杜迎新，林亞維

(武漢理工大學 化學化工與生命科學學院， 武漢 430070)

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基于化學衍生的MALDI質譜法分析唾液酸化聚糖

杜迎新，林亞維

(武漢理工大學 化學化工與生命科學學院， 武漢 430070)

摘要：唾液酸廣泛存在于聚糖的末端，不僅參與細胞信號傳導、細胞粘附以及特異性識別等重要生理過程，還與癌癥等重大疾病密切相關。因此，分析生物體系中的唾液酸化聚糖具有十分重要的意義。MALDI質譜具有靈敏度高、可以直接測定分子量等優(yōu)點，已成為檢測糖鏈的重要工具之一，但該方法直接檢測生物樣品中的唾液酸聚糖鏈仍存在諸多的困難，必須對其進行適當?shù)匮苌?。常用衍生化方法可通過引入帶電基團或疏水基團，以此增強聚糖的色譜分離、離子化效率或防止檢測過程中唾液酸殘基的丟失。本文主要針對從糖綴合物中解離下的唾液酸化聚糖樣品，總結、概述了最近涌現(xiàn)的各種衍生化技術與相應的MALDI質譜檢測方法。

關鍵詞：唾液酸化糖鏈；化學衍生；MALDI質譜法

蛋白質糖基化是基因在轉錄和翻譯后最廣泛、最復雜、最重要的修飾之一，而目前已知真核蛋白中超過半數(shù)的蛋白存在糖基化修飾。這些糖蛋白廣泛分布于細胞、組織及體液中，特別是在細胞膜上和體液中表達廣發(fā)。比如，人們把細胞外壁表達顯著的聚糖稱之為“糖被Glycocalyx”，它們與細胞的易裂、細胞-細胞相互作用等重要行為緊密相關，在細胞分化、發(fā)育、腫瘤發(fā)生與轉移、精卵識別、免疫、傳染及再生中發(fā)揮著重要的作用。因此，糖鏈的分析與檢測已成為當前糖組學研究中最重要的領域之一。

圖1　唾液酸類化合物的結構圖

唾液酸(SA)是一類具有9碳糖結構的神經氨酸衍生物(圖1)，自然界中，通常作為“信號分子”連接在細胞膜寡糖側鏈的非還原末端，形成唾液酸聚糖、唾液酸糖肽和唾液酸糖蛋白；游離的唾液酸主要存在3種形式，即N-乙酰神經氨酸(Neu5Ac)、N-羥乙酰神經氨酸(Neu5Gc)、去氨基神經氨酸(KDN)。唾液酸4號位、7號位、8號位、9號位碳原子上的羥基可被甲基、乳酰基、硫酸和磷酸等基團取代形成多種同系物[1-4]，目前發(fā)現(xiàn)的天然唾液酸已經超過60種。在N-糖鏈與O-糖鏈的末端都發(fā)現(xiàn)有唾液酸單元，它們與糖鏈的聯(lián)接方式為：唾液酸2號碳原子上的羥基可通過α2-(3/4/6)與半乳糖(Gal)相連；通過α2-(3/6)與N-乙酰半乳糖胺(GalNAc)相連；α2-6與N-乙酰葡糖胺(GlcNAc)相連；α2-(8/9)與唾液酸分子相連[5-8]。多種鏈接方式形成不同種類的唾液酸糖鏈和唾液酸聚糖。

早期報道指出，唾液酸化聚糖與癌癥有著密切聯(lián)系：比如，胃癌晚期患者胃液中唾液酸聚糖、結腸癌和淋巴瘤患者血漿中唾液酸聚糖含量高于正常人[9-10]。隨著深入研究發(fā)現(xiàn)，不同病理周期黑色素瘤和肺癌患者血清中唾液酸化聚糖的含量有明顯的變化[11-12]；紅細胞表面唾液酸化聚糖對其生長、衰老、吞噬起重要的調控作用[13-14]；唾液酸羧基的電負性和末端三醇邊鏈結構是維持細胞膜表面Ca2+穩(wěn)態(tài)的重要因素[15-16]，并且與神經突觸的傳遞和神經系統(tǒng)的發(fā)育息息相關[17]。

