磷酸化肽段分离富集方法研究进展

目前磷酸化肽段鉴定主要依赖于质谱技术,但磷酸化肽段的低丰度性以及来自非磷酸化肽段的干扰等因素,影响质谱的分析与鉴定。因此质谱分析前磷酸化肽段的富集,是深入研究磷酸化蛋白质组学的先决条件。该文介绍了磷酸化蛋白质组学中传统的以及新建立的一些磷酸化肽段分离富集方法的原理及优缺点,这些方法包括固相金属离子亲和色谱法(IMAC)、金属氧化亲和色谱法(MOAC)、强阳/阴离子交换色谱法(SCX/SAX)、亲水相互作用色谱法(HILIC)、静电排斥亲水相互作用色谱法(ERLIC)、化学衍生法、MALDI靶盘富集法以及多种富集方法相结合。     

富集分离质谱检测磷酸化肽新材料与分析方法进展

本文对2005年以来,金属离子和金属氧化物亲和色谱材料及其他特别材料结合质谱检测,用于富集分离测定磷酸化肽及磷酸化蛋白的分析方法进行评述。引用文献119篇。     

混合模式色谱材料Click TE-GSH高效富集多磷酸化肽

多磷酸蛋白对于生物体适应内外环境具有重要意义,而明确多磷酸蛋白的磷酸位点功能及其信号转导机制尤为关键. 复杂生物样品中多磷酸化肽的低丰度、低电离的特性,以及非磷酸化肽的抑制作用,决定了质谱分析前进行多磷酸化肽富集是非常必要的步骤. 本工作采用基于巯基-烯烃点击化学法合成的混合模式材料Click TE-GSH进行单磷酸化肽和多磷酸化肽的选择性富集. 我们建立了单磷酸化肽、双磷酸化肽和多磷酸化肽的顺序分段富集方法. 该优化方法能抗干扰,应用于脱脂牛奶时富集到11条多磷酸化肽. 与商品化固化金属亲和色谱(IMAC)材料相比,Click TE-GSH富集多磷酸化肽的选择性更好. 本工作所建立的富集方法为高效富集多磷酸化肽提供新方法和新技术.     

磷酸化蛋白质组学中的分离富集方法研究进展

蛋白质的磷酸化是一种可逆性的翻译后修饰,在细胞的增值、分化、信号转导以及转录与翻译调控、蛋白质复合体的形成、蛋白质降解等方面发挥着极为重要的作用.因此磷酸化蛋白的鉴定成为翻译后修饰研究的重要内容.但由于磷酸化蛋白的丰度较低, 难以用质谱直接检测.为了解决这个问题,改善质谱对磷酸肽的信号响应, 需要对磷酸化蛋白质或磷酸肽进行富集.本文系统地介绍了磷酸化蛋白组学研究中应用较为广泛和最新建立的各种分离富集方法的原理、特点、应用研究进展,包括抗体富集法、激酶特异富集法、亲和富集法、化学修饰法、多种色谱分离富集方法以及MALDI靶盘富集法.     

季铵化磁性壳聚糖复合材料对磷酸化肽的富集研究

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一,蛋白质磷酸化的测定对于全面了解生物过程中的磷酸化途径至关重要。质谱技术是分析蛋白质磷酸化的重要手段,但磷酸化肽固有的低丰度、低电离效率以及与非磷酸化肽共存等特性严重影响质谱对其进行直接分析。为解决此问题,需在质谱分析前对磷酸化肽进行选择性富集。本研究制备了一种基于季铵化磁性壳聚糖的复合材料用于磷酸化肽的富集。此磁性材料具有快速的磁响应性、良好的生物相容性、正电性以及廉价易得等优点。采用β-酪蛋白作为模型蛋白质,结果表明此材料对磷酸化肽具有良好的富集选择性。经过富集后,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,方法的检出限为0.4 fmol。本方法被成功用于脱脂牛奶中磷酸化肽的检测,表明其对复杂样品中磷酸化肽的富集和检测具有良好的应用潜力。     

介孔氧化铁材料快速、高效富集分离磷酸化肽段和蛋白的新方法研究

以0.1μmol/Lβ-casein磷酸化蛋白酶解液为对象,利用在酸性条件下对PO43-能够特异性吸附的氧化铁材料为新载体,对介孔氧化铁材料富集分离磷酸化肽段的孵育液酸含量、孵育液有机溶剂含量和洗脱液选择的条件进行了优化,结果表明:室温条件下,在含有0.1%乙酸和30%乙腈的孵育液中孵育5min后,经1mol/LNH3.H2O溶液的洗脱,介孔氧化铁可有效地将磷酸化肽段从蛋白酶解液中富集分离。本方法也可以选择性地提取α-casein磷酸化蛋白,实现了简单、快速、高效的磷酸化肽段和蛋白的富集分离。同时,通过MALDI-TOF串级质谱分析,成功地完成了在优化条件下分离出的磷酸化肽段磷酸位点的鉴定。     

