磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程,在细胞中磷蛋白含量低,磷酸化位点可变,且磷酸肽的质谱信号常常会受到抑制,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前,质谱仍然是核心的鉴定技术,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

基于高覆盖(磷酸化)蛋白质组对人胚胎干细胞干性维持调控网络的解析

目的:对人胚胎干细胞H9的(磷酸化)蛋白质组进行鉴定和深入分析,探讨维持人胚胎干细胞干性的核心调控网络。方法:利用新近发表的TAFT磷酸化肽段富集策略和高精度质谱鉴定技术,采用无标定量方法对H9细胞(磷酸化)蛋白进行定量分析;结合MOTIFX、iGPS、IPA等多种生物信息学分析软件,分析H9细胞的磷酸化基序-激酶、转录因子-靶基因调控网络。结果:获得了目前高度覆盖的H9细胞(磷酸化)蛋白质组数据集,鉴定到8674种蛋白质,其中3898种能够发生磷酸化修饰,包括13 676条磷酸化肽段,高可信度(class 1)磷酸化位点有11 870种。与已有的H9细胞磷酸化蛋白质组数据相比,其中2247种磷酸化蛋白质和10 025种磷酸化位点是本研究新鉴定到的。基于基序和激酶预测分析,推导出与干性维持相关的已知转录调节因子的新的磷酸化修饰基序以及调控其发生磷酸化的激酶。结合多能性转录因子对靶基因的调控信息,构建了多能性调控分子磷酸化修饰及其转录调控的核心网络。此外,还对特定位点的磷酸化修饰水平及其对应蛋白的总体丰度水平进行了比较及功能分析。基于各自的百分位数,比较磷酸化修饰水平与其对应蛋白的总体丰度,发现具有高丰度但低磷酸化修饰水平的蛋白倾向于参与物质转换或物质运输等生物过程,而低丰度但高磷酸化水平的蛋白质倾向于参与信息转导或调控过程。结论:提供了人胚胎干细胞H9高覆盖的(磷酸化)蛋白质组数据,这些数据有助于深入了解蛋白质磷酸化修饰在胚胎干细胞干性维持中所发挥的重要作用,为胚胎干细胞基础和应用研究提供了宝贵的数据资源。     

蛋白质磷酸化修饰的研究进展

蛋白质磷酸化是最常见、最重要的一种蛋白质翻译后修饰方式,它参与和调控生物体内的许多生命活动。通过蛋白质的磷酸化与去磷酸化,调控信号转导、基因表达、细胞周期等诸多细胞过程。随着蛋白质组学技术的发展和应用,蛋白质磷酸化的研究越来越受到广泛的重视。我们介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白质分析样品的富集及制备、磷酸化蛋白的鉴定及磷酸化位点的预测、蛋白分离后磷酸化蛋白的检测,及蛋白质磷酸化的分子机制,并综述了近年来国内外的主要相关研究进展。     

磷酸化蛋白质组研究中的富集策略

蛋白质的磷酸化与去磷酸化过程,调控着包括信号转换、基因表达、细胞周期等诸多细胞过程。因此,对蛋白质磷酸化修饰的分析是蛋白质组研究中的重要内容。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。目前主要的富集方法包括免疫沉淀、固相金属离子亲和色谱、金属氧化物/氢氧化物亲和色谱等。     

代谢性标记结合二维电泳寻找O-糖基化蛋白的研究

蛋白质的O-糖基化(O-glycosylation)是一种重要的翻译后修饰,参与诸多生理和病理过程。目前,对于蛋白质的O-糖基化的研究进展仍非常缓慢,一个重要的原因就是缺乏高效的对O-糖基化蛋白进行分离和鉴定的技术。创新性地将点击化学反应、二维电泳和质谱技术结合,对寻找细胞内O-糖基化蛋白进行了技术探索性研究。首先利用代谢性标记手段,在人肝癌细胞HCCLM6培养基中加入四乙酰化叠氮半乳糖胺(Ac4GalNAz),对细胞内O-GalNAc糖基化蛋白进行标记;其次通过点击化学将炔基荧光基团连接至标记的O-糖基化蛋白的叠氮基团;应用二维电泳技术对标记蛋白进行分离,并找到13个具有荧光信号的蛋白点;最后对具有荧光信号的蛋白进行质谱鉴定,成功鉴定到7种蛋白,经软件预测后,GRP78蛋白和ANXA1蛋白均具有潜在的O-糖基化位点。这为寻找细胞或生物体中的O-糖基化蛋白奠定了基础,并为高通量筛选O-糖基化蛋白提供技术平台。     