鑒于唾液酸化聚糖的重要性，半個世紀以來，國內外學者對它展開了廣泛研究[18-20]；尤其近十幾年，隨著現(xiàn)代分析測試技術，例如MALDI(基質輔助激光解吸)質譜、液相色譜-質譜聯(lián)用技術( LC-MS)[21-23]、熒光高效液相色譜技術(HPLC-FD)[22]和高效陰離子交換-脈沖安培檢測法(HPAE-PAD)[25-26]、核磁共振法(NMR)[24]等發(fā)展的日趨成熟，唾液酸聚糖研究在分離純化、熒光標記、質譜檢測等方面取得了突破性進展。其中MALDI(基質輔助激光解吸)質譜法以其靈敏度高，能產生具有結構信息的離子碎片，分析速度快等優(yōu)勢，目前已成為檢測唾液酸化聚糖的最主要方法之一。然而，MALDI質譜法直接對唾液酸化糖鏈進行分析，依然存在諸多困難。這可歸咎為以下幾個因素：(1)待測生物樣品中唾液酸的豐度值相對較低，且信號易被其他種類的生物分子或污染物抑制；(2)唾液酸糖苷鍵在MALDI質譜檢測過程中可能發(fā)生斷裂，導致唾液酸化糖鏈中唾液酸殘基的解離或者丟失;(3)唾液酸分子本身結構的特殊性，例如，弱酸性、強極性、基團遷移等決定了在MALDI質譜分析唾液酸化聚糖前可對其進行適當?shù)匮苌托揎棧瑥亩_到唾液酸化聚糖高靈敏度、高選擇性的準確分析[27-29]。本文主要對最近涌現(xiàn)的多種適用于MALDI質譜分析的唾液酸化聚糖衍生方法進行了介紹和總結。其中，唾液酸化聚糖的衍生化方法大致歸為：泛衍生化反應、針對羧基的酯化與酰胺化衍生反應。接下來，我們將重點介紹這幾種方法的研究進展，總結目前適用于MALDI質譜分析的唾液酸化聚糖各種衍生方法，并對評述它們的原理及應用范圍。

1唾液酸化糖鏈衍生化方法

1.1泛衍生化反應

圖2　唾液酸泛甲基化反應

泛苯甲酰基化也是分析唾液酸聚糖樣品中常用的衍生方法，與泛甲基化比較，其優(yōu)點在于可以明顯改善聚糖在反相色譜上的分離性能，便于紫外檢測[36]。Chen等[37]將苯甲酸酐與Neu5Acα2-3、α2-6Gal低聚糖單元在1-甲基咪唑溶液中110°C反應5h，實驗表明，泛苯甲酰基化衍生后的唾液酸殘基在MALDI質譜檢測中的穩(wěn)定性顯著提高，通過清晰MALDI質譜圖可以分析和鑒別皮摩爾水平的α2,3和α2,6連接的唾液酸化聚糖(圖 3)。盡管如此，泛苯甲?；苌鷷r間相對較長，室溫下很難達到理想的衍生效果，且唾液酸羧基未能參與反應，仍需進一步酯化處理，因此這種方法未能被廣泛使用。

圖3　唾液酸泛苯甲?；磻? a.苯甲酸酐與Neu5Acα2,3Gal聚糖的反應；b.苯甲酸酐與Neu5Acα2,6Gal聚糖的反應

2唾液酸羧基的衍生反應

由于末端唾液酸殘基中羧基的存在會導致其糖苷鍵的不穩(wěn)定，在MALDI質譜分析過程中往往會導致唾液酸的解離或丟失[3,38]。電噴霧電離(ESI)中也會降低唾液酸聚糖的離子化效率和質譜信號強度[39,40]。因此，通過對唾液酸羧基化學衍生使唾液酸聚糖呈電中性，從而獲得樣本完整的結構信息是處理唾液酸樣品常用的方法，以下我們將介紹針對唾液酸羧基的幾種主要衍生方法。

2.1羧基酯化反應

圖4　唾液酸羧基酯化反應

羧基酯化反應是提高唾液酸聚糖穩(wěn)定性一種行之有效的方法(圖 4)。Hakomori[41]等最先嘗試用高活性的重氮甲烷與AG50(H+)酸化的唾液酸化聚糖在甲醇-乙醚溶液中反應。反應過程中，首先亞甲基奪取羧基的氫質子，然后帶負電荷的羧基進攻質子化的重氮甲烷，隨即離去一個氮分子，得到甲酯化產物，此反應具有速率高、連續(xù)性強等優(yōu)點,但是由于重氮甲烷的不穩(wěn)定性，使其應用受到局限。

碘甲烷作為常見的親電甲基化試劑，可以使羧基氫質子甲基化。Harvey等[42]將唾液酸聚糖先經過AG50(Na+)離子交換層析柱，轉化為離子態(tài)，然后與碘甲烷在室溫條件下快速反應得到產物，首次成功地將唾液酸甲酯化反應用于MALDI質譜分析中；此方法可以消除不穩(wěn)定質子，抑制唾液酸殘基的丟失，得到更加清晰質譜圖。在此基礎之上，Li等[38]采用“一鍋法”將酶解、純化后的N-聚糖中唾液酸的羧基甲酯化；結果表明，與未衍生的N-聚糖相比，處理后的唾液酸化聚糖檢測靈敏度可以提高約10倍。

Nishimura[43]等使用“糖印跡”(glycoblotting)技術，將1-甲基-3-對甲苯基三氮(MTT)與唾液酸化聚糖的金膠體納米粒子載體發(fā)生“固相甲基化”反應，不僅避免使用易揮發(fā)、毒性強、危險性大的試劑，如碘甲烷和重氮甲烷等，同時減少了MALDI-TOF MS中陽離子種類和數(shù)量，獲得高靈敏度和高分辨率的質譜信號。