Ti-SBA-15介孔材料用于磷酸化肽的高效富集

结合基质辅助激光解吸飞行时间质谱(MALDI-TOF MS)检测技术,考察了Ti-SBA-15介孔材料对β-酪蛋白酶解产物中磷酸化肽的选择性富集性能。实验结果显示,含Ti和Si物质的量比为0.08的Ti-SBA-15介孔材料可选择性地对β-酪蛋白酶解产物中的磷酸化肽进行选择性富集;对于β-酪蛋白和牛血清白蛋白物质的量比为1:100的蛋白质酶解混合液,Ti-SBA-15仍能实现对其磷酸化肽的有效富集。上述结果表明,作为一种多孔、高比表面积的磷酸化多肽的选择性吸附材料,Ti-SBA-15有望在磷酸化蛋白质组的分析中得到广泛的应用。     

智能聚合物基材料富集磷酸化肽和糖肽的研究进展

翻译后修饰是蛋白质组学研究的前沿和重点,它不仅调节着蛋白质的折叠、状态、活性、定位以及蛋白质间的相互作用,也能帮助科学家更全面地了解生物体的生命过程,为疾病的预测、诊断和治疗提供更加强大的支撑和依据。翻译后修饰产物(例如磷酸化肽和糖肽)丰度很低,且存在着强烈的背景干扰,很难直接用质谱进行分析,因此迫切需要开发高效的富集材料和技术来选择性富集翻译后修饰产物。近年来,智能聚合物基材料通过外部物理、化学或生物刺激可逆地改变其结构和功能,实现对磷酸化肽和糖肽高度可控的吸附和脱附,进而衍生开发出一系列新颖的富集方法,极大地吸引研究者们的兴趣。一方面,智能聚合物基材料的响应变化包括材料疏水性的增加或减少、形状和形貌的改变、表面电荷的重新分布以及亲和配体的暴露或隐藏等特性。这些特性使得目标物和智能聚合物基材料之间的亲和力可以通过简单改变外部条件(如温度、pH值、溶剂极性和生物分子等)实现更可控和更智能的精细调节。另一方面,智能聚合物基材料为集成功能模块提供了便捷的可扩展平台,例如特定的识别组件,显著提高了目标物质的分离选择性。智能聚合物基材料在分离方面展现出巨大的潜力,这为蛋白质翻译后修饰产物的分析和研究带来了希望。围绕上述主题,该文依据Web of Science近20年来近50篇代表性文献,概述了智能聚合物基材料在磷酸化肽和糖肽分离及富集中的发展方向。     

血清磷酸化肽的分离富集和质谱定量及其作为潜在肿瘤标志物的评价

血清中磷酸化肽种类和浓度的变化既能反映人体内蛋白质水解酶活性的变化,又能反映蛋白质翻译后磷酸化的水平,业已成为肿瘤标志物寻找和发现的重要目标。因而,血清中磷酸化肽的鉴定及其定量分析在具有临床应用价值的肿瘤标志物的筛选与发现中起着重要作用。由于血清中的内源性磷酸化肽丰度极低,在质谱分析中的离子化效率不高,且受到来自高丰度非磷酸化肽和蛋白质的信号抑制及干扰,血清中磷酸化肽的质谱定量分析是分析化学研究中的一个巨大挑战。文章对血清磷酸化肽的分离富集、质谱定量分析及其作为肿瘤标志物的筛选和评价等3个方面的研究进展进行总结、评述,并展望该领域的未来研究趋势和应用前景。     

新型糖基化多肽富集材料的研究进展

糖基化是生物体内广泛存在的一类蛋白质翻译后修饰,其参与了生命活动的各种调控过程。鉴于糖基化修饰有着重要的生物学意义,近20年来,相关研究逐渐得到重视。因糖蛋白在生物样品中的相对丰度较低且分子结构复杂、离子化能力差,故如何高效地进行样品前处理成为糖蛋白组学研究中所必须面对的一个挑战性课题。除经典的亲水相互作用材料外,近10年来许多新型材料也逐渐被应用于糖肽富集。以材料基质而言,可大体分为壳聚糖材料、有机骨架材料、功能化聚合物材料等。本文总结了这些新兴材料的特点及富集效果,并展望了它们的应用前景。     

Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review

Enrichment, separation and mass spectrometric analysis of biomolecules carrying a phosphate group plays an important role in current analytical chemistry. Application areas range from the preparative enrichment of phospholipids for biotechnological purposes and the separation and purification of plasmid DNA or mRNA to the specific preconcentration of phosphoproteins and -peptides to facilitate their later identification and characterization by mass spectrometry. Most of the recent improvements in this field were triggered by the need for phosphopeptide enrichment technology for the analysis of cellular protein phosphorylation events with the help of liquid chromatography–mass spectrometry. The high sensitivity of mass spectrometry and the possibility to combine this technique with different separation modes in liquid chromatography have made it the method of choice for proteome analysis. However, in the case of phosphoprotein analysis, the low abundance of the resulting phosphopeptides and their low quality fragment spectra interfere with the identification of phosphorylation events. Recent developments in phosphopeptide enrichment and fragmentation technologies successfully helped to overcome these limitations. In this review, we will focus on sample preparation techniques in the field of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules.     

