本文選題：定量蛋白質(zhì)組學(xué) + 離子化源　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文

【摘要】：隨著蛋白質(zhì)組學(xué)技術(shù)和方法研究的深入,蛋白組學(xué)研究已由原來的兩譜三圖三庫進(jìn)一步向蛋白質(zhì)組深度覆蓋和定量研究發(fā)展。蛋白質(zhì)組深度覆蓋是蛋白質(zhì)組注釋基因組、發(fā)現(xiàn)診斷標(biāo)志物、藥物靶標(biāo)和關(guān)鍵蛋白質(zhì)分子的前提和基礎(chǔ),而蛋白質(zhì)組定量信息的獲得有利于蛋白質(zhì)功能的發(fā)現(xiàn)和深度解析。蛋白質(zhì)組深度覆蓋的含義包括蛋白質(zhì)組中蛋白質(zhì)鑒定的深度覆蓋和蛋白質(zhì)序列的長度覆蓋。隨著液相色譜及質(zhì)譜技術(shù)的迅速發(fā)展,液相色譜-質(zhì)譜聯(lián)用技術(shù)已經(jīng)成為實現(xiàn)蛋白質(zhì)組深度覆蓋和蛋白質(zhì)組定量的常用手段。然而由于在蛋白質(zhì)組分析過程中,存在蛋白質(zhì)樣品制備時產(chǎn)生的損耗、樣本的不完全酶解、質(zhì)譜靈敏度低等問題,影響了低豐度蛋白的鑒定,因而限制了蛋白質(zhì)組覆蓋度的提高和更多蛋白定量信息的獲取。另外,采用化學(xué)合成方法制備內(nèi)標(biāo)肽段,價格昂貴、產(chǎn)量低,而體外標(biāo)記SILAC方法和體內(nèi)標(biāo)記iTRAQ或TMT價格昂貴,限制了定量蛋白質(zhì)組技術(shù)的廣泛應(yīng)用。 針對蛋白質(zhì)組學(xué)研究中存在的問題,本論文首先在質(zhì)譜靈敏度方面,研制了一種新型離子源輔助部件,以提高質(zhì)譜靈敏度與穩(wěn)健性;其次,針對實際樣本酶解不完全問題,研究了通過提高蛋白水解酶的量以增加酶切速度和效率的可行性；最后,針對蛋白質(zhì)組絕對定量方法中存在的問題,首先基于金屬標(biāo)簽的價格低廉的特點,和選擇離子監(jiān)測質(zhì)譜高靈敏度的特點,發(fā)展了一種絕對定量新方法。其次發(fā)展了反相色譜填料(C18填料)輔助的18O標(biāo)記定量肽段串聯(lián)體蛋白(quantification concatamer, QconCAT)結(jié)合液-質(zhì)聯(lián)用的蛋白質(zhì)組絕對定量新方法。 本論文由四章組成。第一章概述了蛋白質(zhì)組學(xué)的研究現(xiàn)狀、質(zhì)譜離子源研究現(xiàn)狀以及蛋白質(zhì)酶解效率對蛋白質(zhì)組研究的影響、金屬標(biāo)記及18O標(biāo)記結(jié)合高效液相色譜-質(zhì)譜定量方法的最新進(jìn)展,并在目前研究背景下提出了本課題的研究內(nèi)容。 在第二章,研制了一種離子源輔助部件,不僅提高了目前使用電噴霧離子源的流速,并且增加了靈敏度和噴霧的穩(wěn)定性。輔助部件設(shè)計時,首先通過電動力學(xué)方法模擬對影響離子源靈敏度的兩個因素進(jìn)行優(yōu)化,然后通過制作輔助部件提高了離子化效率及傳輸效率,進(jìn)而提高了質(zhì)譜的靈敏度。首先用多場耦合分析計算軟件對離子源輔助部件形狀進(jìn)行設(shè)計,之后用標(biāo)準(zhǔn)肽段樣品對輔助部件性能進(jìn)行檢驗。裝有輔助部件的離子源流速設(shè)為1-2μL/min后,信號比未使用輔助部件離子源提高了5.6倍,即信噪比增加為原來的2.8倍；定量限比未使用輔助部件納升源降低65%,且流速保持在在1-2μL/min,增加了質(zhì)譜分析的穩(wěn)定性。 在第三章,我們將金屬標(biāo)簽與選擇離子監(jiān)測技術(shù)集合起來,建立了一種蛋白質(zhì)絕對定量新方法。首先考察了多反應(yīng)離子監(jiān)測質(zhì)譜技術(shù)和選擇離子監(jiān)測質(zhì)譜檢測的靈敏度,表明在分析組成簡單的樣品時選擇離子檢測質(zhì)譜技術(shù)有更好的靈敏度；其次考察了金屬標(biāo)簽用于定量蛋白質(zhì)組學(xué)的可行性,對標(biāo)記效率、標(biāo)記歧視性、標(biāo)記穩(wěn)定性和金屬標(biāo)記肽段色譜保留行為進(jìn)行了考察。實驗結(jié)果表明金屬標(biāo)記可以用于絕對定量。采用建立的蛋白質(zhì)絕對定量新方法對騰沖嗜熱菌蛋白進(jìn)行定量,結(jié)果表明絕對定量方法的靈敏度高,定量限為1fmol;線性范圍為1fmol～500finol時,線性范圍內(nèi)R2值大于0.99,具有良好的線性；測定的標(biāo)準(zhǔn)肽段回收率為117.01%,說明該方法具有較高的準(zhǔn)確度；將該方法應(yīng)用于騰沖嗜熱菌中烯醇酶蛋白的定量分析,相對標(biāo)準(zhǔn)偏差為5.47%,表明定量結(jié)果可信。結(jié)果表明該定量方法可以用于蛋白質(zhì)組絕對定量分析,是簡單樣本的一種新的方法選擇 在第四章,我們發(fā)展了一種通過增大酶量的蛋白質(zhì)樣本快速、高效酶切方法,并基于這種酶切方法發(fā)展了18O標(biāo)記QconCAT結(jié)合液-質(zhì)聯(lián)用的目標(biāo)蛋白質(zhì)組絕對定量新方法。在溶液狀態(tài)下,通過增加蛋白酶量至酶與底物質(zhì)量比為1：1時,可在37℃下2小時內(nèi)實現(xiàn)對蛋白樣品的完全酶切,且未發(fā)生自切現(xiàn)象。對酶切后的多肽混合物,通過反相色譜柱分離,實現(xiàn)蛋白酶及酶切肽段的分離,并回收了蛋白酶。進(jìn)一步通過對回收后的蛋白酶酶切效率進(jìn)行考察,發(fā)現(xiàn)蛋白質(zhì)的酶切效率沒有降低,可重復(fù)使用。使用該方法的優(yōu)點是與一般酶切方法相同,容易掌握,操作簡單,酶切時間短、效率高�；谠摲椒ǖ�180標(biāo)記方法不僅標(biāo)記時間短,且不產(chǎn)生回標(biāo)現(xiàn)象。將這種蛋白質(zhì)酶切方法與18O標(biāo)記方QconCAT結(jié)合液-質(zhì)聯(lián)用技術(shù)進(jìn)行整合,建立了一種用于目標(biāo)蛋白質(zhì)組絕對定量的分析方法,并對人肝微粒體中的重要藥物代謝酶進(jìn)行了定量分析。
[Abstract]:With the rapid development of protein group , it has been found that the technique of liquid chromatography - mass spectrometry has been used as a common means to realize the deep covering of protein and the quantitative information of protein .

