改进的~(18)O同位素标记法标记蛋白质多肽

针对18O同位素标记反应两个重要影响因素——肽段分散度和胰酶灭活方法,进行了标记条件的改进和灭活方法的优化。在H218O中加入RapigestTM SF助溶剂并微波辅助加热,使α-酪蛋白胰酶酶切肽段的标记效率得到明显改进(18O/16O峰面积比值>99%)。标记后,对胰酶进行还原烷基化化学修饰彻底灭活,使标记后的肽段稳定性显著提高,放置6d不发生回交反应。对标准蛋白质甲状腺球蛋白酶切肽段混合物标记后的质谱实验结果表明:优化的标记方法能快速稳定地标记蛋白质酶切多肽。     

18O稳定同位素标记定量蛋白质组研究技术的建立与优化

建立了18O稳定同位素标记方法,用于复杂体系蛋白质相对定量分析。对影响蛋白质标记稳定性的实验条件进行了比较和优化。结果表明,采用酶切后标记的方法,酶切肽段在胰酶催化下,在pH 5.0的K2HPO4/KH2PO4缓冲体系中,37℃18O标记反应16 h,绝大部分肽段即可达到100%的标记效率。对多个16O/18O成对肽段峰强度的动态范围及定量准确度进行了考察。结果表明,18O标记方法是一种简便、稳定、可靠的相对定量方法,10倍动态范围内,标记率相对标准偏差在18.4%以内,16O/18O峰强度呈很好的线性关系。本实验考察了标记后的肽段在不同溶液体系中的稳定性,为复杂样品的预处理和预分离的溶液条件提供了依据。     

金属标记结合高效液相色谱-选择离子监测质谱的蛋白质绝对定量新方法

建立了金属标记结合高效液相色谱-选择性离子监测质谱(SIM)的蛋白质绝对定量新方法。实验考察了金属标记效率、金属标记的稳定性、标记后肽段的色谱保留和质谱行为、新定量方法的线性范围和准确度。实验结果表明金属标记具有标记效率高,稳定性好,色谱保留行为一致等优点。另外,金属标记-选择离子监测质谱绝对定量方法灵敏度高,其定量限低至1 fmol,线性范围为1~500 fmol,线性范围内R2值大于0.99,具有良好的线性关系;经过测量,标准肽段的回收率为117.01%,说明该方法具有较高的准确度。将该方法应用于腾冲嗜热菌中烯醇酶蛋白的定量分析,相对标准偏差为5.47%,表明该方法的精密度高。以上结果表明该方法可以用于生物样本中的蛋白质的绝对定量分析,为比较简单的生物样本中蛋白质的绝对定量方法提供了一种新的选择。     

高效液相色谱-串联质谱法结合稳定同位素标记肽段同时测定稻米及其制品中3种过敏蛋白质

基于稳定同位素标记特征肽段和液相色谱-质谱联用仪建立稻米及制品中3种过敏蛋白质的同时定量方法。稻米及制品样品经盐溶液提取,赖氨酰基内切酶(Lys-C)和胰蛋白酶依次水解,C18-SD柱净化后,采用纳升高效液相色谱-线性离子阱-静电场轨道阱(NanoLC-LTQ-Orbitrap)采集和Protein Discovery软件鉴定,NCBI和Uniprot数据库的基本局部搜索比对工具(BLAST)筛选验证,最终获得表征稻米及制品中α-淀粉酶/胰蛋白酶抑制剂类蛋白质(seed allergenic protein RAG2, RAG2)、乙二醛酶Ⅰ活性蛋白(glyoxalase Ⅰ)和α-球蛋白(19 kDa globulin)3种过敏蛋白质的特异性肽段。3个特异性肽段经液相色谱梯度洗脱,在Poroshell色谱柱上实现完全分离,由三重四极杆质谱仪分析。实验通过优化多反应监测(MRM)质谱参数,比较不同溶剂体系、水解酶种类和酶量等酶解条件,结合内标法定量,实现对稻米及制品中3种蛋白质的绝对定量。实验结果表明,当酶解溶剂中含1 g/L十二烷基硫酸钠,采用Lys-C和胰蛋白酶组合消化策略,可有效提高3种蛋白质的酶切效率至65.7%~97.3%。该方法在1~200 nmol/L范围内线性关系良好,相关系数均大于0.9972, 3种蛋白质的检出限和定量限分别为3 mg/kg和10 mg/kg。3种蛋白质在空白稻米制品基质中3个水平下的加标回收率为80.6%~103.7%,日间和日内精密度均小于11.5%。该方法稳定性好,检测灵敏度高,操作简便,在分析各类稻米及制品中3种过敏蛋白质含量具有广泛的应用前景。     