糖肽中的唾液酸化聚糖具有強親水性，抑制了其在MALDI質譜中的電離能力，為了增強天然唾液酸糖鏈在質譜中的離子化效率，Amano[44]等以DMSO為溶劑，羧基作為親核試劑進攻強疏水性試劑1-芘基二氮甲烷(PDAM)上疊氮相連的活性C原子，同時離去一分子氮，生成帶有芘基的疏水性唾液酸衍生物。衍生化處理后， MALDI-MS能夠檢測出源自1ng前列腺特異性抗原中的唾液酸糖肽衍生物。進一步研究發(fā)現(xiàn)[45]，PDAM衍生之后的α2,3和α2,6唾液酸聚糖異構體在MALDI產生不同CID離子碎片，這種特異性電離模式，可用于快速區(qū)別兩種不同連接方式的唾液酸聚糖。

Hincapie等[46]首先使用4-(4,6-二甲氧基三嗪-2-基)-4-甲基嗎啉鹽酸鹽(DMT-MM)為偶聯(lián)劑，N-聚糖與甲醇溶液在80°C 下反應1 h，可得到高轉化率的甲酯化衍生產物。最近，Reiding等[47]選用1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽，1-羥基苯并三唑(EDC/HOBt)為羧基活化劑，使唾液酸化聚糖與乙醇的酯化反應溫度降低一半，酯化產率更高。值得強調的是，此類反應具有高度區(qū)域專一性，α2-6連接的唾液酸化聚糖與乙醇發(fā)生酯化反應(圖5 a)，而α2-3連接唾液酸聚糖的唾液酸分子與相鄰的半乳糖(Gal)4號位羥基發(fā)生酯化反應，形成六元環(huán)內酯 (圖5 b)，這種特異性的酯化方式可以提高唾液酸化聚糖在親水相互作用色譜法(HILIC)中的分離效率，并且由于衍生產物分子量的差異，從而能準確的測定糖鏈中唾液酸的連接方式，具有很大的應用價值及前景。

圖5　唾液酸與乙醇的酯化反應: a. α2-6連接的唾液酸化聚糖與乙醇發(fā)生酯化反應; b. α2-3連接唾液酸聚糖與相鄰的半乳糖(Gal)4號位羥基反應，形成六元環(huán)內酯。

2.2羧基酰胺化反應

酯類化合物在強酸或強堿性條件易發(fā)生水解反應，其穩(wěn)定性遠遠不如酰胺結構。酰胺鍵的形成能夠穩(wěn)定唾液酸與多糖之間的糖苷鍵，抑制MALDI過程中唾液酸殘基的丟失。諸多報道指出，唾液酸乙酰氧基易發(fā)生基團遷移或者解離[48,49]，同酯化反應一樣，酰胺化(圖 6)也是唾液酸衍生中重要方法。

圖6　唾液酸羧基的酰胺化反應

Sekiya等[50]以DMT-MM為縮合劑，唾液酸化聚糖與NH4Cl在弱酸性條件下反應，生成酰胺；研究表明，酰胺反應能夠抑制Neu5Acα2-6Gal糖苷鍵源內裂解(ISD)、源后裂解(PSD)、碰撞誘導解離(CID)過程中的斷裂，明顯提高質譜檢測中聚糖的電離效率。Novotny等[51]綜合運用酰胺化反應和泛甲基化兩種衍生方法。研究表明，α2-3連接的唾液酸聚糖經過六元環(huán)內酯中間產物最終在泛甲基化條件下開環(huán)得到羧酸甲酯，而α2-6連接的唾液酸聚糖先由酰胺化反應生成酰胺基最后經過泛甲基化得到二甲基酰胺結構，兩種糖類異構體經過衍生之后在MALDI-TOF MS中質荷比相差13 Da。通過上述方法很容易鑒別出乳癌患者血清中兩種不同連接方式的唾液酸化糖肽的相對含量。

最近，Wuhrer[52]等將血漿中免疫球蛋白G(IgG)經酶解、分離、純化后得到的唾液酸糖肽與二甲胺在EDC/HOBt 的DMSO溶液中反應(Table 2b)；MALDI-TOF-MS表明，α2-3連接的唾液酸化糖肽在1號位形成六元環(huán)內酯，而α2-6的連接的唾液酸化糖肽形成二甲基酰胺結構，這種新的衍生方法可以有助于分析醫(yī)療性抗體中糖基化位點和唾液酸特異性的連接方式。

