2010全國質譜大會大會報告（四）

中國科學院大連化學物理研究所 許國旺研究員

　　來自中國科學院大連化學物理研究所的許國旺研究員做了題為“液相色譜-質譜用于代謝標志物的發現和鑒定”的報告。許老師提出，現在是一個組學的時代，代謝組學作為最主要的組學之一，目前在疾病研究、藥物研發及職務和微生物等領域均得到重視。

　　代謝組學是研究小分子（相對分子質量小于1, 000）一個十分有用的工具，它以組織、體液或細胞為研究對象。一般來說，代謝組學的研究包括樣品采集、預處理、代謝組數據采集、多變量數據分析、標志物發現和識別及最終的生物解釋幾方面。由于缺乏數據庫樣本及標樣，代謝標志物的識別是研究的一個瓶頸。

　　許老師在報告中，講述了基于色譜-質譜聯用技術的集成識別策略，包括多變量數據分析、精確分子量測定、質譜碎片裂解規律、色譜保留規律、餾分微制備、親和色譜、酶解等。并列舉了藥物作用機理和疾病標志物的代謝組學研究作為實例闡述了這一集成策略。

Wisconsin-Madison（威斯康星-麥迪遜）大學 Li,Lingjun(李靈軍)教授

　　來自Wisconsin-Madison（威斯康星-麥迪遜）大學的Li,Lingjun(李靈軍)教授，做了題為“Probing Neuronal Communication via Novel Mass Spectral Strategies”（用新型質譜技術探索神經通訊）的報告。她首先介紹了神經肽在細胞間通訊中至關重要的作用，這些聚合多肽在化學信使調節，和在神經電路中測定它們的功能中具有重要作用。甲殼動物具有較簡單和已被充分研究的神經系統，可作為很好的模型系統來發展分析方法，并觀測神經肽豐富的全部細節是如何來精細調節神經電路，并在細胞和神經網絡水平產生多種輸出。使用高靈敏度的基于質譜的多肽圖和從頭測序方法，發現了大量的新型多肽，并發現即使一個相對簡單的神經元網絡，同樣包含了未被預期的豐富多樣的神經肽。另外，質譜成像技術和活體微透析取樣工具，可史無前例地對神經肽分布和分泌的細節進行追蹤。對于完成對生物活性的神經肽的功能發現的目標，發展了建立在同位素甲醛標記和多同位素標記（N,N-二甲基亮氨酸）的新型定量技術基礎上的方法，可對不同生理條件下的神經肽組進行區分顯示。李教授介紹了神經肽在攝食行為和環境壓力下的調節。總而言之，這種多肽組學結合生理學的研究，有助于闡釋在調節神經網絡可塑性方面，神經肽扮演的功能。以下是英文摘要：

　　Neuropeptides make up the largest and the most complex signaling molecules used in intercellular communication. Because of critical roles that these polypeptides play in the regulation of chemical messengers and determine their functions in the neural circuitry. The simpler and well-characterized crustacean nervous system provides an excellent model system to facilitate analytical method development and to investigate how a rich repertoire of neuropeptides can fine tune a well-defined neural circuit that produces multiple outputs at the cellular and network levels. Using a highly sensitive mass spectrometry-based peptide profiling and de novo sequencing strategy, a large number of novel peptides have been discovered, revealing that even a relatively simple neural network contains an unexpectedly-rich diversity of neuropeptides. Furthermore, both mass spectrometric imaging techniques and in vivo microdialysis sampling tools have been implemented to follow neuropeptide distribution and secretion in unprecedented details. Towards the goal of functional discovery of bioactive neuropeptides, novel quantitative schemes based on isotopic formaldehyde labeling and multiplexed isobaric labeling based on N.N-dimethylated leucine have been developed to produce differential display of neuropeptidomes under different physiological. Example of neuropeptide regulation of feeding behavior and environmental stress will be described in this presentation. Collectively, these combined peptidomic and physiological studies will help to elucidate the function roles that neuropeptides play in regulating neural network plasticity.

