新型等点聚焦系统用于蛋白质组样本的预分离方法研究

对蛋白质或多肽的高效分离,将有利于降低肽段信号之间的干扰,保证规模化的蛋白质组学深度覆盖鉴定,并提高定量蛋白质组分析的准确性。本研究采用一种新型的等电聚焦预分离系统(OFFGEL),考察系统对293T细胞在蛋白质与肽段水平的分级分离效果,在OFFGEL系统中聚焦后的蛋白质或肽段可以从溶液中直接回收,与下游的LC-MS/MS肽段鉴定流程兼容。实验结果表明,肽段的分离效果明显优于蛋白质的分离,分级分离后各馏分对应的等电点位置与肽段的理论等电点分布高度一致,每个馏分中单独鉴定的肽段比例超过90%,显示了该系统对肽段的高分辨分离能力,结合生物质谱技术,在293T细胞中实现6727个蛋白质的规模化鉴定,表明该系统在复杂体系蛋白质组研究中的应用潜力。     

肽段化学衍生技术及其在质谱高灵敏度鉴定中的应用进展

鸟枪法串联质谱蛋白质鉴定是蛋白质组学研究中广泛采用的策略。然而,蛋白质组样品的高复杂性、宽动态范围分布使得低丰度肽段以及难于离子化肽段的质谱检测仍然存在着巨大的挑战。为了提高质谱检测灵敏度,通过化学衍生技术在多肽中引入易于离子化的小分子标签的方法获得了广泛关注。本文综述了近年来应用于多肽及其翻译后修饰的化学衍生试剂,侧重其在高灵敏度质谱分析中的应用进展,并展望了化学衍生技术的发展方向及其在蛋白质组学中的应用前景。     

毛细管反相液相色谱-串联质谱用于肽的鉴定和相对定量分析

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。     

反相高效液相色谱法测定蟾酥中的3种蟾毒内酯

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。。     

基于四极杆-飞行时间质谱的非标记蛋白定量检测方法学研究

以四极杆飞行时间质谱检测的蛋白肽段的质谱信息为基础,开发了一种简单、准确的蛋白定性、定量检测方法,并用于牛血清白蛋白的定性、定量检测。对酶解肽段的梯度洗脱条件和流速等液相色谱条件进行了优化;进行蛋白样品的酶解及质谱检测,利用MassHunter定量软件对数据进行分析;为减少假阳性结果的出现,实验优化了数据处理软件中的设置参数,并对方法的重现性进行了考察。结果显示,重复检测的8个肽段峰面积的RSD均在3.0%以下。该方法对牛血清白蛋白的检出限为100 ng。实验发现,样品中蛋白的含量与检测到的肽段匹配率呈对数关系,并从检测到的丰度较高的8个肽段中确定可用于BSA蛋白定性检测的肽段为:HLVDEPQNLIK,定量检测的肽段为:ATEEQLK,方法的回收率为106%。该方法操作简便,检测快速,成本较低,专一性强,可用于蛋白特异性肽段的筛选及复杂生物样品中蛋白的检测。     

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合¹⁸O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和¹⁸O标记效率,并对QconCAT蛋白质结合¹⁸O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。     

纳升液相色谱-质谱分析方法的重现性对尿液多肽组分析结果的影响

多肽组学是蛋白质组学技术的延伸和扩展,在医学和生物学研究中的应用日益广泛,但是,多肽组鉴定方法的重现性对实验结果的影响目前尚不清楚.本研究利用纳升液相色谱-高分辨质谱对健康人的尿液多肽组进行了7次平行分析,考察图谱数目、图谱利用率、鉴定的肽段数目、蛋白质数目、样品总离子强度和肽段保留时间等指标的变化,以揭示重复实验之间分析结果的可变性和稳定性.7次测定的肽段数目平均值为208,标准偏差为38;7次结果合并后,得到了归属于114个蛋白质的426个肽段,肽段和蛋白质数目均显著增加;而35个蛋白质的109个肽段在所有7次实验中均被检出,表明多肽组的单次分析结果既具有一定的随机性,又具有相对的稳定性.增加平行实验次数会扩大多肽组数据集,但测定3次以上后增加幅度减小.相比于肽段,多肽组的结果在蛋白质水平上更为稳定,提示利用蛋白质为多肽组的生物标志物更为稳健.     