α2-3連接的唾液酸化聚糖羧基與相鄰單糖的羥基的空間位阻效應和分子內氫鍵使得酸性條件下其更趨于發(fā)生分子內酯化反應，酰胺化反應活性遠遠低于α2-6連接的唾液酸聚糖[53]?；诖耍琇i等[54]選用一種高性能磷正離子PyAOP作為酰胺縮合劑，室溫條件下，唾液酸羧基與甲胺脫水縮合，生成甲胺化衍生產物。這種衍生方法不僅解決了MALDI-TOF過程中唾液酸殘基丟失和乙酰氧基遷移等問題，同時對α2,3、α2,6連接的唾液酸聚糖均表現(xiàn)出很高的反應活性。Nishikaze等[55]用類似的方法，實現(xiàn)了甲胺鹽酸鹽與唾液酸糖肽的甲胺化，這種衍生方法一方面防止質譜檢測中唾液酸殘基的損失，減少唾液酸化糖肽和非唾液酸化糖肽之間離子化效率的差異，從而以正離子CID測定肽鏈序列與糖鏈結構，負離子CID提供聚糖糖基化位點信息；另一方面，甲胺化衍生還能夠固定唾液酸聚糖在液相色譜中的保留時間、改善其色譜峰的峰形以及ESI-MS中重現(xiàn)性和檢測靈敏度。經過甲胺化衍生，Liu等[56]使用納升級液質聯(lián)用技術NanoLC-MS以PGC為固定相從正常人血清樣品中找出293種不同的N-糖鏈和7種二天線唾液酸糖鏈的異構體。

Zhang[57]等用EDC為偶聯(lián)劑，在pH=4.5條件下，對甲苯胺作為酰胺化試劑固定唾液酸殘基；實驗證明，此衍生反應不僅可以有效地增強聚糖在MALDI-TOF質譜中的離子化效率，而且提高了唾液酸聚糖的疏水性，有利于其在C18柱中良好地分離。

唾液酸化糖蛋白樣品一般前處理方法是酶解法釋放出唾液酸化糖鏈，然而游離的聚糖末端醛基也可以與酰肼類化合物反應，以致不能產生具有穩(wěn)定專一結構的目標衍生物。VanCott等[58]在弱酸性條件下讓唾液酸化糖蛋白的羧基先與乙酰肼Ah發(fā)生衍生反應，然后再用PNGase F將其酶解成特殊位點修飾的聚糖，避免游離糖鏈干擾衍生反應，使得質譜檢測過程中離子化信號更加穩(wěn)定清晰。

Terabayashi等[59]用2-(2-吡啶)乙胺二鹽酸鹽(PAEA) 熒光試劑修飾唾液酸乳糖，運用HPLC-FD、MALDI-MS等檢測技術，唾液酸乳糖異構體(3-sialyllactose, 6-sialyllactose等)在HPLC-FD檢出限達到皮摩爾水平，一級質譜中相關分子離子和二級質譜(MS/MS)中B/Y型離子信號明顯增強。

此外， Nohta[60-61]等創(chuàng)造性地將商業(yè)化全氟烷基胺試劑十七氟十一胺(HFUA)和唾液酸低聚糖在室溫條件下快速進行酰胺化反應，實現(xiàn)唾液酸低聚糖異構體(3，-SL, 6，-SL) 等在全氟烷基為固定相的色譜柱中良好的分離，其檢測限在二級質譜中低至埃摩爾，此方法為唾液酸糖綴合物的化學衍生提供了新方向。該類衍生化方法使用到的衍生化試劑及重要反應溶劑列表見表1。

2.3唾液酸羧基的環(huán)化反應

聚唾液酸對脊椎動物神經系統(tǒng)的發(fā)育和生理功能的調控發(fā)揮關鍵的作用[62,63]。在弱酸性條件下，唾液酸羧基與相鄰唾液酸分子的羥基形成六元環(huán)內酯(圖 7)。Geyer等[64]在室溫條件下、將聚唾液酸用0.5%正磷酸處理30min，實現(xiàn)了聚唾液酸兩個相鄰分子之間六元環(huán)酯化反應：α2-8連接O-乙酰聚唾液酸的羧基與相鄰的唾液酸9

表1唾液酸羧基常用衍生試劑。其中,DMT-MM：4-(4,6-二甲氧基三嗪-2-基)-4-甲基嗎啉鹽酸鹽；EDC：1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽；HOBt：1-羥基苯并三唑；PyAOP：(3H-1,2,3-三唑并[4,5-b]吡啶-3-氧基)三-1-吡咯烷基鏻六氟磷酸鹽。

衍生試劑名稱反應溶劑或縮合劑相關文獻重氮甲烷甲醇-乙腈[41]碘甲烷二甲基亞砜[42]1-甲基-3-甲苯基三氮二甲基亞砜-乙腈[43]1-芘基二氮甲烷二甲基亞砜[44,45]甲醇DMT-MM[46]乙醇EDC/HOBt[47]氯化銨DMT-MM[41]二甲胺二甲基亞砜[50,51]甲胺PyAOP[52]對甲苯胺EDC[54,55]乙酰肼EDC[57]2-(2-吡啶)二胺DMT-MM[58]十七氟十一胺DMT-MM[59]

號位羥基成環(huán)，而α2-9連接O-乙酰聚唾液酸的羧基與8號位羥基成環(huán)。這種特異性分子間環(huán)化方式解決了質譜檢測中聚唾液酸的羧基引起的檢測問題，極大增加聚唾液酸離子碎片的質荷比。必須強調的是，酯化后的聚唾液酸溶解性相對較差，導致其在ESI質譜中很難被檢測。