加拿大Alberta大學 Le,Chris（樂曉春）教授

　　來自加拿大Alberta大學的Le,Chris（樂曉春）教授，做了題為“Mass Spectromety and Affinty Chromatography Techniques for Studying Arsenic-binding Proteins in Human Cells”（在研究人類細胞中砷結合蛋白中的質譜和親和色譜技術）的報告。砷中毒對于疾病、環境等的影響眾所周知，但砷中毒的機理還不清楚，可能是三價砷結合了蛋白的硫基團，因此改變了蛋白的構象抑制了蛋白功能。為研究與蛋白作用的砷，課題組開發了一種親和選擇技術并與串聯質譜聯用，從大量的細胞蛋白中選擇和鑒定特定的與砷結合的蛋白。受控實驗使用包含游離半胱氨酸或非活性半胱氨酸的蛋白，顯示砷親和柱特異捕獲游離的半胱氨酸。該方法被應用到牛膽綠素還原酶B、A549人肺癌細胞亞細胞器組分等研究中。使用親和色譜-串聯質譜方法鑒定大量的砷結合蛋白，因為其重要的生物功能引起科學家的關注。實驗證實砷可以結合細胞提取物的蛋白質，而砷如何影響生物系統中的蛋白功能，需要繼續研究活體細胞中砷和蛋白的相互作用才能確認。以下是英文摘要：

　　 Arsenic is one of the most important environmental agents in causing chronic human disease. Elevated levels of arsenic drinking water may affect >100 million people around the world .A wide variety of adverse health effects, most seriously, cancers of bladder, lung, urinary tract, and skin, have been attributed to chronic exposure to arsenic . However, the biochemical mechanisms responsible for these effects caused by arsenic remain unclear ,but may be mediated by the binding of trivalent arsenicals to thiol groups in proteins, thereby changing the conformation of these proteins and inhibiting their functions. If some of the affected proteins are responsible for cellular repair of DNA damage, for example, the inhibition of these proteins could lead to carcinogenesis.

　　 To study interaction of arsenic with proteins, we have developed an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or inactive cysteine showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind tu arsenic .The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurty in the commercial preparation of carbonic anhydrase II ,demonstrate the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the analysis of subcellular fraction ,and 24 proteins in the membrane/ organelle fraction that could bind to arsenic .This added substantially to the current list of only a few known arsenic-binding proteins.

　　 A mumber of arsenic-binding proteins identified using the affinity chromatography tandem mass spectrometry approach were of particular interest because of their important biological functions .For example ,DNA-dependent protein kinase, ATP-dependent helicase II (Ku 70),and topoismerase 2 alpha , are involved in DNA repair and maintaining genome stability .several other proteins modulate the redox status of cells , e.g . peroxiredoxin-1 and thioredoxin ,and apoptosis ,e.g.,lamin A and heat shock cognate protein .this work shows that arsenic can bind to these proteins in cells extracts。How arsenic affects the function of these proteins in biological systems will have to be confirmed by studying arsenic interaction with proteins in living cells.

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臺灣Soochow大學化學系 Ming-Ren Fuh(傅明仁)教授

　　來自臺灣Soochow大學化學系的Ming-Ren Fuh(傅明仁)教授，做了題為“Microfluidic chip-based nano-liquid chromatography-tandem mass spectrometry for bioanalysis”（基于微流芯片的納升液相色譜-串聯質譜用于生物分析）的報告。在報告中，傅教授先介紹了基于微流控芯片的商品化產品，這些納升級液相系統已經顯著提高了納升級液相分析的穩定性。基于芯片的納升級液相-串聯質譜已被證明可提高液相分離的分辨率，并增強質譜檢測的靈敏度。傅教授介紹了課題組應用基于芯片的納升級液相-串聯質譜分析用于藥物代謝物、生物標志物和臨床診斷的生物體液的神經遞質的結果，給出了包括重現性、檢出限、線性和精密度等分析性能；還討論了分析人尿的基質效應和離子抑制效應，展示了基于芯片的微流控納升級液相-串聯質譜在臨床診斷中的應用能力。以下是英文摘要：