等电聚焦分离与18O稳定同位素标记联用的磷酸化蛋白质组学定量方法研究

磷酸化蛋白质组学定量分析,要对磷酸化修饰富集技术和定量技术进行研究。基于此,本研究采用18O稳定同位素标记技术对胰蛋白酶酶解肽段混合物进行标记,并对其标记时间和标记后胰蛋白酶的变性条件进行优化。结果表明:在pH=4～5的KH2PO4缓冲体系中,37℃,标记反应持续19～24h,除了C-端肽之外,几乎所有的肽段都可达到100%标记;采用TCEP可以有效地抑制16O-18O回标现象。建立了与18O标记技术兼容性良好的IPG-IEF技术对磷酸化肽段进行选择性富集,富集后共从HepG2细胞中鉴定到491个磷酸化位点、362个磷酸化肽段和356个磷酸化蛋白,表明IPG-IEF在大规模磷酸化肽段分离富集中是有效的;最后与高准确度高灵敏度高分辨率的LTQ-FTICR质谱仪联用,建立了基于18O-IPG-IEF-LTQ-FTICR的磷酸化蛋白质组定量技术。实验结果表明,该技术可以实现磷酸化肽段的有效定性和定量。本研究为磷酸化蛋白质组学定量研究提供了实用技术。     

基于蛋白质组学的细胞色素P450和葡萄糖醛酸转移酶亚型绝对定量分析

利用蛋白质组学方法,将大鼠肝微粒体样品进行胰蛋白酶水解;再利用液相色谱-串联质谱法(LCMS/MS),采用多反应监测模式(MRM),通过测定蛋白质水解后产生的特征酶切肽段,实现同时对大鼠肝微粒体内药物代谢酶P450和UGT的绝对定量。本实验首先建立标准工作曲线,对肝微粒体样品中P450和UGT进行定量,在线性范围内,相关系数r>0.995,线性关系良好,定量限≤10 nmol/L;以合成的稳定同位素标记特征肽段作为内标,对UGT1A1进行定量分析。结果表明,同位素标记特征肽段与未标记肽段色谱行为与质谱响应一致,在基质溶液中同位素标记肽段线性关系良好,利用标准曲线法和稳定同位素稀释法测得UGT1A1含量分别为17.30和18.23 nmol/g,两种方法所得结果基本一致,但稳定同位素稀释法操作简便,更适用于复杂样品的高通量测定。     

氨基酸-稳定同位素稀释质谱法用于合成肽段准确含量分析

建立了氨基酸同位素稀释液相色谱-串联质谱法准确测定合成肽段绝对含量的方法。实验中对合成肽段的纯度进行了表征,色谱纯度表征结果为99%以上,质谱纯度为90%以上。在肽段溶液中加入13C标记的氨基酸后进行酸溶液水解时间的优化,水解后的氨基酸直接经液相色谱分离和质谱检测,结果表明肽段中的被测氨基酸在150 ℃、6 mol/L HCl溶液水解4～6 h就可以达到水解平衡。每个肽段选择两个或两个以上的被测氨基酸,测得随机选择的5种合成肽段的绝对含量为62.07%～88.18%,测定结果的相对标准偏差小于8%,相对误差小于5%,均满足定量要求。除常用的被测氨基酸苯丙氨酸、缬氨酸、异亮氨酸外,还考察了选择赖氨酸和精氨酸作为被测氨基酸的可行性,实验结果表明增加精氨酸为被测氨基酸是可行的,从而进一步增加了方法的普适性。该方法的建立避免了色谱法定量时氨基酸衍生化处理带来的副反应影响及操作繁琐等问题,提高了肽段含量测定的准确度和精密度,为肽段含量的准确测定提供了一种新的方法。     

用于痕量监测Zr4+、Cr2O72-、Fe3+、HPO42-和指纹识别的功能化Eu3+/Tb3+配位聚合物荧光探针的构筑

采用芳香族π共轭及含氮原子有机连接剂,合成同构铽、铕发光配位聚合物(CPs){[Eu(PLIA)_1.5(H₂O)₂]·H₂O}_n (1)和{[Tb(PLIA)_1.5(H₂O)₂]·H₂O}_n (2),其中H₂PLIA=5-((吡啶-4-基甲基)氧基)苯-1,3-二甲酸。对合成的配合物进行了结构测定、表征和荧光痕量识别实验研究。2个同构配合物具有理想的三维框架结构,π…π堆积及氢键等弱相互作用增强了其化学稳定性;表征显示配位聚合物1和2具有良好的荧光性质、结晶性、热力学稳定性及结构完整性,可作为荧光传感的材料。1和2对水溶液中的Zr⁴⁺、Cr₂O₇^2-和Fe³⁺、HPO₄^2-具有选择性好、灵敏度高的荧光识别能力,其检出限分别为0.139 μmol·L^-1(1,Zr⁴⁺)、0.626 μmol·L^-1(1,Cr₂O₇^2-)、0.430 μmol·L^-1(2,Fe³⁺)、1.36 μmol·L^-1(2,HPO₄^2-)。探究了1和2作为探针的荧光猝灭机理。更有趣的是,1和2具有指纹识别性能,其荧光指纹纹路清晰连贯,细节明显,可被清晰观察。     

