串联飞行时间质谱中亚胺离子的断裂特征及其在肽段鉴定中的作用

基质辅助激光解吸电离-串联飞行时间质谱(MALDI-TOF/TOF)产生的亚胺离子可以提供丰富的肽段组成信息,该方法通常用于基于数据库搜索的蛋白质鉴定,或者结合化学衍生法用于从头测序,因而在一定程度上限制了对亚胺离子的认识及应用。本研究利用239个串联质谱探索MALDI-TOF/TOF中亚胺离子的断裂特征以及它们在肽段鉴定中的应用,发现在高能碰撞诱导解离条件下组氨酸等14种氨基酸可产生较强的亚胺离子信号(>50%阳性率),氨基酸的化学结构、位置效应和氨基酸残基个数是影响碎片离子强度的主要因素。此外,探讨了亚胺离子应用过程中的假阳性问题,提出亚胺离子相对强度的比较可以降低假阳性和提高肽段鉴定确定度,有助于完善目前的数据库搜索算法和辅助从头测序分析。     

耐药相关果糖二磷酸醛缩酶C的生物质谱分析与鉴定

醛缩酶C是生命代谢过程中重要糖酵解同工酶之一. 应用二维凝胶电泳分离卵巢癌细胞中高表达的果糖二磷酸醛缩酶C, 并通过基质辅助激光解吸电离飞行时间串联质谱对其进行分析与鉴定, 结果表明应用高分辨二维凝胶电泳分离, 结合生物质谱联用技术分析和鉴定未知蛋白具有简单快捷的特点.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

亲和标记-基质辅助激光解吸电离飞行时间质谱用于蛋白质相对定量方法的研究

将亲和标记技术和基质辅助激光解吸电离飞行时间串联质谱联用,建立了蛋白质相对定量方法,以牛血清白蛋白为实验对象,考察了该方法的准确度、重现性等指标;又以数种标准蛋白混合物为研究对象,考察了该方法的动态范围及相应的标准偏差,为实际生物样品中差异蛋白质分析奠定了基础。     

高效液相色谱-基质辅助激光解吸电离串联飞行时间质谱分离鉴定牛乳铁蛋白素

建立了高效液相色谱-基质辅助激光解吸电离串联飞行时间质谱(MALDI-TOF/TOF MS)分离鉴定牛乳铁蛋白素(bovine lactoferricin, LfcinB)的方法。采用胃蛋白酶酶解牛乳铁蛋白,酶解液离心后取上清液,经过离子交换色谱、反相液相色谱、透析等技术分离,对分离得到的目标产物进行抗菌活性分析、蛋白含量测定和MALDI-TOF/TOF MS鉴定。分离得到高活性的LfcinB,其相对分子质量为3124.89,蛋白含量为18.20 μg/mL。本方法具有精度高、分析速度快和分辨能力强等优点,是其他传统的分析鉴定LfcinB方法所无法比拟的,为进一步研究LfcinB奠定了基础。     

基于MIL-100(Fe)的磁性纳米碳材料的制备及其在内源性肽富集中的应用

以Fe₃O₄为核，采用层层自组装的方法合成核壳型Fe₃O₄@MIL-100（Fe）磁性纳米材料，经高温煅烧得到具有较大孔径（17.78 nm）、高含碳量（6.79%）的磁性纳米材料Fe₃O₄@MC。将该材料用于多肽的富集，并通过基质辅助激光解吸电离飞行时间质谱仪（MALDI-TOF MS）进行鉴定。Fe₃O₄@MC材料从牛血清白蛋白（BSA）酶解液中富集得到33条肽段，且具有较高的选择性（BSA酶解液与BSA的质量比为1：400），用于人血清中内源性肽段的分离富集也取得了较好的效果。     

一系列阳离子性卟啉化合物的基质辅助激光解吸质谱行为

应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)研究了一系列阳离子性卟啉化合物的质谱行为.结果表明,阳离子性卟啉与非离子性卟啉化合物的激光解吸电离方式有明显不同.对于四-氮R基(R=甲基,乙基,丙基,苄基)吡啶基卟啉,吡啶基氮上的侧链基团(R)可明显影响该类化合物在MALDI-TOF-MS测试过程中离子形成方式;R基团的增大以及平衡阴离子半径的增大可增加平衡阴离子与卟啉环阳离子之间相互作用力的共价成分,因此在MALDI-TOF-MS测定过程中能够得到卟啉环阳离子与多个平衡阴离子结合在一起的较高质量数的离子峰.另外,还初步探讨了阳离子性卟啉化合物的激光解吸电离机理.     