　　Recent commercially available microfluidic chip-based nano-liquid chromatography system has greatly improved the robustness of nano-LC analysis. Chip-based nano-LC-tandem mass spectrometry(nano-LC-tandem MS) has been proven to enhance the resolution of LC separation and the sensitivity of MS detection. In this paper, we will discuss the utilization of chip-based nano-LC-tandem MS for the analyses of drug metabolite (7-aminoflunitrazepam),biomarker(8-isoprostane) and neurotransmitters in biological fluids for clinical diagnosis. The analytical performance (reproducibility, detection limit, linearity and precision) for various analytical compounds will be presented. In addition, matrix effect and ion suppression effect of human urine will be discussed. The applicability of microfluidic chip-based nano-LC-tandem MS for clinical diagnosis will be demonstrated.

中國科學院北京基因組研究所 劉斯奇研究員

　　來自中國科學院北京基因組研究所的劉斯奇研究員做了題為“Protein biomarker in clinical assay using mass spectrometry: Possibility and difficulities（質譜在研究蛋白質生物標記物臨床鑒定中的應用：可能性和難點）”劉老師總結了近段時間閱讀的文獻，與大家分享。蛋白質組帶有基因組和代謝組所不具有的特性，如表達的動態性和空間性、后反應修飾等。

　　研究蛋白質生物標記物的優點在于，樣品收集相對簡單，來自于體液；大多數蛋白生物標記物均是翻譯后修飾；蛋白直接與病理性功能相關。因此，對蛋白質組的研究是熱點。研究成果有ELISA、蛋白活性和蛋白芯片等。其中，劉老師重點講了ELISA。

　　目前研究的方法可以鑒定1000多種蛋白質，但被FDA批準的鑒定方法中沒有一個是用蛋白質組學的方法。因此，在發現新的蛋白待測物方面，蛋白質組學的發展并沒有為生物標記物的發現做出貢獻。劉老師指出，在臨床化學中，亟需一種蛋白診斷的方法。從定性到定量、從發現到目標化將成為今后蛋白質組學的兩大研究趨勢。

臺灣國立清華大學生物工程和環境科學系 Yuh-chang Sun(孫毓章)教授

　　來自臺灣國立清華大學生物工程和環境科學系的Yuh-chang Sun(孫毓章)教授，做了題為“Development of photocatalyst-based ICP-MS coupling techniques for determination of trace elements and their species”（研發基于光催化的ICP-MS聯用技術，測定痕量元素及其形態）的報告。課題組研究了對超痕量（亞ug/L）金屬元素及其形態分析的方法，使用前處理手段、色譜分離和ICP-MS聯用，可克服生物或環境分析中的背景基質干擾并提高靈敏度。他們開發了幾種聯用系統，使用了光氧化還原納米二氧化鈦的接口裝置，把包含無機和有機金屬的各種形態均轉化為利于ICP-MS分析的氣相產物，證明該方法對于分析環境和生物背景下的痕量元素及其形態是有效的。以下是英文摘要：

　　It is well known that the speciation, or chemical form, of metal governs its fate, toxicity, mobility, and bioavailability in environmental and biological systems. To assess these chemical properties and to accurately gauge their impact on human health and the environment, metal need to be characterized at the atomic level. To attain new information about environmental and biological effects of trace elements, new methodologies or modify conventional analytical methods is deemed as vital factor for the progress of bio -and environmental -studies. In view of the limitation on the analytical capability of single instrumental technique, analytical chemists can seldom rely on a single instrumental technique to analyzed a sample with complicated matrix and analyte species with a variety of phsico-chemical form. It is thus necessary to develop a technique which can fulfill ultratrace analyses of metal species down to the sub-μg/L concentration range in complicated samples.