Preparation of Cerium Dioxide Functionalized Magnetic Layered Double Hydroxides for High-efficiency Phosphopeptide Enrichment

In this work, we prepared a material with magnetic nanoparticles (Fe₃O₄) as core, layered double hydroxides(LDHs) as affinity shell, and cerium dioxide(CeO₂) as functional molecules(denoted as Fe₃O₄@LDH-CeO₂). On the basis of combined immobilized metal ion affinity chromatography(IMAC) and metal oxide affinity chromatography(MOAC), Fe₃O₄@LDH-CeO₂ was used to enrich phosphopeptides with high efficiency. The material exhibited high selectivity(α-casein:β-casein:BSA=1:1:5000, mass ratio), high recovery(95.87%), and good reusability of 10 times adsorption- desorption experiments. The feasibility of Fe₃O₄@LDH-CeO₂ was further investigated by extracting phosphopeptides from biological samples(nonfat milk, serum, saliva, and A549 cell lysate).     

Phosphopeptide enrichment by IEF

In our efforts to improve the identification of phosphopeptides by MS we have used peptide IEF on IPG strips. Phosphopeptides derived from trypsin digests of single proteins as well as complex cellular protein mixtures can be enriched by IEF and recovered in excellent yields at the acidic end of an IPG strip. IPG peptide fractionation in combination with MS/MS analysis has allowed us to identify phosphopeptides from tryptic digests of a cellular protein extract.     

基于离心式富集装置的酪氨酸磷酸肽分析新方法

酪氨酸磷酸化及其相应激酶活性的研究在抗肿瘤药物靶点的研发中具有重要意义.由于酪氨酸磷酸化仅占蛋白质总磷酸化含量的不足0.1%,因此规模化的酪氨酸磷酸化鉴定面临着重大技术挑战.本研究构建了TiO2串联C18反相填料的离心式富集装置,结合抗体免疫沉淀法,建立了酪氨酸磷酸肽的富集策略.此新型富集装置由吸头、适配器和离心(EP)管组成,将TiO2富集磷酸肽和C18填料反相分离磷酸肽有机结合,以离心的方式进行样品的上样、清洗、洗脱和分离,再通过抗酪氨酸磷酸化抗体进一步特异性富集酪氨酸磷酸肽,从而实现了酪氨酸磷酸肽的高效富集和大规模质谱鉴定.通过离心式富集装置简化了实验步骤,减少了样品损失和人为因素干扰;而且离心式、平行化的样品处理方式可显著提高分析通量.将此策略成功用于小鼠肝脏蛋白质酪氨酸磷酸化肽段的富集和质谱鉴定,在5 mg鼠肝蛋白中共鉴定出967个酪氨酸磷酸化位点,对应545个蛋白质,显示了其在蛋白质组学研究中的应用潜力.     

Matrix interference free determination of perchlorate in urine by ion association-ion chromatography-mass spectrometry

Quantitative measurement of perchlorate in biological fluids is of importance to assess its toxicity and to study its effects on the thyroid gland. Whenever possible, urine samples are preferred in toxicologic/epidemiologic studies because sample collection is non-invasive. We present here a pretreatment method for the determination of perchlorate in urine samples that lead to a clean matrix. Urine samples, spiked with isotopically labeled perchlorate, are exposed to UV to destroy/decompose organic molecules and then sequentially treated with an H⁺-form cation exchange resin to remove protolyzable compounds, with ammonia to raise the pH to 10-11 and finally passed through a mini-column of basic alumina to remove the color and other organic matter. After filtration through a 0.45 μm syringe filter, the sample thus prepared can be directly injected into an ion chromatograph (IC). We use ion association-electrospray ionization-mass spectrometry (ESI-MS) to detect and quantify perchlorate. The proposed sample preparation method leads to excellent limits of detection (LOD's) for perchlorate since there is essentially no dilution of sample and the matrix effects are eliminated. Results of urine samples from both men and women volunteers are reported for perchlorate, as well as for iodide and thiocyanate, which are generally present at much higher concentrations and for which a “dilute and shoot” approach is adequate. The limit of detection (S/N = 3) for iodide, thiocyanate and perchlorate by the present method was 0.40, 0.10 and 0.080 μg l⁻¹, respectively.     