基于MALDI-TOF MS的串联富集策略在磷酸化蛋白组学中的应用

提出一种除盐-富集串联用于磷酸肽富集研究的思路。选用C18柱和铈(Ⅳ)修饰的壳聚糖材料进行脱盐实验,以制备的基于聚合物基体螯合Fe3+的亲和色谱材料为富集材料。将直接富集和串联策略应用到标准品和血清中,研究结果表明,该富集材料具有高选择性和高灵敏度(1.6 fmol),铈(Ⅳ)修饰的壳聚糖材料前提下的串联策略能明显降低样品的复杂性。相比直接富集方法,能够提高磷酸化肽的覆盖率。     

硅锆复合氧化物的制备、表征及其在磷酸化肽段富集中的应用

采用溶胶-凝胶法制备了氧化硅-氧化锆复合氧化物(SiO2-ZrO2),并对其物理与化学性质进行了详细地表征.以α-酪蛋白的酶解产物为探针,比较了氧化硅-氧化锆复合氧化物与氧化锆对磷酸化肽段的富集能力.结果表明: 在pH<2时富集,SiO2-ZrO2对磷酸化肽的选择性要优于ZrO2;使用SiO2-ZrO2材料时,多磷酸化肽段的回收率更高.结合MALDI-TOF MS检测技术,SiO2-ZrO2成功地应用于脱脂牛奶酶解产物中磷酸化肽的分离和富集.     

多金属氧酸盐/壳聚糖磁性复合材料的制备及其用于磷酸化肽的富集

建立了一种基于多金属氧酸盐磁性材料富集磷酸化肽的方法。采用层层自组装技术制备多金属氧酸盐/壳聚糖磁性材料,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,用于磷酸化肽的富集。该磁性材料具有快速磁响应、亲水性、正电性等优点,对磷酸化肽具有高的富集选择性。实验用β-酪蛋白作为模型蛋白质,通过富集后,方法的检出限为0.02 fmol,说明合成的磁性材料对微量蛋白样品分析具有很高的应用潜力。     

基于强阳离子交换色谱分离的磷酸化肽段富集策略

为了在短时间内获得相对含量高的磷酸化肽段,以标准磷酸化蛋白质为模型对强阳离子交换色谱（SCX）分离磷酸化肽段体系的缓冲溶液和梯度设置进行了研究,并用酵母酶切肽段混合物考察了该路线在较复杂的样品中的应用。实验结果表明优化后的体系能够在30 min内分离出磷酸肽段,而且非磷酸化肽段的干扰很少,这样便相对提高了磷酸化肽段在质谱仪中的响应强度,重要的是该体系可以对复杂样品进行很好的分离。这说明SCX用于规模化磷酸化肽段富集的策略是可行的。本研究为磷酸化蛋白质组学规模化分析提供了实用技术。     

3-氨基-9-乙基咔唑衍生化寡糖混合物的高效液相色谱分离及激光解吸电离飞行时间质谱分析

建立了以3-氨基-9-乙基咔唑(AEC)为衍生化试剂对寡糖的标记方法。寡糖的还原端与AEC的伯氨基反应生成烯胺,再被NaBH3CN还原为二级胺,使得寡糖被AEC标记。衍生物通过反相高效液相色谱分离纯化,采用的色谱柱为Waters Symmetry C18柱(3.9 mm×150 mm,5 μm),乙腈和乙酸铵水溶液(pH 4.5)为流动相,梯度洗脱,在254 nm波长处检测,并以基质辅助激光解吸电离飞行时间质谱进行分析。在此衍生化条件和色谱条件下,葡寡糖衍生物分离良好,并且AEC衍生可显著提高葡寡糖的质谱检测灵敏度。该方法适用于寡糖的分离纯化和结构分析,并与生物质谱具有良好的兼容性,表明该方法在微量寡糖链分析方面有广阔的应用前景。     