　　Accordingly, the most important features of an analytical tool suitable for meeting the requirement of modern bio-analytical works are shorter temporal resolution, good selectivity and high sensitivity. For ultratrace elements measurements, ICP coupled with Mass Spectrometry (ICP-MS) has been considered as first priority option. Although the analytical sensitivity has been significantly improved by the technical advances in ICP-MS, instrumental limitations, such as difficulties in differentiating elemental species and removing matrix interferences caused by the concomitant salt, still remain in advanced analytical technologies. To satisfy the analytical requirements, the potency of hyphenating analytical separation techniques to mass spectrometers has been recognized. Basically, according to Histchfeld, the advantages brought about by coupling techniques are increasing the differentiating and separating power of analytical methods and synergism between methods. However,the design of the analytical system is difficult, owing to the complex composition of the real-world sample, the diversity of physicochemical forms of the element, their lability and low concentrations. For overcoming the abovementioned problems, attempts to couple ICP-MS with various types of chromatography for separation, as well as on-line sample pretreatment techniques for signal enhancement and matrix removal have been made. To expand the analytical capability, in this study, we developed several hyphenated systems by integrating the alternative photo-redox characteristic of nano-TiO2 into the interfacing device to convert both inorganic and organic metal-containing species to gaseous products that are favor for ICP-MS determination. Based on our experimental results, this presentation will describe the studied hyphenated methods which have been proven feasible for the analyses of trace elements and their chemical species in environmental and biological systems.

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中國科學院上海有機化學研究所 郭寅龍研究員

　　來自中國科學院上海有機化學研究所的郭寅龍研究員做了題為“基于MALDI-FTMS 的酶活檢測方法研究”的報告。郭老師介紹，MALDI離子化方式具有以下優勢：MALDI-MS可耐高濃度鹽、緩沖液和其他非揮發性成分，具有較大的樣品處理能力和樣品分析速度。傅立葉變化質譜檢測器又具有以下優勢：分辨率高、質量范圍寬、準確度高、靈敏度不受分辨率影響；不需要外部設備，用軟件執行就可以實現多級質譜串聯；與軟電離方式(ESI和MSLDI)相容性好。

　　郭老師介紹了，實驗中建立的簡單快速的牛奶中β-內酰胺酶的MALDI-MS檢測方法，即通過檢測β-內酰胺酶催化的酶反應來反映β-內酰胺酶的活性，采用高速離心的方法可以減少牛奶中干擾小分子檢出的物質。傅立葉變換質譜的高分辨和高質量準確性使其可以排除復雜的樣品基質干擾，該方法可以擴展用于更為復雜的體系。質譜儀器的高選擇性，可以在檢測β-內酰胺酶的同時，對牛奶樣本中存在的其他殘留進行檢測。

美國哥倫比亞Missouri醫學院 Gu, Zezong(顧澤宗)教授

　　來自美國哥倫比亞Missouri醫學院的Gu, Zezong(顧澤宗)教授，做了題為“Quantitative Profiling of S-Nitrosylated Proteins in Parkinson’s Disease Paradigms with the Effects of Botanical Phenolics”（在植物酚效應下定量研究帕金森病病例中的S-亞硝基化蛋白）的報告。在報告中，顧教授先介紹了跟衰老相關的神經疾病如帕金森病（PD）的起因，是由自由基活性的氮氧化物（RNS/ROS）引起的，會導致神經細胞死亡，和發病機制相關。自由基氮氧化物（NO）是一種廣泛調節從發展到疾病的細胞功能的信號分子。課題組研究了其機理，并開發了基于凝膠的蛋白質組學方法，命名為NitroDIGE，在全局范圍去定量研究蛋白S-nitrosylation（S-亞硝基化）。應用該方法，課題組鑒定了以帕金森病的體內和體外模型的S-亞硝基化蛋白的一個子集，并測定了在細胞帕金森病模型中是否S-亞硝基化蛋白會被不同的植物酚類物質（如來源于綠茶的兒茶素、來源于胡黃連的夾竹桃麻素等）的調控。NitraoDIGE方法顯示用植物化合物治療后，能夠減少過量的S-亞硝基化蛋白，表明植物酚類物質能夠有效減輕硝化壓力和帕金森病相關的困擾。以下是英文摘要：

