，可以直接給出元素組成，這更有助于寡糖碎片的結(jié)構(gòu)分析[10-14]。

本研究將利用HCD對均一結(jié)構(gòu)的褐藻膠寡糖，包括甘露糖醛酸寡糖和古洛糖醛酸寡糖進行二級質(zhì)譜分析，并與傳統(tǒng)的CID方式的結(jié)果進行比較，探索兩種不同的碎裂方式在褐藻寡糖結(jié)構(gòu)分析中的差異，以擴展高分辨質(zhì)譜儀LTQ Orbitrap XL的高能碰撞解離模式在褐藻寡糖結(jié)構(gòu)分析中的應用。

1 實驗部分

1.1 儀器和試劑

LTQ-Orbitrap XL線性離子阱靜電場軌道阱高分辨組合式質(zhì)譜儀：美國Thermo Fisher公司產(chǎn)品。寡糖標準品：由中國海洋大學醫(yī)藥學院趙峽課題組提供，溶于50%乙腈水溶液中；乙腈(色譜純)：美國Honeywell公司產(chǎn)品；實驗用水：Milli-Q制備的超純水。

1.2 質(zhì)譜條件

電噴霧離子源(ESI)，噴霧電壓3 kV，離子傳輸毛細管溫度275 ℃；干燥氣為氮氣，壓力0.05 MPa；CID和HCD碰撞氣均為氦氣，負離子模式檢測；CID歸一化碰撞能量采用15%～25%，HCD碰撞能量采用45～60 eV。

2 結(jié)果與討論

2.1 甘露糖醛酸及古洛糖醛酸二糖、三糖的二級質(zhì)譜解析

分析了較復雜的褐藻膠三糖——甘露糖醛酸三糖和古洛糖醛酸三糖，其二級質(zhì)譜解析示于圖1[10]。甘露糖醛酸三糖的分子離子峰為m/z545，糖苷鍵斷裂產(chǎn)生m/z369、193和175碎片離子，分別記做 C2/Y2、C1/Y1和B1/Z1，它們是源于還原端和非還原端的離子碎片，是吡喃糖醛酸三糖的特征碎片離子。

本研究還分析了甘露糖醛酸三糖CID和HCD譜圖的差異，其二級質(zhì)譜采集信息示于圖2。將產(chǎn)生跨環(huán)斷裂碎片的區(qū)域放大10倍后，CID MS/MS給出了所有的糖苷鍵斷裂碎片C2/Y2(m/z369)、B2/Z2(m/z351)、C1/Y1(m/z193)和 B1/Z1(m/z175)。糖環(huán)斷裂僅顯示了0,2A(m/z309和m/z485)，而HCD MS/MS除了給出糖苷鍵斷裂碎片外，還給出了所有的A型碎片離子,如0,2A(m/z309和m/z485)、2,5A(m/z291和m/z467)、0,4A3(m/z471)和2,4A(m/z235和m/z411)，以及甘露糖醛酸三糖區(qū)別于古洛糖醛酸三糖特殊的Zint脫羧碎片m/z307[10]，其中2,4A(m/z235和m/z411)是首次被檢測到。

對比甘露糖醛酸三糖和古洛糖醛酸三糖的CID和HCD譜圖的主要對照特征峰和峰高可以發(fā)現(xiàn)，兩種寡糖的CID譜圖沒有明顯差異，所有差異均顯示在HCD譜圖中。除Z2脫羧碎片m/z307外，在古洛糖醛酸三糖的HCD中未見碎片2,4A3(m/z411)、2,5A3(m/z467)和0,4A3(m/z471)，這可作為區(qū)別兩種寡糖的特殊碎片。

通過比較甘露糖醛酸二糖和古洛糖醛酸二糖的CID和HCD二級質(zhì)譜圖發(fā)現(xiàn)，同一二糖的CID和HCD之間無明顯差異。這是因為在二糖中沒有中間糖環(huán)，在原三糖HCD譜中發(fā)現(xiàn)的脫羧碎片也就不存在了。

2.2 甘露糖醛酸和古洛糖醛酸四、五糖的二級質(zhì)譜解析

從實驗數(shù)據(jù)可以看出，四糖和五糖具有相同的結(jié)果，本研究以甘露糖醛酸五糖和古洛糖醛酸五糖為例進行解析，結(jié)果示于圖3。甘露糖醛酸五糖和古洛糖醛酸五糖的CID和HCD譜圖示于圖4。通過比較甘露糖醛酸五糖的CID和HCD譜圖可以發(fā)現(xiàn)：HCD二級質(zhì)譜圖給出了通過糖苷鍵斷裂產(chǎn)生的所有表征它們直鏈連接方式的碎片離子m/z175/193、m/z351/369、m/z527/545和m/z703/721；而在CID譜圖中，因1/3效應缺失了m/z175/193。但是，在CID譜圖中可見跨環(huán)斷裂產(chǎn)生的碎片離子0,2A5(m/z837)、0,4A5(m/z823)和2,5A5(m/z819)，而在HCD譜圖中未見這3個特征峰。與三糖相同，甘露糖醛酸區(qū)別于古洛糖醛酸殘基的Z離子脫羧碎片Zint-CO2(m/z307和m/z483)僅出現(xiàn)在HCD譜圖中。還可以看到，HCD在低質(zhì)荷比端提供了大量的碎片離子，彌補了CID的1/3效應，HCD譜比CID譜在低質(zhì)荷比區(qū)域的信號和峰度高，CID譜則反之，這是由于HCD高能裂解的特點。