圖7　聚唾液酸環(huán)化反應

3結語與展望

糖基化是闡釋蛋白質功能的重要生理過程。然而，處于現(xiàn)代分子生物學的發(fā)展初期，科學家們在分子層面對聚糖的功能解析仍處于相對滯后的階段。因此，從分子水平上開展聚糖的研究(糖基化位點的鑒別、糖鏈結構的檢測等)是亟待解決和發(fā)展的難題；唾液酸化聚糖異于其它種類的聚糖，在糖鏈分析中占有舉足輕重的地位，不斷嘗試新的衍生、富集、分離、檢測技術以求對唾液酸更深層次的了解是目前糖生物學領域科研工作者的主流話題。

參考文獻:

[1] Angata T.，Varki A. Chemical Diversity in the Sialic Acids and Related r-Keto Acids: An Evolutionary Perspective[J]. Chemical Reviews, 2002, 102(2): 439-469.

[2] Lamari F. N.，Karamanos N. K. Separation methods for sialic acids and critical evaluation of their biologic relevance[J]. Journal of Chromatography, 2002, 718(1-2): 3-19 .

[3] Nie H.，Li Y.，Sun XL. Recent advances in sialic acid-focused glycomics[J]. Journal of Proteomics, 2012, 75(11): 3098-3112.

[4] Andersen M. T,. Packer N. H., Larsen M. R., et al. Structural analysis of glycoprotein sialylation-Part I:pre-LC-MS analytical strategies[J]. RSC Advances, 2013, 3(45): 22683-22705.

[5] Takasaki S.，Suzuki K.，Kobata A., et al.Paulson J C. The sugar chains of cold-insoluble globulin. A protein related to fibronectin[J]. The Journal of Biological Chemistry, 1979, 254(17): 8548-8553.

[6] Iwasaki M.，Inoue S.，Inoue Y., et al. Novel oligosaccharide chains on polysialoglycoproteins isolated from rainbow trout eggs. A unique carbohydrate sequence with a sialidase-resistant sialyl group, DGa1NAc beta 1 leads to 4(NeuGc2 leads to 3)DGa1NAc[J]. Biochemistry, 1984, 23(2): 305-315.

[7] Gerd R.，Jasna P. K.，Hanisch F. G., et al. Identification of a new ganglioside from the starfish Asterias rubens[J]. Carbohydrate Research, 1992, 236(15): 321-326.

[8] Tsuji S., Datta A. K., Paulson J. C. Systematic nomenclature for sialyltransferases[J]. Glycobiology, 1996, 6(7): V-VII.

[9] Wada T., Anzai T., Fujimoto T., et al.Studies on gastric juice protein. VI. On pathological-physiological characteristics of visible mucus of gastric juice in disorders of the stomach[J]. Gann, 1962, 53: 275-283.

[10] Dnistrian A.M.，Schwartz M.K. Plasma lipid-bound sialic acid and carcinoembryonic antigen in cancer patients[J]. Clinical Chemistry, 1981, 27 (10): 1737-1739.

[11] Silver H.K., Karim K.A., Salinas F. A., et al. Serum sialic acid and sialyltransferase as monitors of tumor burden in malignant melanoma patients[J]. Cancer Resear, 1979, 39(12):5036-5042.

[12] Satoh H.，Ohtsuka M.，Hasegawa S.，et al. Serum sialyl lewis X-i antigen levels in non-small cell lung cancer: correlation with distant metastasis and survival[J]. Clinical Cancer Research, 1997, 3(4):495-499.

[13] Gattegno L.，Bladier D.，Cornillot P.，et al. Physiological ageing of red blood cells and changes in membrane carbohydrates[J]. Biomedicine, 1979, 30(4): 194-203.

[14] Huang Y.X.，Wu Z.J.，Mehrishi J.，et al. Human Red Blood Cell Aging: Correlative changes in surface charge and cell properties[J]. Journal of Cellular and Molecular Medicine, 2011, 15(12): 2634-2642.

[15] Hudgin R. L., Ashwell G.，Morell A. G.，et al. The isolation and properties of a rabbit liver binding protein specific for Asialoglycoproteins[J]. Biological Chemistry, 1974, 249(17):5536-5543.

[16] Langer G.A.，F(xiàn)rank J.S.，Seraydarian K.，et al. Sialic acid: effect of removal on calcium exchangeability of cultured heart cells[J]. Science, 1976, 193(4257): 1013-1015.

[17] Wang B.，Petocz P.，Brand-Miller J.，et al. Dietary sialic acid supplementation improves learning and memory in piglets[J]. The American Journal of Clinical Nutrition，2007，85(2): 561-569.

[18] Blix F. G., Gottschalk A., Klenk E. Proposed nomenclature in the field of neuraminic and sialic acids[J]. Nature, 1957, 179(4569):1088.

[19] Colman P.M., Varghese J. N., Laver W.G., et al. Structure of the catalytic and antigenic sites in influenza virus neuraminidase[J]. Nature, 1983, 303(5912): 41-44.