LC-MS analysis in the aquatic environment and in water treatment – a critical review

LC-MS has become an invaluable technique for trace analysis of polar compounds in aqueous samples of the environment and in water treatment. LC-MS is of particular importance due to the impetus it has provided for research into the occurrence and fate of polar contaminants, and of their even more polar transformation products. Mass spectrometric detection and identification is most widely used in combination with sample preconcentration, chromatographic separation and atmospheric pressure ionization (API). The focus of the first part of this review is directed particularly toward instruments and method development with respect to their applications for detecting emerging contaminants, microorganisms and humic substances (HS). The current status and future perspectives of 1) mass analyzers, 2) ionization techniques to interface liquid chromatography (LC) with mass spectrometry (MS), 3) methods for preconcentration and separation with respect to their application for water analysis are discussed and examples of applications are given. Quadrupole and ion trap mass analyzers with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are already applied in routine analysis. Time-of-flight (TOF) mass spectrometers are of particular interest for accurate mass measurements for identification of unknowns. For non-polar compounds, different ionization approaches have been described, such as atmospheric pressure photoionization (APPI), electrochemistry with ESI, or electron capture ionization with APCI. In sample preconcentration and separation, solid phase extraction (SPE) with different non-selective sorbent materials and HPLC on reversed-phase materials (RP-HPLC) play the dominant role. In addition, various on-line and miniaturized approaches for sample extraction and sample introduction into the MS have been used. Ion chromatography (IC), size-exclusion chromatography (SEC), and capillary electrophoresis (CE) are alternative separation techniques. Furthermore, the issues of compound identification, matrix effects on quantitation, development of mass spectral libraries and the topic of connecting analysis and toxicity bioassays are addressed.     

Urine sample preparation for proteomic analysis

Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross‐reactions that should be taken into consideration. The incorporation of new post‐translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery.     

Optimization of a rapid capillary electrophoresis ESI-IT tandem mass spectrometry method for the analysis of short-chain carnitines in human plasma

A capillary electrophoresis (CE) method coupled to electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) is described for the rapid analysis of carnitine, acetylcarnitine, and propionylcarnitine in human plasma. Optimization of the procedure was achieved by a reduced sample pretreatment and after examining several physicochemical parameters that influence both the CE separation and the MS analytes detection. The analysis of total carnitine in human plasma after hydrolysis of short-chain metabolites is also shown. The analysis of carnitine and metabolites was obtained in less than 10 min using a 200 mM ammonium formate buffer, pH 2.5, with high sensitivity and specificity using the MS detection in product ion scan mode. The method was tested for quantitative recovery using dialyzed human plasma as matrix and showed linearity in the concentrations ranges 20–160, 1–32, and 0.25–8 μM for carnitine, acetylcarnitine, and propionylcarnitine with (squared) correlation coefficients of 0.9984, 0.9995, and 0.9991, respectively. The intraday and intermediate analysis repeatability and accuracy are within 15% of relative standard deviation (RSD) at low, medium, and high concentration and within/or slight exceeding 20% at the lower limit of quantitation (LLOQ). The method is sensitive for determining carnitine and its metabolites in human plasma with high specificity.     

磁性纳米材料在样品前处理中的应用进展

磁性纳米材料作为一种新型功能复合材料,因其具有吸附能力强、表面可修饰、易分离和良好的生物相容性等特点,已广泛应用于生物传感器、药物传导和医学成像方面。由于磁性纳米材料分离速度快且吸附性能好,因此在分析化学样品前处理中的应用也日益受到人们的关注。本文简略介绍了磁性纳米材料的特性、分类及制备方法,综述了磁性纳米材料在分离和富集生物大分子、有机物和无机物中的应用,并展望了磁性纳米材料的应用前景。     

环境水样及固相样品中全氟化合物分析方法研究进展

全氟化合物(Perfluorinated compounds,PFCs)是一类持久性有机污染物,在环境中普遍检出,严重危害人体健康,已成为环境科学和毒理学研究的热点之一.PFCs含有较多同系物及同分异构体,且在环境中通常为痕量水平(ng/g或μg/L),迫切需要建立灵敏可靠的样品前处理和检测技术.国内外已针对环境样品基质中痕量PFCs的检测开展了大量研究,但有关复杂环境样品中PFCs前处理方法、分析方法、基质效应等方面的系统评述还相对较少.本文综述了环境样品(水、沉积物/污泥、土壤、植物)中PFCs的前处理方法、检测方法(尤其是同分异构体)及环境样品基质效应对其检测的影响,以期为相关研究提供参考.