圖1 甘露糖醛酸三糖(a)和古洛糖醛酸三糖(b)的二級質(zhì)譜解析Fig.1 MS/MS analysis of trimannuronate (a) and triguluronate (b)

圖2 甘露糖醛酸三糖和古洛糖醛酸三糖的CID(a, c)和HCD(b, d)譜圖Fig.2 CID (a, c) and HCD (b, d) MS/MS spectrum of trimannuronate and triguluronate

圖3 甘露糖醛酸五糖(a)和古洛糖醛酸五糖(b)的質(zhì)譜解析Fig.3 MS/MS analysis of pentamannuronate (a) and pentaguluronate (b)

圖4 甘露糖醛酸五糖和古洛糖醛酸五糖的CID(a,c)譜和HCD(b,d)譜Fig.4 CID (a, c) and HCD (b, d) MS/MS spectrum of pentamannuronate and pentaguluronate

比較甘露糖醛酸五糖和古洛糖醛酸五糖的CID以及HCD差異，發(fā)現(xiàn)與三糖的解析結(jié)果有相同的規(guī)律，M5和G5的CID譜圖沒有明顯差異，M5的HCD譜圖較G5僅多了Zint-CO2(m/z307和m/z483)，進一步驗證了Zint-CO2碎片離子是區(qū)別甘露糖醛酸寡糖與古洛糖醛酸寡糖的重要標志。通過比較發(fā)現(xiàn)，需要將CID和HCD結(jié)合起來才能得到五糖完整的解析信息。聚合度為2～5的兩種類型寡糖的CID MS/MS和HCD MS/MS詳細二級碎片信息列于表1。

表1 8種寡糖在CID和HCD方式下的質(zhì)譜碎片峰比較Table 1 Comparison of CID and HCD for 8 oligosaccharides

2.3 高聚合度甘露糖醛酸及古洛糖醛酸的二級質(zhì)譜解析

對于聚合度高于5的寡糖，二級質(zhì)譜采集選擇雙電荷或三電荷離子作為母離子。本研究以七糖為例進行解析，甘露糖醛酸七糖和古洛糖醛酸七糖的CID和HCD譜圖示于圖5。M7選擇m/z624雙電荷離子作為母離子，從圖中可以看出，HCD譜給出的信息遠多于CID譜，在糖苷鍵斷裂的碎片中有豐富的糖環(huán)斷裂碎片，尤其是Zint-CO2離子(m/z307、483、659、835、1 011)的信號在M7的HCD譜中很明顯，但是在G7的HCD譜中未見，因此可以很容易地區(qū)分這兩種寡糖。對于高聚合度的寡糖，HCD的優(yōu)勢明顯高于CID。

圖5 甘露糖醛酸七糖和古洛糖醛酸七糖的CID譜和HCD譜Fig.5 CID and HCD MS/MS of heptamannuronate and heptaguluronate

3 結(jié)論

通過分析褐藻膠寡糖的HCD和CID二級質(zhì)譜數(shù)據(jù)發(fā)現(xiàn)：對于聚合度較低的寡糖，HCD較CID能夠給出更多的糖環(huán)斷裂碎片和低質(zhì)荷比碎片等信息，這是寡糖結(jié)構(gòu)解析的關鍵；對于聚合度為4和5的寡糖，在高質(zhì)荷比區(qū)域的CID信息較HCD信息更為豐富，因此需要綜合兩種裂解模式提供的信息進行解析；對于聚合度高于5的寡糖，HCD提供的信息遠遠多于CID模式。從數(shù)據(jù)分析發(fā)現(xiàn)，HCD通過檢測Zint-CO2離子可以清晰地判斷寡糖類型。對于褐藻膠寡糖的二級質(zhì)譜分析，將HCD高能裂解的特點應用到寡糖結(jié)構(gòu)解析，可以提供更多的糖環(huán)斷裂碎片與高質(zhì)量精度和高靈敏度的二級質(zhì)譜數(shù)據(jù)，并且避免了CID技術中低質(zhì)荷比數(shù)據(jù)缺失，有助于褐藻膠寡糖的結(jié)構(gòu)解析。

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Comparison and Application of Collision Induced Dissociation and High Energy Collision Induced Dissociation for Alginate Oligosaccharides Analysis

SONG Ni, ZHANG Xiu-li, WANG Cong, LV Zhi-hua, REN Su-mei

(KeyLaboratoryofMarineDrugs,SchoolofMedicineandPharmacy,OceanUniversityofChina,Qingdao266003,China)

O657.63

A

1004-2997(2017)05-0551-08

10.7538/zpxb.youxian.2016.0063

2016-06-16;

2016-08-23

中央高?；究蒲袠I(yè)務費實驗室基金項目(201551012)資助

宋 妮(1975—)，女(漢族)，山東青島人，工程師，從事有機質(zhì)譜分析研究。E-mail: nisong1975@ouc.edu.cn

任素梅(1963—)，女(漢族)，山東青島人，教授級高級工程師，從事有機質(zhì)譜分析研究。E-mail: rensumei@ouc.edu.cn

時間：2016-12-28；網(wǎng)絡出版地址：http:∥www.cnki.net/kcms/detail/11.2979.TH.20161228.0935.020.html