　　A convergent feature for most aging-related neurological diseases, such as Parkinson’s Disease (PD), is excessive generation of free radicals-reactive nitrogen and oxygen species (RNS/ROS), which can contribute to neuronal cell death and link to the disease pathogenesis. Free radical nitric oxide (NO) is a signaling molecule involving in the regulation of a wide range of cellular functions from development to disease. Emerging evidence suggests that nitrosative stress due to NO over-production induces post-translation modifications of protein cysteine and modulates protein enzymatic activity in cells. S-Nitrosylation, the covalent adduction of NO to specific protein cysteine thiol, is considered as a predominant, redox-based prototypical mechanism for cell signaling .Previously, endogenous protein S-nitrosylation was detected by the biotin switch assay. Taking the advantages of both biotin switch assay and differential in-gel electrophoresis (DIGE),we developed a gel-based proteomics method, named as NitroDIGE, to globally and quantitatively investigate protein S-nitrosylation. Using this method we identified a subset of S-nitrosylated proteins from both in vitro and in vivo models of Parkinsonism including pesticide rotenone-induced PD-models could be modulated by different botanical phenolic compounds, including epigallocatechin galllate(EGCC) from green tea, and apocynin from Picrorhiza kurrooa, a herbal plant grown in the Himalayan. The NitroDIGE results demonstrated that the treatment of botanical compounds could reduce excessive S-nitrosylated proteins in SH-SY5Y cells exposed to rotenone,indicating that these botanical phenolics could serve as effective scavengers to attenuate nitrosative stress and PD-relevant insults.

北京大學 劉虎威教授

　　來自北京大學的劉虎威教授做了題為“在線正反相二維液相色譜四級桿飛行時間質譜聯用方法及其在大鼠腹膜表層脂質輪廓分析中的應用”的報告。報告中，劉教授介紹了有關動物體內磷脂的分析。與磷脂有關的疾病有動脈粥樣硬化、糖尿病、肥胖證、帕金森綜合癥等。磷脂的分類中，極性的結構適合用正向色譜分離，長鏈的脂肪酸適用于反向色譜分離，只用一種色譜進行分離，達不到很好的效果。如果將正向與反向聯用，可以提高分離效率。但是，正向色譜的流動相是有機相，而反向色譜的流動相是水相，從第一維向第二維切換的過程中容易造成堵塞，因此，改進接口技術是關鍵。

　　劉教授利用改進的接口技術并將將液相色譜四級桿飛行時間質譜聯用，對于大鼠腹膜表層脂質進行分析，結果顯示，檢測到1萬多個化合物，而之前用毛細管電泳與質譜聯用的時，只檢測到96個化合物，劉教授提出，復雜的數據處理仍是需要解決的問題。

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輝瑞-惠氏（Pfizer/Wyeth） Feng, Xidong（馮喜東）教授

　　來自輝瑞-惠氏（Pfizer/Wyeth）的Feng, Xidong（馮喜東）教授做了題為“Structure Elucidation of Natural Product Cyclic Peptides with Labile Side-Chain Modifications Using CID, IRMPD and ECD FTMS/MS（基于CID, IRMPD 和ECD FTMS/MS的側鏈改性研究天然產物環肽的結構鑒定）”的報告。

　　馮教授在報告中介紹了，通過Multi-CHEF、SORI-CID、iRMPO和ECO實驗以及基因交替的方法，使用FTMS/MS，用不穩定的側鏈改性的方法闡明未知天然產物環肽的結構。結果表明，具有ppm檢測精度的FTMS適合于研究不常見的氨基酸殘基的序列。