Aiming at the problems existing in the study of proteomic analysis , this paper first developed a new ion source auxiliary component in the aspect of mass spectrum sensitivity to improve the sensitivity and robustness of mass spectrometry ; secondly , the feasibility of increasing the amount of proteolytic enzyme to increase the enzyme cutting speed and efficiency was studied .
In the end , based on the characteristics of low price of metal label and high sensitivity of choosing ion monitoring mass spectrum , a new method of absolute quantification is developed , which is based on the characteristics of low cost of metal label and selection of high sensitivity of ion monitoring mass spectrometry .

This thesis consists of four chapters . The first chapter summarizes the research status of proteomic research , the research status of mass spectrometer ion source and the effect of protein enzymolysis efficiency on the research of proteome , the latest development of the quantitative methods of metal marker and 18 O labeled with high performance liquid chromatography - mass spectrometry , and the research contents of this subject in the current research background .

In chapter 2 , an ion source auxiliary component is developed , which not only improves the current flow rate of the electric spray ion source , but also increases the sensitivity and the spray stability .
The quantitative limit is 65 % lower than that of the auxiliary member nanoliter source , and the flow rate is maintained at 1 - 2 . m u.L / min , which increases the stability of the mass spectrometry analysis .

In chapter 3 , we combine the metal label with the selective ion monitoring technology to establish a new method for absolute quantification of protein . First , the sensitivity of multi - reactive ion monitoring mass spectrometry and selective ion monitoring mass spectrum detection is studied . It is shown that the choice of ion detection mass spectrometry has better sensitivity in the analysis of sample with simple composition .
The feasibility of using metal tags for quantitative proteomic analysis was investigated . The results showed that the metal markers could be used in absolute quantification . The results showed that the metal markers could be used for absolute quantification . The results showed that the absolute quantification method was used to quantify the proteins of Tengchong thermophilic bacteria . The results show that the absolute quantitative method has a high sensitivity , a limit of 1fmol , and a linear range of 1fmol ~ 500finol , the R2 value in the linear range is more than 0.99 , and has good linearity ;
The recovery rate of the standard peptide segment was 117.01 % , which indicates that the method has higher accuracy .
The method is applied to the quantitative analysis of enolate in Tengchong thermophilic bacteria , and the relative standard deviation is 5.47 % , indicating that the quantitative results are authentic . The results show that the quantitative method can be used for absolute quantitative analysis of proteome , and is a new method for simple sample .

In chapter 4 , we have developed a new method for the determination of the protein sample by increasing the amount of enzyme . The enzyme digestion efficiency of the protein can be completely digested by adding protease to the ratio of the enzyme to the mass ratio of 1 : 1 . The method has the advantages that the enzyme digestion efficiency of the protein is not reduced and the protease can be reused . The method has the advantages of short labeling time and no return phenomenon .

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R917

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李楠楠;周廉淇;毛心麗;張姣;衛(wèi)軍營;林虹君;李佳斌;田芳;張養(yǎng)軍;錢小紅;;~(18)O同位素標(biāo)記定量肽段串聯(lián)體蛋白質(zhì)結(jié)合同位素稀釋-多反應(yīng)監(jiān)測質(zhì)譜的蛋白質(zhì)絕對定量新方法[J];色譜;2013年06期

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本文編號：1873614