色谱技术分离蛋白酶解和蛋白/多肽的研究进展

本综述点评了最近十年应用色谱技术解决蛋白酶解和蛋白/多肽分离的一些最新进展,涉及的研究工作大多来自中国科学家的报道.所评论的研究进展主要包括有微酶解反应器技术、先分离蛋白酶解混合肽段的bottom-up技术和先分蛋白再分离酶解后肽段的top-down技术.简而言之,微酶解反应器的新技术能够将传统的几小时的酶解时间缩短到几秒钟;而多维多模式或阵列色谱分离的新技术可以非常有效地分离蛋白质组混合物.这些新技术的进步很大程度上促进了蛋白质组学中蛋白/多肽的分离研究.     

新型亲水聚合物修饰硅胶颗粒固定化酶的制备及在蛋白质组鉴定中的应用

采用原子转移自由基聚合法制备了亲水聚合物修饰的硅胶颗粒作为一种新型固定化酶载体,在实现胰蛋白酶高密度固定的同时,显著降低了载体材料非特异性吸附导致的样品损失。因此,该固定化酶材料兼具高酶解效率和高回收率的特性。以标准蛋白质牛血清白蛋白(BSA)为样本,使用该固定化酶1 min即可完成酶解,鉴定到肽段对BSA的氨基酸序列覆盖率可达90%以上。该固定化酶材料成功应用于酵母菌全蛋白质复杂样本的酶解,从3 min酶解产物中鉴定到666个蛋白质,超过同样条件下溶液酶解12 h的鉴定结果。     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

苯基桥键型介孔材料的制备与表征

以1,4-二(三乙氧基硅基)-苯为硅源,聚氧乙烯-聚氧丙烯-聚氧乙烯三嵌段共聚物为模板剂,十六烷基三甲基溴化铵为共模板剂,乙醇为共溶剂,在酸性条件下合成了球形的苯基桥键型有序介孔材料。X射线衍射和透射电镜表征结果表明,该材料具有有序的二维六方相介观结构;傅立叶红外变换、13C和29S i固体核磁共振表征证实硅胶骨架中成功引入了苯基桥键,且在合成和模板移除过程中未发生S i—C键断裂;元素分析表明材料含碳量为34%~39%;热重分析说明材料稳定温度可达300℃;氮气吸附脱附揭示了材料有较高的比表面积(500~600 m2/g)和窄的孔径分布(3.21~3.95 nm)。将该苯基材料不经化学改性直接用作反相高效液相色谱固定相,并与商品键合硅胶苯基色谱柱比较,发现桥键型苯基材料对芳香类化合物具有很好的分离选择性,残留硅羟基明显减少,作为一种新的液相色谱填料具有很好的应用前景。     

集成化蛋白质组定量分析平台的构建及应用

为提高蛋白质组定量分析的准确度、通量和自动化程度,构建了由微升级混合离子交换色谱、亲水型固定化酶反应器（hIMER）和纳升级反相色谱-电喷雾串级质谱（nanoRPLC-ESI-MS/MS）组成的集成化蛋白质定量分析平台。该平台实现了二甲基化标记蛋白质样品在线分离、酶解、肽段分离鉴定和定量分析。采用质量比为1：1的轻、重标记的蛋白质样品考察该平台的定量性能,发现蛋白质水平二甲基化标记效率为90%;蛋白质经hIMER在线酶解10 min产生的漏切及酶解产物在hIMER柱上的非特异性吸附对定量准确度的影响较小,所有定量到的重/轻标记的蛋白质质量比的平均值为1.01。最后将该平台应用于小鼠腹水型肝癌淋巴道高、低转移细胞系差异蛋白质的分析,发现了12种蛋白质在高转移细胞系中低表达,15种蛋白质在高转移细胞系中高表达。以上结果证明了该平台可以实现高准确度和高通量的蛋白质组定量分析。     

介孔材料MCFs的合成及组装青霉素酰化酶的性质研究

介孔材料由于具有纳米级规则孔道和巨大的比表面积而在催化、吸附及分离等方面存在较大的应用价值．近年来,由介孔分子筛如MCM-41和SBA-15州等组装功能性材料已成为研究的热点．酶作为高效催化剂有许多优点,但在溶液中易失活,使用后无法回收,有的酶在溶液中还存在自水解问题：将酶组装在介孔材料中制成固定化酶则可解决上述问题．目前已成功地将辣根过氧化物酶     