加拿大Alberta大學實驗室醫學和病理學系分析和環境毒理室 Li,Xing-Fang教授

　　來自加拿大Alberta大學實驗室醫學和病理學系分析和環境毒理室的Li,Xing-Fang教授，做了題為“Mass Spectrometry Techniques for Discovery of Carcinogens in Drinking Water and Studies of Health Effects”（用質譜技術發現飲用水中的致癌原和對健康影響的研究）的報告。在報告中，李教授首先介紹了飲用水污染消毒中現存的一些問題。飲用水的微生物污染仍是全球范圍內水引起的疾病的重要原因，而飲用水消毒中會引入消毒副產物（DBPs），是消毒劑（如氯和氯胺）和水中的天然有機物（如NOM）反應產生的。為了控制引用水質，在很多國家，如三鹵代甲烷（THMs）和鹵代乙酸（HAAs）的消毒副產物已被替代。流行病學的研究表明，飲用氯代的水會增加患膀胱癌的風險。然而，從過去30年積累的證據表明，已被調節的DBPs不是膀胱癌風險增加的原因，或在生殖研究中觀察到其反作用。還有其它不明物毒性更大，會在更低水平上生成DBPs。為了應對飲用水安全在分析和生物分析方面的挑戰，課題組發展了一系列液相色譜-串聯質譜技術用于分析飲用水中超痕量的致病原。課題組發現了一些以前未知的DBPs，可能是膀胱癌的致病原，比如飲用水中的亞硝胺類、鹵代苯醌、氯代phanazines。該報告舉例了用串聯質譜法對DBP-DNA結合的研究，可作為基因毒性測試的工具，有助于在立法中考慮DBPs的優先級。以下是英文摘要：

　　Microbial contamination of drinking water is still the major cause of water-borne disease affecting millions of people around the world. Disinfection of drinking water is the most effective public health measure to disinfect pathogens to eradicate water-borne diseases. However, disinfection of water unintentionally results in formation of disinfection byproducts (DBPs) from the reactions between disinfectants (e.g. chlorine and chloramines) and natural organic matters (NOM) in water. In order to control drinking water quality, surrogate DBPs such as several trihalomethanes (THMs) and haloacetic acids (HAAS) are currently regulated in the most countries. Epidemiological studies have shown potential association of drinking chlorinated water and increased risk of developing bladder cancer. However, accumulating evidence from past 30 years suggests that the regulated DBPs are not the cause of the increased bladder cancer risk or adverse reproductive effects that have been observed in population studies. Other unidentified, yet more toxic, DBPs may be produced at much lower levels. To address the analytical and bioanalytical challenges in drinking water safety, we have developed a set of liquid chromatography and tandem mass spectrometry techniques for ultra sensitive detection of carcinogens in water. We have discovered some previously unknown DBPs that are likely bladder carcinogens, such as nitrosamines, haloquinones, and chloro-phanazines in drinking water. In the present study, we report tandem mass spectrometry study of DBP-DNA binding as a tool for genotoxicity testing to assist prioritization of DBPs for regulatory consideration.

美國普渡大學癌癥研究中心 Tao,Andy(陶緯國)教授

　　來自美國普渡大學癌癥研究中心的Tao,Andy(陶緯國)教授，做了題為“In-Depth Phosphoproteome Analyses Using PolyMAC”（用PolyMAC深入分析磷酸化蛋白質組）的報告。在報告中，陶教授介紹了課題組發展的一種磷酸化肽的富集方法，稱為PolyMAC（基于聚合物的金屬離子親和富集）。該試劑的核心部分是水溶性并容易從聚合物中獲得的。課題組對PolyMAC和TiO2方法在專一性、重現性和靈敏度方面進行了比較；并比較了用幾種金屬離子功能化的PolyMAC試劑。富集后的磷酸化肽用納升級液相色譜-串聯質譜聯用（nLC-MS/MS）方法分析。液相為Eksigent公司的ultra 2D LC系統，質譜用LTQ Orbitrap velos，數據用Proteome Discovery軟件的SEQUEST方法檢索。結果顯示，僅使用50 ug的溶胞物，一次實驗，PolyMAC富集后即可鑒定1000個磷酸化肽，具有95％以上的選擇性。總體上，PolyMAC方法顯示了優秀的選擇性，出眾的回收率和高重現性，是今日最有效的磷酸化肽富集技術。課題組還利用該方法研究了乳腺癌體系。以下是英文摘要：

　　Protein phosphorylation plays critical roles in the regulation of many cellular functions. Analysis of phosphoproteomes by mass spectrometry depends on an efficient method to enrich phosphopeptides from complex mixtures. The current prevailing enrichment methods lack required efficiency and reproducibility due to complex sample conditions. Here, we utilize a novel soluble nanopolymer-based phosphopeptide enrichment approach termed PolyMAC (Polymer-based Metal ion Affinity Capture) for in-depth phosphoproteome analysis.