赖氨酸胍基化对蛋白质组分析的影响

赖氨酸胍基化在蛋白质组学定性和定量研究中起着重要作用,本文系统分析了胍基化前后,HeLa细胞蛋白质经胰蛋白酶酶解产生的3种不同类型肽段的质谱鉴定情况,并探讨了不同肽段质谱响应改变的内在原因。发现赖氨酸在侧链能选择性地发生胍基化反应（其选择性达到96.8%）,转化为高精氨酸,碱性增强。因此在正离子质谱模式下,C端为赖氨酸的肽段产生了更多的y离子,提供了许多新的离子碎片信息。在鉴定结果中,此类肽段所占总肽段的比例由原来的51.7%上升为57.3%,并且有1015条新的肽段被检测到。对于不含有赖氨酸的肽段,其鉴定结果在胍基化前后基本没有变化。结果表明,胍基化可以在一定程度上提高质谱鉴定的灵敏度和互补性,提高蛋白质分析的覆盖率。     

等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定

分泌蛋白质组(secretome)是指在特定的时空条件下,细胞、组织等分泌的全部蛋白质。分泌蛋白质组可能包含了大量的疾病诊断生物标志物,因此其相关研究越来越受到重视。分泌蛋白质组的组成高度复杂且浓度范围宽,这对分析方法提出了挑战。建立有效的蛋白质或肽段预分离策略,将有利于分泌蛋白质的高覆盖率鉴定。本研究以肝癌细胞系MHCC97L的无血清培养分泌蛋白质为研究对象,采用一种新型等电聚焦预分离(OFFGEL)系统,考察了肽段水平的分级对蛋白质鉴定结果的影响。结果表明,分离后各馏分中肽段的等电点分布与理论预测基本一致,每个馏分中单独鉴定的肽段比例接近80%,显示了该系统对肽段的高分辨分离能力。结合生物质谱技术,在肝癌细胞分泌系统中鉴定了2995个蛋白质,显示了该系统在复杂体系蛋白质组研究中的应用潜力。     

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

Investigation of the pyrolysis products of methionine-enkephalin-Arg-Gly-Leu using liquid chromatography-tandem mass spectrometry

Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC-UV/Vis-MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides.     

Top-down approach of completely sequencing a 4.9 kDa recombinant peptide using quadrupole time-of-flight mass spectrometry

A recombinant peptide (near the C-terminal region of head involution defective protein) of 4.9 kDa has been completely sequenced and characterized using medium-resolution mass spectrometry (QTOF). The observed difference in the experimental mass and the theoretical mass is due to beta-mercaptoethanol adduct formation on the cysteine residue. The fragmentation pattern is correlated with the primary structure of the protein. Top-down sequencing of the peptide was extended to small proteins like barstar of mass 10.3 kDa.     

Prediction of product ion isotope ratios in the tandem electrospray ionization mass spectra from the second isotope of tryptic peptides: Identification of the variant <Emphasis Type="Italic">β</Emphasis>131 Gln→Glu,hemoglobin Camden

Many human hemoglobin variants occur in heterozygotes; that is, the variant and normal hemoglobins are present in the same sample. In a procedure for rapidly identifying such variants by mass spectrometry, mutations that increase the mass by 1 Da require a special approach. One of the steps in this procedure involves digesting the denatured hemoglobin with trypsin and analyzing the resulting peptide mixture by mass spectrometry to identify the mutant peptide. Generally the mutant peptide ion can then be selected as the precursor and sequenced by tandem mass spectrometry to identify or confirm the mutation. However, with heterozygotes in which the mass of the variant is 1 Da higher than normal, the first isotope of the mutant peptide occurs at essentially the same mass as the second isotope of the normal peptide, precluding analysis of the mutant peptide on its own. Product ions from the second isotope of a peptide are doublets, 1 Da apart. The way in which the relative abundance of the components in these doublets varies with the elemental composition of the product ions was predicted from the isotopic abundance of the elements and agreed well with experimental data. These results were applied to the identification of a variant that increases the mass by 1 Da in a heterozygote-that is, beta 131 Gln-->Glu, hemoglobin Camden.     

Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.     

Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.     

[M + Fe − 5H]2− peptide ion composition verified by Fourier transform mass spectrometry accurate mass and tandem mass spectrometry analyses

Previously, the unusual ion composition [M + Fe - 5H]2- had been proposed as the major species observed when a gamma-carboxy glutamate-containing glyco-peptide was analyzed with electrospray ionization in the negative ionization mode. The sequence assignment of this highly post-translationally modified peptide was based on the mass analysis using a quadrupole ion trap together with information from both Edman and DNA sequencing. Because there was little precedent for the loss of five protons from a ferric cationized peptide, we utilized Fourier transform mass spectrometry accurate mass and tandem mass spectrometry analyses to verify the peptide ion composition.     