[20]Varki A. Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins[J]. Nature, 2007, 446(7139): 1023-9.

[21] Kozak R.P.，Royle L.， Gardner R.A. ，et al. Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition[J]. Analytical Biochemistry, 2014, 453(3): 29-37.

[22] Timothy G.K.，Krueger J.，Gerardy-Schahn R.，et al. A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases[J]. Analytical Biochemistry, 2012, 427(2): 107-115.

[23] Wuhrer M., Deelder A.M., Hokke C.H.，et al. Glycoproteomics based on tandem mass spectrometry of glycopeptides. Analytical Chemistry , 2007, 849(1-2):115-128.

[24] Cristina D.M.,Wallace C.E., Geringer S.A. Synthesis and Elimination of C-3-Labeled Thiosialosides[J]. Organic Letters, 2014, 16(10): 2676-2679.

[25] Hurum D.C., Rohrer J.S. Five-minute glycoprotein sialic acid determination by high-performance anion exchange chromatography with pulsed amperometric detection[J]. Analytical Biochemistry, 2011, 419(1): 67-69.

[26] Zhang Z., Chess E.K., Szabo C.M., et al. Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection[J]. Analytical Chemistry, 2012, 84(9): 4104-10.

[27] Klein A.，Diaz S., Manzi A.E., et al. New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones[J]. Glycobiology, 1997, 7(3): 421-432.

[28] Liu X.，Johnson S.，Li J.，et al. O-acetylation of sialic acids in N-glycans of Atlantic salmon (Salmo salar) serum is altered by handling stress[J]. Proteomics, 2008, 8(14): 2849-2857.

[29] Wheeler S.F.，Domann P.，Harvey D.J. Derivatization of sialic acids for stabilization in matrix-assisted laser desorption/ionization mass spectrometry and concomitant differentiation of alpha(2→3)- and alpha(2→6)-isomers. Rapid Communications in Mass Spectrometry, 2009, 23(2):303-312.

[30] Hakomori S. A Rapid Permethylation of Glycolipid, and Polysaccharide Catalyzed by Methylsnlfinyl Carbanion in Dimethyl Sulfoxide[J]. The Journal of Biochemistry, 1964, 55(2):205-208.

[31] Kerek F. A simple and rapid method for the perm- ethylation of carbohydrates[J]. Carbohydrate Research, 1984, 131(2): 209- 217.

[32] Kang P.，Mechref Y.，Novotny M.V., et al. Solid-phase permethylation of glycans for mass spectrometric analysis[J]. Rapid Communications in Mass Spectrometry, 2005, 19(23):3421-3428.

[33] Kang P.，Mechref Y.，Novotny M.V. High-throughput solid-phase permethylation of glycans prior to mass spectrometry. Rapid Commun Mass Spectrom[J].Analytical Chemistry, 2008, 22(5):721-734.

[34] Zhou H., Warren P.G., Lee R.S., et al. Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS[J]. Analytical Chemistry, 2014, 86(13): 6277-6284.

[35] Liu X.，Afonso L. Is permethylation strategy always applicable to protein N-glycosylation study?: A case study on the O-acetylation of sialic acid in fish serum glycans[J]. Methods in Molecular Biology , 2010, 18 (600): 259-268.

[36] Daniel P.F. Separation of benzoylated oligosaccharides by reversed-phase high-pressure liquid chromatography: application to high-mannose type oligosaccharides[J]. Methods in Enzymology, 1987, 138: 94-116.

[37] Chen P.，Wiesler D.，Novotny M.V., et al. Mass Spectrometric Analysis of benoylated Sialooligosaccharides and Differentiation of Terminalα2-3 andα2-6 Sialogalactosylated Linkages at Subpicomole Levels[J].Analytical Chemistry, 1999, 71 (21): 4969-4973.

[38] Liu X.，Sawyer M.B.，Patrick P.，et al. “One-Pot” Methylation in Glycomics Application:Esterification of Sialic Acids and Permanent Charge Construction[J]. Analytical Chemistry, 2007, 79(10): 3894-3900.

[39] Palmisano G.，Larsen M.R., Andersen M.T., et al. Structural analysis of glycoprotein sialylation - part II:LC-MS based detection[J]. RSC Advances, 2013, 3(45): 22706-22726.

[40] Zaia J. Mass spectrometry of oligosaccharides[J]. Mass spectrometry reviews, 2004, 23(3): 161-227.

[41] MacDonald D.L.，Patt L.M.，Hakomori S. Notes on improved procedures for the chemical modification and degradation of glycosphingolipids[J]. Journal of Lipid Research, 1980, 21(5): 642-647.

[42] Powell A.K.， Harvey D.J. Stabilization of Sialic Acids in N-linked Oligosaccharides and Gangliosides for Analysis by Positive Ion Matrix-assisted Laser Desorptiod Ionization Mass Spectrometry[J]. Rapid Communications in Mass Spectrometry, 1996, 10(9): 1027-1032.