　　The central piece of PolyMAC reagents lies in a water soluble and highly accessible polymer support. PolyMAC reagents are synthesized from polyamidoamine (PAMAM) dendrimers functionalized with metal ions to specifically capture O-phosphorylated peptides through bidentate chelation chemistry. An extensive comparison was made between PolyMAC reagents and the TiO2 method, in terms of the specificity, reproducibility and sensitivity. We also compared different types of PolyMAC reagents functionalized with several metal ions. The enriched phosphopeptides were analyzed by nanoflow liquid chromatography tandem mass spectrometry (Nlc-MS/MS). The MS spectra were acquired on an Eksigent ultra 2D LC system that is coupled to hybrid liner ion trap orbitrap mass spectrometer (LTQ Orbitrap velos, Thermo Fisher). Data were searched using the SEQUESTTM algorithm within the Proteome Discoverer software. Using only 50 ug of total cell lysate, PolyMAC enrichment has led to the identification of over 1000 phosphopeptides with over 95% selectivity in a single run. Overall, the PolyMAC method demonstrated excellent selectivity, outstanding recovery yield and high reproducibility, thus rendering it one of the most effective phosphopeptide isolation techniques to date.

　　The PolyMAC enrichment approach has been applied to examine the differences in signaling pathways in breast cancer cell lines modulated by a tumor suppressor,spleen tyrosine kinase (Syk). MDA-MB-231 human breast cancer cell line was transfected with a tetracycline-inducible Syk vector. Proteins from cells with or without Syk induction were processed and phosphotyrosine peptides were enriched using the combination of pTyr peptide immunoprecipitation and PolyMAC to obtain the phosphorylation profiles. We identified 794 sites of tyrosine phosphorylation in malignant breast cancer cells,514 of which are dependent on the expression of Syk. Proteins with changes in pTyr phosphorylation were manually validated and a number of them confirmed through immunoprecipitation-Western blot experiments. They were mapped in a variety of major signaling networks including cell migration and apoptosis, therefore offering numerous leads to future breast cancer studies.

清華大學 林金明教授

　　來自清華大學的林金明教授做了題為“紙基電噴霧質譜常態離子源直接測定復雜樣品”的報告。質譜是測定復雜樣品的有效手段，能夠提供待測物的分子信息。目前的質譜分析通常需要對復雜樣品進行預處理和預分離，無法實現快速和高效分析。為解決復雜樣品直接檢測過程中的樣品前處理難題，林教授的課題組提出了一種稱為紙基電噴霧的新型常態離子源。將樣品直接加載到三角形的色譜紙上，接通高壓電后即可直接生成電噴霧，能夠進行直接的質譜檢測。

　　與Nano-ESI的對比實驗表明，紙基電噴霧比Nano-ESI能保留更多的分子離子，其電離能力更軟。計算表明，紙基電噴霧產生的分子離子具有的內能比Nano-ESI產生的分子離子內能低0.4電子伏左右，因此更適合生物分子樣品的質譜分析。

　　這一離子源能夠適用于大多數的化合物，包括小分子有機物、肽和蛋白質等。利用在紙上進行液體干燥后檢測的方法還可以直接測定血樣和尿樣中的各種藥物和代謝物。利用紙的采樣能力能夠直接分析固相表面的樣品，測定快速，靈敏度高。將紙基電噴霧與便攜式質譜儀聯用，能夠拓寬在非實驗室環境下的復雜樣品檢測能力。最后，林教授指出，紙基電噴霧具體的機理還有待于進一步的實驗證明。