A peptidomic approach for monitoring and characterising peptide cyanotoxins produced in Italian lakes by matrix-assisted laser desorption/ionisation and quadrupole time-of-flight mass spectrometry

In recent years, the occurrence of cyanobacterial blooms in eutrophic freshwaters has been described all over the world, including most European countries. Blooms of cyanobacteria may produce mixtures of toxic secondary metabolites, called cyanotoxins. Among these, the most studied are microcystins, a group of cyclic heptapeptides, because of their potent hepatotoxicity and activity as tumour promoters. Other peptide cyanotoxins have been described whose structure and toxicity have not been thoroughly studied. Herein we present a peptidomic approach aimed to characterise and quantify the peptide cyanotoxins produced in two Italian lakes, Averno and Albano. The procedure was based on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry mass spectrometry (MALDI-TOF-MS) analysis for rapid detection and profiling of the peptide mixture complexity, combined with liquid chromatography/electrospray ionisation quadrupole time-of- flight tandem mass spectrometry (LC/ESI-Q-TOF-MS/MS) which provided unambiguous structural identification of the main compounds, as well as accurate quantitative analysis of microcystins. In the case of Lake Averno, a novel variant of microcystin-RR and two novel anabaenopeptin variants (Anabaenopeptins B(1) and Anabaenopeptin F(1)), presenting homoarginine in place of the commonly found arginine, were detected and characterised. In Lake Albano, the peculiar peptide patterns in different years were compared, as an example of the potentiality of the peptidomic approach for fast screening analysis, prior to fine structural analysis and determination of cyanotoxins, which included six novel aeruginosin variants. This approach allows for wide range monitoring of cyanobacteria blooms, and to collect data for evaluating possible health risks to consumers, through the panel of the compounds produced along different years.     

Mass spectrometry in India

This review emphasizes the mass spectrometry research being performed at academic and established research institutions in India. It consists of three main parts covering the work done in organic, atomic and biological mass spectrometry. The review reveals that the use of mass spectrometry techniques started in the middle of the 20th century and was applied to research in the fields of organic, nuclear, geographical and atomic chemistry. Later, with the advent of soft and atmospheric ionization techniques it has been applied to pharmaceutical and biological research. In due course, several research centers with advanced mass spectrometry facilities have been established for specific areas of research such as gas-phase ion chemistry, ion-molecule reactions, proscribed chemicals, pesticide residues, pharmacokinetics, protein/peptide chemistry, nuclear chemistry, geochronological studies, archeology, petroleum industry, proteomics, lipidomics and metabolomics. Day-by-day the mass spectrometry centers/facilities in India have attracted young students for their doctoral research and other advanced research applications.     

Collision induced dissociation-based characterization of nucleotide peptides: Fragmentation patterns of microcin C7–C51, an antimicrobial peptide produced by <Emphasis Type="Italic">Escherichia coli</Emphasis>

Covalent protein-nucleic acid conjugates form an original class of compounds that occur in nature or can be generated in vitro through cross-linking to investigate domains involved in protein/nucleic acid interactions. Their mass spectrometry fragmentation patterns are poorly characterized. We have used electrospray-ionization mass spectrometry (ESI-MS) combined with collision-induced dissociation (CID) to characterize microcin C7-C51, an antimicrobial nucleotide peptide that targets aspartyl-tRNA synthetase and inhibits translation. The fragments of microcin C7-C51 were analyzed in positive- and negative-ion modes and compared with those of the corresponding unmodified heptapeptide and to the derived aspartyl-adenylate. The positive- and negative-ion mode fragments of microcin C7-C51 provided information on both the nucleotide and peptide moieties. Accurate mass measurement obtained using an LTQ Orbitrap instrument was a key factor for a comprehensive interpretation of the fragments. The experimental results obtained permitted the proposal of stepwise fragmentation pathways involving ion-dipole complexes. The data provide a better understanding of nucleotide peptide fragmentation in the gas phase.     

Contributions of mass spectrometry to structural immunology

Mass spectrometry has made important contributions to the field of immunology in the past decade. A variety of mass spectrometric-based techniques have been applied to study the structures of macromolecules that play a vital role in the immune response. These include traditional molecular mass measurements to identify post-translational modifications and structural heterogeneity, mass mapping of proteolysis products, sequencing by tandem mass spectrometry and conformational analysis. Antigen-antibody and other immune complexes have been detected by mass spectrometry, providing an avenue to study macromolecular assemblies that are important to immune function. By virtue of the ability of mass spectrometry based techniques to analyze complex biological mixtures, mass spectrometry has also been employed to identify and sequence protein epitopes important in both the humoral and cellular immune responses. This has been achieved through a combination of immunoaffinity and mass spectrometric techniques, and the coupling of high-performance chromatographs to mass spectrometers. These approaches are important for the identification of pathogens and show promise for the early diagnosis of disease associated with viral and bacterial infection and malignancy. These investigations will enable the mechanisms associated with normal and impaired immune function to be elucidated. Mass spectrometry has been utilized to characterize the structure of peptide mimics, multiple antigenic peptides and other constructs in the design of synthetic immunogens. Information derived from these studies will aid in the development of novel therapeutics and vaccines.