同源异型框蛋白Msx1的两个新的磷酸化位点的鉴定

同源异型框蛋白Msx1的同系物Msx2中已鉴定出磷酸化修饰位点,并且发挥着重要的生物学功能。但是对Msx1的磷酸化修饰情况还不清楚,为了对Msx1的磷酸化修饰情况进行研究。利用生物信息学的方法对Msx1的磷酸化修饰位点进行预测,进一步利用免疫沉淀和质谱技术对其进行具体实验验证。C2C12细胞中的Msx1蛋白复合物经胰蛋白酶消化后,通过高效液相色谱和质谱技术进行分离和分析。结果发现两个新的磷酸化修饰位点,分别是152位的丝氨酸和160位的丝氨酸,而且两个磷酸化位点在人和小鼠中是高度保守的。进一步,制备针对这两个磷酸化位点的特异性磷酸化抗体。磷酸化位点的鉴定以及特异性抗体的获得为进一步研究Msx1的生物学功能提供了良好的条件。     

质谱技术解析磷酸化蛋白质组

蛋白质磷酸化是生物体内存在的一种普遍的调节方式,在细胞信号传递中占有极重要的地位.质谱已逐渐被人们认为是挑战这一领域的有利工具.综述了目前利用质谱技术分析磷酸化蛋白质的方法,包括利用固定化的金属亲和层析柱、抗体和化学标签技术富集目的分子,肽片段质量图和前体离子扫描（precusor ion scans）等技术检测磷酸化肽段,串联质谱对磷酸化肽段测序鉴定磷酸化位点,以及引入质量标签对蛋白质的磷酸化水平进行定量等.虽然现在已经有很多可行的方法用于分析磷酸化蛋白质,但要达到从少量生物样品中解析其全部磷酸化蛋白质仍需要有很多技术上的突破.     

蛋白质O-GlcNAc糖基化修饰对tau蛋白磷酸化修饰的影响

蛋白质的O位N-乙酰葡萄糖胺(O-GlcNAc)糖基化修饰是一种新近发现的广泛存在于细胞核蛋白与细胞浆蛋白的蛋白质翻译后修饰.其性质与经典的膜蛋白和分泌蛋白的糖基化修饰不同,而与蛋白质磷酸化修饰更相似.O-GlcNAc糖基化和磷酸化均修饰tau蛋白的丝氨酸和苏氨酸残基,通过改变O-GlcNAc糖基化供体底物浓度以及其关键酶活性等方法,改变分化后成神经细胞样的PC12细胞中的蛋白质O-GlcNAc糖基化修饰水平,然后用特异性识别不同位点磷酸化的tau蛋白抗体,进行蛋白质印迹分析来检测tau蛋白磷酸化水平的变化.结果发现细胞内蛋白质O-GlcNAc糖基化对tau蛋白磷酸化的影响,在不同的磷酸化位点其影响不同.增加蛋白质O-GlcNAc糖基化修饰导致tau蛋白大多数磷酸位点的磷酸化水平降低,反之亦然.这些结果说明,tau磷酸化在大多数位点受到O-GlcNAc糖基化修饰的负性调节.这一研究为阐明调节tau蛋白磷酸化水平的机理和阿尔茨海默病脑中tau异常过度磷酸化的分子机制提供了新的线索.     

有丝分裂期MPM-2磷蛋白家族及其调控因子

由于有丝分裂磷蛋白单克隆抗体(mitotic phosphoprotein monoclonal antibogy-2,MPM-2)识别很大一组在M期特异磷酸化的蛋白质,因而引起研究者的极大兴趣。该抗体亦成为研究M期功能性磷蛋白的重要工具。多个MPM-2磷蛋白已经定位在各种有丝分裂装置上,如中心体、着丝粒、纺锤体、染色体骨架及中体等,显示MPM-2磷蛋白在调控有丝分裂进程方面所扮演的重要角色。结合作者的工作和这一领域的相关研究,概述了MPM-2磷蛋白的细胞周期分布特点及其在细胞有丝分裂期进程中的重要功能,重点介绍MPM-2磷酸化位点的研究状况以及到目前为止已鉴定的MPM-2磷蛋白家族成员的基本情况,并总结了MPM-2磷蛋白相关调控因子的研究进展。     

生物质谱在细胞信号转导研究中的应用

近几年快速发展起来的生物质谱技术 ,依靠 (酶解后肽段 )精确质量数测定和随机肽序列标签分析 ,实现了对蛋白质高通量的鉴定 ,并被成功地用于蛋白质相互作用和蛋白质磷酸化等翻译后修饰研究。与传统的研究手段相比 ,上述技术能够在一次实验中对多信号通路中所有磷酸化的蛋白质分子及其磷酸化位点进行鉴定 ,已成为蛋白质组学最新发展中令人关注的一个热点。简要综述质谱技术应用于上述工作中的 3种策略     