[43] Miura Y.，Shinohara Y.，Nishimura S.I.，et al. Rapid and Simple Solid-Phase Esterification of Sialic Acid Residues for Quantitative Glycomics by Mass Spectrometry[J]. Chemistry, 2007, 13(17):4797-4804.

[44] Amano J.，Nishikaze T., Mizuno M.，et al. Derivatization with 1-Pyrenyldiazomethane Enhances Ionization of Glycopeptides but Not Peptides in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry[J]. Analytical Chemistry, 2010, 82(20): 8738-8743.

[45] Jinmei H., Nishikaze T. , Amano J.，et al. Negative-ion MALDI-MS2 for discrimination of α2,3- and α2,6-sialylation on glycopeptides labeled with a pyrene derivative[J]. Journal of Chromatography B, 2011, 879(17-18): 1419-1428.

[46] Tousi F.;Jonathan B.; Hincapie M.，et al. Differential Chemical Derivatization Integrated with Chromatographic Separation for Analysis of Isomeric Sialylated Glycans: A Nano-Hydrophilic Interaction Liquid Chromatography-MS Platform[J]. Analytical Chemistry, 2013, 85(17): 8421-8428.

[47] Reiding K.R.，Deelder A.M., Manfred W.，et al. High-Throughput Profiling of Protein N?Glycosylation by MALDITOF-MS Employing Linkage-Specific Sialic Acid Esterification[J]. Analytical Chemistry , 2014, 86(12): 5784-5793.

[48] Kamerling J.P., Schauer R., Vliegenthart J.F. Migration of 0-acetyl groups in N, o-acetylneuraminic acids[J]. European Journal of Biochemistry, 1987, 162(3): 601-607.

[49] Varki A, Diaz S.. The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of O-acetyl groups[J]. Analytical Biochemistry, 1984, 137(1): 236-247.

[50] Sekiya S.，Yoshinao W.，Tanaka K.，et al. Derivatization for Stabilizing Sialic Acids in MALDI-MS[J]. Analytical Chemistry , 2005, 77(15): 4962-4968.

[51] Alley WR Jr, Novotny M.V. Glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins[J]. Journal of Proteome Research, 2010, 9(6): 3062-3072.

[52] de Haan N., Reusch D., Wuhrer M.，et al. Linkage-Specific Sialic Acid Derivatization for MALDI-TOF-MS Profiling of IgG Glycopeptides[J]. Analytical Chemistry, 2015, 87(16): 8284-91.

[53] Toyoda M，Matsuno YK, Narimatsu H, et al. Quantitative Derivatization of Sialic Acids for the Detection of Sialoglycans by MALDI MS[J]. Analytical Chemistry, 2008, 80(13): 5211-5218.

[54] Liu X.，Chen W., Li J，et al. Methylamidation for Sialoglycomics by MALDI-MS:A Facile Derivatization Strategy for Both α2,3- and α2,6-Linked Sialic Acids[J]. Analytical Chemistry, 2010, 82(19): 8300-8306.

[55] Nishikaze T.，Kawabata S.，Tanaka K.，et al. In-Depth Structural Characterization of N-LinkedGlycopeptides Using Complete Derivatization for Carboxyl Groups Followed by Positive- and Negative-Ion Tandem Mass Spectrometry[J]. Analytical Chemistry, 2014, 86(11): 5360-5369.

[56] Zhang Q，Liu BF，Liu X，et al. Methylamidation for isomeric profiling of sialylated glycans by nanoLC-MS[J]. Analytical Chemistry, 2014, 86(15): 7913-7919.

[57] Shah P.，Yarema K.J.，Zhang H., et al. Mass spectrometric analysis of sialylated glycans with use of solid-phase labeling of sialic acids[J]. Analytical Chemistry, 2013, 85(7):3606-3613.

[58] Gil G.C., Velander W.H., Van Cott K.E., et al. High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry[J]. Analytical Chemistry, 2010, 82(15): 6613-6620.

[59] Endo S.，Maeda T. , Terabayashi T.，et al. Fluorescent labeling of a carboxyl group of sialic acid for MALDI-MS analysis of sialyloligosaccharides and ganglioside[J]. Biochemical and Biophysical Research Communications, 2009, 378(4): 890-894.

[60] Hayama T.，Todoroki K. , Nohta H.，et al. Fluorous derivatization combined with liquid chromatography/tandem mass spectrometry: a method for the selective and sensitive determination of sialic acids in biological samples[J]. Rapid Communication in Mass Spectrometry, 2010, 24(19): 2868-2874.

[61] Sakaguchi Y.，Todoroki K. , Nohta H.，et al. Liquid chromatography/tandem mass spectrometry with fluorous derivatization method for selective analysis of sialyl oligosaccharides[J]. Rapid Communication in Mass Spectrometry, 2014, 28(23): 2481-2489.

[62] Schnaar R.L.，Gerardy-Schahn R., Hildebrandt H. Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration[J]. Physiological Reviews, 2014, 94(2):461-518.

[63] Bushman J.，Mishra B., Loers G.，et al. Tegaserod mimics the neurostimulatory glycan polysialic acid and promotes nervous system repair[J]. Neuropharmacology,2014,79: 456-466.