Quantitative analysis of 5 alpha-reductase inhibitors in DU145 cells using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry

N-(Dicyclohexyl)acetylpiperidine-4-benzylidene-4-carboxylic acid (1) is an excellent in vitro inhibitor of 5 alpha-reductase (5 alpha R). Compound 1 showed, however, much lower inhibition activity of 5 alpha R in vivo than in vitro, which might be caused by poor membrane permeability. The methyl ester of 1 (1a) was therefore tested as a model prodrug to see if it has better permeability properties than the corresponding acid 1. It was also monitored that this methyl ester was cleaved into the active compound 1 within the DU145 cells. Quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) methods were established with reliable linearity factors (0.996 for MALDI-TOFMS and 0.998 for HPLC/MS/MS) and reproducibility (relative standard deviation = 6.5% for MALDI-TOFMS and 2.8% for HPLC/MS/MS). The samples for MS analysis were effectively prepared from the cell homogenates using solid-phase extraction, with a high recovery of 90% on average. The intracellular amount of 1a (1.7 nmol) was much higher than that of 1 (0.032 nmol) in DU145 cells after 6 h of incubation. After incubation with the ester (1a), the cleaved acid (1) was detected within the cells. The concentration of acid 1 (0.045 nmol) in this experiment was higher than the acid content (0.032 nmol) after direct incubation with 1. Surprisingly, high amounts of the cleaved compound 1 were found outside the cells after 6 h of incubation with 1a.     

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.     

Combination of extraction tip and MALDI-TOF-MS for efficient separation and analysis of cysteine-containing peptides

In our work,a new extraction tip with gold-modified polymer is developed.The simple,self-made and extremely economical tips were successfully applied to capture cysteine-containing peptides.The loading capacity of a tip(column bed:0.3 mm diameter,5 mm length)is 2–4μg peptides.We can make one tip in 30 s and each costs less than 0.1 cent.The use of these tips can achieve a stable analysis with less background interference,even for 10 ng target peptides.Compared with other separation techniques,our method can save much time and energy while providing a means to selectively capture cysteine-containing peptides from complex analyte due to the strong interaction.All results showed that our new extraction tips have minimal cost and perfect selectivity;thus they have great potential in sample pretreatment systems for proteomics.     

Identification of proteins by combination of size-exclusion chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and comparison of some desalting procedures for both intact proteins and their tryptic digests

Separation of a protein mixture by size-exclusion chromatography (SEC) was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Identification of proteins in the collected fractions was performed both as intact proteins by MALDI-TOFMS and using peptide mass fingerprinting (PMF) after their digestion with trypsin. The presence of salts mostly disturbs the MALDI-TOFMS signal and, therefore, proper purification or desalting procedures must be employed. Four desalting procedures (desalting column packed with Sephadex G-100, on-target washing, centrifugal filter devices and ZipTip C(18)) for purification of fractions of proteins separated by SEC and their tryptic digests prior to determination of their exact molecular masses by MALDI-TOFMS were compared. In the case of intact proteins, the experiments showed that the best desalting procedures are the use of ZipTip C(18) pipette tips and Ultrafree CL centrifugal filter devices. The peptide digests can be purified by using ZipTip C(18) pipette tips or on-target washing when both of these procedures provide similar results. On-target washing can be used as a simple procedure to improve the mass spectra of salt-containing samples. Analyses of the droplets collected after the on-target washing show losses of sample and matrix caused by dissolution of these compounds during this procedure. Further, it was found that protein identification based on PMF is more sensitive than analyses of intact proteins and that multiple on-target washing is very advantageous for analyses of peptide mixtures with a high content of salts.     

On-line size-exclusion chromatography/mass spectrometry of low molecular mass heparin

Heparin and low molecular mass heparin (LMMH) consists of complex mixtures of sulphated linear oligosaccharides that are difficult to analyse. An on-line size exclusion chromatographic/electrospray ionization (ESI) mass spectrometric method that allows the determination of more than 60 components in an LMMH preparation is presented. The experimental setup includes on-line cation exchange in order to prevent massive adducting in the ESI interface.     

Solid-state glycation of beta-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques

The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on beta-lactoglobulin (beta-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of beta-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75).     

Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.