Characterization of a TiO₂ enrichment method for label-free quantitative phosphoproteomics

Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates. Although different techniques to enrich for phosphopeptides have been reported, there are limited data on their suitability for direct quantitative analysis by MS. Here we report a TiO₂ based enrichment method compatible with large-scale and label-free quantitative analysis by LC–MS/MS. Starting with just 500 μg of protein, the technique reproducibly isolated hundreds of peptides, >85% of which were phosphorylated. These results were obtained by using relatively short LC–MS/MS gradient runs (45 min) and without any previous separation step. In order to characterize the performance of the method for quantitative analyses, we employed label-free LC–MS/MS using extracted ion chromatograms as the quantitative readout. After normalization, phosphopeptides were quantified with good precision (coefficient of variation was 20% on average, n = 900 phosphopeptides), linearity (correlation coefficients >0.98) and accuracy (deviations <20%). Thus, phosphopeptide ion signals correlated with the concentration of the respective phosphopeptide in samples, making the approach suitable for in-depth relative quantification of phosphorylation by label-free LC–MS/MS.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Integrating titania enrichment,iTRAQ labeling,and Orbitrap CID‐HCD for global identification and quantitative analysis of phosphopeptides

Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site‐specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong‐cation exchange, the peptides were analyzed by LC‐MS/MS on an Orbitrap mass spectrometer, which collects CID and high‐energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.     

生物质谱结合IMAC亲和提取和磷酸酶水解分析蛋白质磷酸化修饰

蛋白质磷酸化是生物体内非常重要的翻译后修饰方式 ,对磷酸化蛋白质的分析及磷酸化位点的确定有助于理解与其相关的生物功能。基质辅助激光解吸 /电离飞行时间质谱和电喷雾 四极杆 飞行时间质谱这两种生物质谱仪在蛋白质鉴定和翻译后修饰分析中发挥着重要作用。固相金属亲和色谱可选择性亲和提取肽混合物中的磷酸肽 ,结合磷酸酶水解实验和基质辅助激光解吸 /电离飞行时间质谱分析可确定肽混合物中的磷酸肽 ,最后用电喷雾 四极杆 飞行时间串联质谱分析磷酸肽的序列 ,结合数据库检索确定磷酸化位点。     

Methodologies for characterizing phosphoproteins by mass spectrometry

Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and "top-down" proteomics to phosphoprotein analysis.     

Methodologies for Characterizing Phosphoproteins by Mass Spectrometry

Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and “top-down” proteomics to phosphoprotein analysis.     

Enhanced detection and identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS

The analysis of PTMs such as phosphorylation has become an important field in MS because they can directly indicate protein states and interactions. Whereas the characterization of singly and doubly phosphorylated peptides has almost become routine, identifying phosphorylation events at multiple residues within a small region of a protein is still problematic. The identification of multiple modifications can be further hampered by low sequence information due to multiple neutral losses from phosphorylated side chains. Here we present a strategy for the analysis of complex phosphopeptides that combines peptide enrichment by titanium dioxide, separation by RP separation on monolithic columns and MS using high energy HE-CAD in a MALDI TOF/TOF analyser. Using synthetic phosphopeptides our approach is compared to multistage activation (MSA) MS/MS and the recently described electron transfer dissociation (ETD) method using an ESI-LTQ mass spectrometer.     

Catch me if you can: mass spectrometry-based phosphoproteomics and quantification strategies

Phosphorylation of proteins is one of the most prominent PTMs and for instance a key regulator of signal transduction. In order to improve our understanding of cellular phosphorylation events, considerable effort has been devoted to improving the analysis of phosphorylation by MS-based proteomics. Different enrichment strategies for phosphorylated peptides/proteins, such as immunoaffinity chromatography (IMAC) or titanium dioxide, have been established and constantly optimized for subsequent MS analysis. Concurrently, specific MS techniques were developed for more confident identification and phosphorylation site localization. In addition, more attention is paid to the LC-MS instrumentation to avoid premature loss of phosphorylated peptides within the analytical system. Despite major advances in all of these fields, the analysis of phosphopeptides still remains far from being routine in proteomics. However, to reveal cellular regulation by phosphorylation events, not only qualitative information about the phosphorylation status of proteins but also, in particular, quantitative information about distinct changes in phosphorylation patterns upon specific stimulation is mandatory. Thus, yielded insights are of outstanding importance for the emerging field of systems biology. In this review, we will give an insight into the historical development of phosphoproteome analysis and discuss its recent progress particularly regarding phosphopeptide quantification and assessment of phosphorylation stoichiometry.