[64] Galuska S.P.，Bleckmann C., Geyer H., et al. Mass spectrometric fragmentation analysis of oligosialic and polysialic acids[J]. Analytical Chemistry, 2010,82(5): 2059-2066.

[65] Kobayashi K.，Imanari T，Tanabe S, et al.Fluorometric Determination of N-Acetyl and N-Glycolyl Neuraminic Acids by High Performance Liquid Chromatography as Their 4’-Hydrazino-2-stilbazole Derivatives[J]. Analytical Sciences, 1985, 1(1):81-84.

[66] Hara S, Ohkura Y, Nakamura M, et al. Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection[J]. Journal of Chromatography, 1986,377: 111-119.

[67] Shamberger R J. Serum sialic acid in normals and in cancer patients, 1984, 22(10):647-651.

[68] Hara S, Takemori Y, Ohkura Y, et al. Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids[J]. Anal Biochem, 1987, 164(1):138-145.

[69] Stehling P., Fitzner R.，Reutter W., et al. Rapid analysis of O-acetylated neuraminic acids by matrix assisted laser desorption/ionization time-of-flight mass spectrometry[J]. Glycoconjugate, 1998, 15(4): 339-344.

[70] Galuska S.P.，Kontou M., Geyer H., et al. Quantification of nucleotide-activated sialic acids by a combination of reduction and fluorescent labeling[J]. Analytical Chemistry, 2010, 82(11): 4591-4598.

[71] Li H.W.，F(xiàn)an X.D. Quantitative analysis of sialic acids in Chinese conventional foods by HPLC-FLD[J]. Open Journal of Preventive Medicine, 2014, 4(2): 57-63.

[72] Anumula K.R. Rapid quantitative determination of sialic acids in glycoproteins by high-performance liquid chromatography with a sensitive fluorescence detection[J]. Analytical Biochemistry, 1995, 230(1): 24-30.

[73] Wang T., Nam P.K., Ma Y., et al. Compositional Monosaccharide Analysis of Transgenic Corn Glycoproteins by HPLC with Fluorescence Detection and LC-MS with Sonic Spray Ionization[J]. Journal of Chromatographic Science, 2007, 45(4): 200-206.

[74] 王蕾，李曉東. 高效液相色譜法檢測嬰兒配方奶粉中唾液酸[J]. 中國乳品工業(yè)，2013, 41(5): 52-55.

[75] 錢振杰，張恒，陳敬. 嬰幼兒配方乳粉中唾液酸含量的測定[J]. 西北大學學報( 自然科學版)，2014, 44(4): 584-587.

[76] Sun S.M.，Li G.S., Yu G.L., et al. Fluorescence Labeling Strategies for Different Structural Carbohydrates[J]. Analytical Chemistry,2012,28(1): 18-27.

[77] Szabo Z., Bones J., Karger B.L., et al. Sialic acid speciation using capillary electrophoresis: optimization of analyte derivatization and separation[J]. Analytical Chemistry, 2012, 84(18):7638-7642.

[78] Galeotti F., Galeazzi T., Volpi N., et al. On-line high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry profiling of human milk oligosaccharides derivatized with 2-aminoacridone[J].Analytical Chemistry, 2012, 430(1):97-104.

[79] Wang D.，Zhou X.，Sun X.L., et al. Quantification of free sialic acid in human plasma through a robust quinoxalinone derivatization and LC-MS/MS using isotope-labeled standard calibration[J]. Journal Of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2014, 944: 75-81.

Analysis of Sialylated Glycans by MALDI Mass Spectrometry Based on Chemical Derivatization

DU Ying-xin, LIN Ya-wei

(Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070, China)

Abstract:Sialic acid, mostly at the end of glycans, is not only widely involved in many important biological processes such as cell signaling, cell adhesion, and specific recognition, but also concerns with critical diseases like cancer. Therefore, it is of vital importance to analyze sialylated glycans from biological samples to understand their functions. Currently, Matrix-assisted laser desorption/ionization (MALDI) mass pectrometry, with its high sensitivity and ability to directly measure molecular weights, has been a useful tool for characterization of sialylated glycans. However, there still remain many difficulties while detecting sialylated glycans in biological samples. As a result, derivatizations are often needed prior to MS analysis to introduce ionizable or hydrophobic groups, so as to increase the chromatographic separation of glycans, enhance ionization efficiency, and stabilize the sialic acid residues of sialylated glycans. Mainly dealing with sialylated glycan samples dissociated from glycoconjugates, this paper introduced and summarized the latest derivatization strategies and related MALDI-MS detection method.

Key words:sialylated glycans; chemical derivatization; Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

收稿日期：2016-01-05

基金項目：國家自然科學基金資助項目(青年)(21405117)

作者簡介：林亞維(1979-)，女，江西景德鎮(zhèn)人，副教授，博士，研究方向為分析化學質譜分析。

中圖分類號：O657.6

文獻標識碼：A

文章編號：1674-344X(2016)02-0004-08