基于生物质谱的乙酸酐稳定同位素标记定量蛋白质组学方法的建立与优化

建立了一种基于生物质谱的乙酸酐稳定同位素标记,定量蛋白质组学研究方法,优化了影响标记效率的各种条件。在pH8.0的Na2B4O7/H3BO3缓冲体系中,当乙酸酐摩尔浓度25倍过量于肽段摩尔量,22℃反应30 min时,标记即可完全。对多对H6/D6-乙酸酐标记肽段在基质辅助激光解吸电离质谱中的动态范围及定量准确度进行了考察,并通过串联质谱分析确定了乙酰化位点。结果表明:在10倍和30倍动态范围内,线性关系良好(r=0.99,r=0.98),理论值和观测值的偏差分别为0.5%和20%。     

立方Nd2O3上过氧物种光诱导生成的18O同位素示踪考察

采用原位显微Raman光谱和¹⁸O同位素示踪技术,以325 nm激光为激发光源,对立方Nd₂O₃上过氧物种的光诱导生成过程进行了详细表征,进一步证实过氧源于分子氧对晶格氧的氧化反应. 结果还表明,325 nm激光在室温下即可诱导过氧的生成,在实验条件下,生成的过氧物种可与Nd₂O₃的晶格氧发生快速的氧交换反应,位于Nd₂O₃体相的晶格氧也可迁移至样品表层进而参与过氧的生成. 325 nm激光照射有助于促进晶格氧的迁移以及晶格氧与分子氧之间的氧交换反应.     

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Decision peptide-driven: A free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse ¹⁸O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse ¹⁸O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse ¹⁸O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using ¹⁸O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.     

Incorporation of 18Oxygen into Peptide Mixtures and Analysis with Multi‐Dimensional Chromatography and Mass‐Spectroscopy

Abstract

Enzymatic labeling with ¹⁸oxygen has the potential to become a widely applied method of isotope labeling for differential protein expression analysis by mass spectrometry because it is not amino acid specific and the reagents are cost‐effective and readily available. In this work, we investigate experimental parameters that affect efficient ¹⁸O incorporation with a model bovine serum albumin protein system and then use optimized chemistries for labeling the c‐terminus of peptides in a yeast proteome. Additionally, the role of sample handling, including the use of liquid chromatography was examined. An analytical methodology was developed which demonstrates the application of multi‐dimensional chromatography in conjunction with enzymatic labeling.     

Latest developments in sample treatment for O-isotopic labeling for proteomics mass spectrometry-based approaches: A critical review

Nowadays isotopic ¹⁸O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using ¹⁸O-labeling. In this critical review we comment on the different ways in which ¹⁸O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage.     

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification

The relative quantification and identification of proteins by matrix‐assisted laser desorption ionization time‐of‐flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N‐terminal derivatization with 4‐sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while ¹⁸O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4‐sulphophenyl isothiocyanate derivatization with ¹⁸O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R² value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.     

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics

We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the epsilon-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of beta-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes.     

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling

Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification. Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest and scope of elemental mass spectrometry techniques for quantitative proteomics. In this work one of the first methodologies for absolute quantification of peptides and proteins using MeCAT labeling is presented. Liquid chromatography (LC) interfaced to ICP-MS has been used to separate and quantify labeled peptides while LC coupled to electrospray ionization mass spectrometry served for identification tasks. Synthetic-labeled peptides were used as standards to calibrate the response of the detector with compounds as close as possible to the target species. External calibration was employed as a quantification technique. The first step to apply this approach was MeCAT-Eu labeling and quantification by isotope dilution ICP-MS of the selected peptides. The standards were mixed in different concentrations and subjected to reverse-phase chromatography before ICP-MS detection to consider the column effect over the peptides. Thus, the prepared multi-peptide mix allowed a calibration curve to be obtained in a single chromatographic run, correcting possible non-quantitative elutions of the peptides from the column. The quantification strategy was successfully applied to other labeled peptides and to standard proteins such as digested lysozyme and bovine serum albumin.