基于温敏型材料的固定化酶的制备及其在蛋白质组快速分析中的应用

蛋白质组学通过规模化鉴定、分析从细胞、组织或有机体中提取的蛋白质,从而获得蛋白表达、修饰、组成和定量的变化信息。在目前最为有效的“鸟枪法”蛋白质组学策略中,固定化酶试剂基质常用固相载体材料,该固定化酶试剂在酶解蛋白质时为异相体系,存在固液界面传质阻力和空间位阻,限制了酶解效率和样品处理通量。针对这一技术瓶颈,本研究利用温敏聚合物对外界温度变化的响应能力,制备了一种新型的基于可溶性温敏聚合物的固定化胰蛋白酶试剂。该固定化酶特有的温度敏感特性,使其具有“高温均相酶解,低温异相分离”的特色,且兼具酶切时间显著缩短、酶可重复利用的优势。 BSA 1 min固定化酶解产物肽段的氨基酸序列覆盖率可达94%,高于传统溶液酶解12 h所得覆盖率为(74%)。进一步将该固定化酶试剂应用于HeLa细胞全蛋白质组的酶解,其酶解效果与相同条件下溶液酶解12 h相当。该固定化酶试剂对复杂蛋白质的快速、高效酶解充分证明其在蛋白质组学研究中的应用潜力。     

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

中空介孔硅铝球形分子筛的制备及催化裂解性能

利用沸石前驱体溶液和介孔硅球(MSS)为原料,通过水热法成功制备了具有中强酸性介孔壳的中空介孔硅铝球形分子筛(HMAS)。利用透射电镜(TEM)、扫描电镜(SEM)、X射线衍射(XRD)、N2-吸脱附、27Al核磁共振(27Al NMR)及NH3程序升温脱附(NH3-TPD)对材料的结构和性能进行了表征。研究结果表明,在MSS的中空过程中伴随有物质再分配和介孔结构的逐渐演变。MSS介孔孔道中的十六烷基三甲基溴化铵(CTAB)分子,一方面保护MSS免遭强碱性沸石前驱体溶液的溶蚀,另一方面作为形成HMAS介孔壳层的模板剂。在此CTAB分子的作用下,沸石前驱体结构单元被引入到HMAS的介孔球壳上。所得材料具有介孔结构和中强酸性,在催化裂解1,3,5-三异丙苯反应中表现出优异的催化性能。     

毛细管整体柱在线二维分离系统应用于人体软骨的蛋白质组分析

将已建立的 7 cm 柱长的磷酸基团强阳离子交换富集整体柱与85 cm柱长的C12烷基反相整体柱结合的在线二维分离平台应用于软骨提取蛋白的蛋白质组分析。对20 μg软骨提取蛋白的酶解产物进行14个盐梯度的分级,然后对14个馏分进行反相色谱梯度分离及串联质谱鉴定,成功地鉴定得到了7434个独立肽段对应的1901个非冗余蛋白质。对所鉴定到的蛋白质进行定位分类,结果表明鉴定到的大部分蛋白质是来自于软骨细胞内部的低丰度蛋白质,这对于许多关节类疾病的研究有重要意义。     

An analysis of protein abundance suppression in data dependent liquid chromatography and tandem mass spectrometry with tryptic peptide mixtures of five known proteins

Reverse phase liquid chromatography (RPLC) has been widely used in proteomics research for peptide separation. When protein samples are separated by RPLC and identified with electrospray ion trap mass spectrometry (ESI-MS), the signals of high-abundance proteins may suppress those of low-abundance proteins, a phenomenon known as abundance suppression. To what degree the abundance suppression correlates to the number of tryptic peptides in the high-abundance proteins has not been carefully investigated. We tried to answer this question by studying the mixtures digested from five known proteins. The numbers of identified tryptic peptides (longer than five amino acids) of the five proteins ranged from 12 to 47. Four different peptide mixtures with 10- to 100-fold abundance differences of five known proteins were separated by RPLC and identified by ESI-MS. Our results showed that abundance suppression was related to the tryptic peptide numbers in the high-abundance protein. Within a 100-fold protein abundance difference range, tryptic peptide number in the low-abundance proteins could be suppressed up to seven times by high-abundance proteins. The procedure we suggest here can help to identify low-abundance proteins co-purified with their high-abundance binding protein. The result can also help to identify specific high-abundance proteins for removal by immunoaffinity.     

Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography

This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange. identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2'-dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol (pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species between the two samples. Peptides obtained from proteins that were up-regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

Purification of RhlFN- in Bacteria Bodies with Acetic Acid-Water as Mobile Phase in RPLC

The extract of E.Coli containing recombinant human intcrferon-(rhIFN-)with 7.0 mol/L guanidine hydrochloride(GullCl)w a S directly injeeted into a column of reverse phase liquid chromatography(RPLC)to scparatc and purify rhIFN- with acetic acid-water as mobile phase. GuH Cl and most impure proteins can be scparatcd by this way.Compared with the usual dilution method,the bioactivity recovery of the purified rhIFN- was found to be over 500%.In addition, compared to common organic solvents cmploycd in RPLC,acetic acid has higher freezing point, and therefore,it is easy to concentrate the aim-irotcin by frcczc-drying when acetic acid-water is used as mobile phase in RPLC.     

Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.     

尺寸排阻-反相液相色谱-质谱联用技术在大鼠肾脏翻译后修饰蛋白质鉴定中的应用

LC-MS联用技术在蛋白质组学研究中具有重要的作用,但是在复杂的生物体系中,由于样品的高度复杂性和其中蛋白质含量的巨大差异,执行全面且无倾向的蛋白质组分析是一项挑战。因此,在液相色谱分离中采用基于不同原理的色谱分离方法来降低蛋白质样本的复杂度,并对微量蛋白质进行富集,对后续采用质谱方法进行信息的采集和深入分析至关重要。在这里我们开发了一种基于尺寸排阻色谱(SEC)与反相液相色谱(RPLC)结合的新方法来进行复杂体系蛋白质的分离和鉴定,特别是对于微量蛋白质的分析。首先使用SEC对蛋白质进行分离和富集,并酶解成多肽,再通过RPLC-MS联用的方法对酶解后的多肽进行分离和鉴定。结果显示使用上述方法可以有效降低蛋白质样本的复杂度,并有效提高微量蛋白质的鉴定能力,可从大鼠肾脏鉴定出23621个肽段及1345个蛋白质,比常规的二维强阳离子交换-反相液相色谱法(2D SCX-RPLC)鉴定到的肽段及蛋白质分别多出69%及27%。此外,该方法对肾脏翻译后修饰(PTM)蛋白质的鉴定显示出更多的优势,翻译后修饰的多肽鉴定率显著增加,特别是磷酸化肽段的鉴定效率可达到靶向富集策略的水平。在此展示的SEC-RPLC-MS可以更好地了解蛋白质翻译后修饰对肾脏的影响,最终将有助于增加我们对正常的生理性肾功能以及病理过程机制的理解。     

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.     

Development and validation of rapid ion-pair RPLC method for simultaneous determination of certain B-complex vitamins along with vitamin C

A rapid, simple, and accurate ion-pair RPLC method has been developed for simultaneous analysis of vitamin C and major B-complex vitamins. An RP C18 column thermostated at 30 degrees C was used with gradient elution of mobile phase comprising 10 mM potassium dihydrogen phosphate buffer (containing 3 mM sodium hexane-1-sulfonate, adjusted to pH 2.80 with o-phosphoric acid) and methanol at a flow rate of 1.0 mL/min to achieve the best possible separation and resolution of all vitamins in about 11.00 min. The detection was performed at 274 nm. The method has been implemented successfully for simultaneous determination of vitamins present in 12 multivitamin/multimineral pharmaceutical preparations, as well as in human urine. Typical validation characteristics were evaluated in accordance with International Conference on Harmonization guidelines. Good linearity over the investigated concentration levels was observed. Intraday repeatability was < or = 2.0%, and interday variation was < or = 2.6%, for all vitamins. The method can be used for assay of these vitamins over a wide concentration range with good precision and accuracy; hence, it would be appropriate for routine QC as well as in clinical analysis.     

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.     

Polarity‐based fractionation in proteomics: hydrophilic interaction vs reversed‐phase liquid chromatography

During recent decades, hydrophilic interaction liquid chromatography (HILIC) ahs been introduced to fractionate or purify especially polar solutes such as peptides and proteins while reversed‐phase liquid chromatography (RPLC) is also a common strategy. RPLC is also a common dimension in multidimensional chromatography. In this study, the potential of HILIC vs RPLC chromatography was compared for proteome mapping of human peripheral blood mononuclear cell extract. In HILIC a silica‐based stationary phase and for RPLC a C₁₈ column were applied. Then separated proteins were eluted to an ion trap mass spectrometry system. Our results showed that the HILIC leads to more proteins being identified in comparison to RPLC. Among the total 181 identified proteins, 56 and 38 proteins were fractionated specifically by HILIC and RPLC, respectively. In order to demonstrate this, the physicochemical properties of identified proteins such as polarity and hydrophobicity were considered. This analysis indicated that polarity may play a major role in the HILIC separation of proteins vs RPLC. Using gene ontology enrichment analysis, it was also observed that differences in physicochemical properties conform to the cellular compartment and biological features. Finally, this study highlighted the potential of HILIC and the great orthogonality of RPLC in gel‐free proteomic studies. Copyright © 2015 John Wiley & Sons, Ltd.