蛋白质高效分离两维色谱柱的选择优化

为了构建高效的离子交换/反相二维液相色谱(IEC/RPLC)分离平台系统,提高复杂蛋白质样品的分离效率,对色谱柱进行了评价与筛选。通过对实际人肝蛋白质样品的分离效果的比较,选择确定了TSKgel DEAE-5PW弱阴离子交换色谱柱(WAX)作为第一维色谱分离柱;考察了同一规格的10支代表性反相色谱柱(250 mm×4.6 mm, 5 μm, 30 nm, C4、C8或C18),通过评价其对尿嘧啶、硝基苯、萘和芴的分离性能以及对3种标准蛋白质样品的非特异性吸附、对人肝蛋白质样品的WAX馏分的分离效果,最终确定以Jupiter 300 C4反相色谱柱作为第二维色谱分离柱。对两维色谱柱的选择优化为蛋白质高效分离二维液相色谱平台的搭建提供了可靠基础。     

Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics. Application to the human Jurkat T-cell proteome

A dual-purpose sample-trapping column is introduced for the capacity enhancement of proteome analysis in on-line two-dimensional nanoflow liquid chromatography (strong cation-exchange chromatography followed by reversed-phase liquid chromatography) and tandem mass spectrometry. A home-made dual trap is prepared by sequentially packing C18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm x 200 microm I.D. for SCX, 0.7 cm x 200 microm for RP) ended with a home-made frit and is connected to a nanoflow column having a pulled tip treated with an end frit. Without having a separate fraction collection and concentration process, digested peptide mixtures were loaded directly in the SCX part of the dual trap, and the SCX separation of peptides was performed with a salt step elution initiated by injecting only 8 microL of NH4HCO3 solution from the autosampler to the dual trap. The fractionated peptides at each salt step were directly transferred to the RP trap packed right next to the SCX part for desalting, and a nanoflow LC-MS-MS run was followed. During the sample loading-SCX fractionation-desalting, flow direction was set to bypass the analytical column to prevent contamination. The entire 2D-LC separation and MS-MS analysis were automated. Evaluation of the technique was made with an injection of 15 microg peptide mixtures from human Jurkat T-cell proteome, and the total seven salt step cycles followed by each RPLC run resulted in an identification of 681 proteins.     

Automated clean-up, separation and detection of polycyclic aromatic hydrocarbons in particulate matter extracts from urban dust and diesel standard reference materials using a 2D-LC/2D-GC system

A multidimensional, on-line coupled liquid chromatographic/gas chromatographic system was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs). A two-dimensional liquid chromatographic system (2D-liquid chromatography (LC)), with three columns having different selectivities, was connected on-line to a two-dimensional gas chromatographic system (2D-gas chromatography (GC)). Samples were cleaned up by combining normal elution and column back-flush of the LC columns to selectively remove matrix constituents and isolate well-defined, PAH enriched fractions. Using this system, the sequential removal of polar, mono/diaromatic, olefinic and alkane compounds from crude extracts was achieved. The LC/GC coupling was performed using a fused silica transfer line into a programmable temperature vaporizer (PTV) GC injector. Using the PTV in the solvent vent mode, excess solvent was removed and the enriched PAH sample extract was injected into the GC. The 2D-GC setup consisted of two capillary columns with different stationary phase selectivities. Heart-cutting of selected PAH compounds in the first GC column (first dimension) and transfer of these to the second GC column (second dimension) increased the baseline resolutions of closely eluting PAHs. The on-line system was validated using the standard reference materials SRM 1649a (urban dust) and SRM 1975 (diesel particulate extract). The PAH concentrations measured were comparable to the certified values and the fully automated LC/GC system performed the clean-up, separation and detection of PAHs in 16 extracts in less than 24 h. The multidimensional, on-line 2D-LC/2D-GC system eliminated manual handling of the sample extracts and minimised the risk of sample loss and contamination, while increasing accuracy and precision.

A comprehensive two-dimensional normal-phase × reversed-phase liquid chromatography based on the modification of mobile phases

A comprehensive orthogonal two-dimensional liquid chromatography (2D-LC) based on the modification of mobile phases was developed with a sample loop–valve interface. To improve the compatibility of mobile phases and analysis speed, some special solvents were chosen as the mobile phases, and the column temperature was elevated to decrease the viscosity of mobile phases of reversed-phase liquid chromatography (RPLC). Based on this principle, the first dimension was normal-phase liquid chromatography (NPLC) with a SiO₂ column, and the second dimension was reversed-phase liquid chromatography containing two tandem C18 columns. 1,4-Dioxane was used in the NPLC mobile phase, and isopropyl alcohol was employed in the RPLC mobile phase. Moreover, the elevated column temperature enabled the reduction of the backpressure and using tandem C18 columns to improve the resolving power in RPLC. The new comprehensive 2D-LC system and applied strategy offered a novel idea for construction of 2D-LC system. A traditional Chinese medicine, Zhengtian pill, was used as the test sample to evaluate the constructed 2D-LC system. 876 peaks were detected, and the peak capacity reached 1740.     

Integrated strong cation exchange/capillary reversed-phase liquid chromatography/on-target digestion coupled with mass spectrometry for identification of intact human liver tissue proteins

We present a comprehensive method for proteome analysis that integrates both intact protein separation and proteolytic fragment characterization mass spectrometric approaches. Strong cation exchange chromatography (SCX) was used as the first separation dimension and capillary reversed-phase liquid chromatography (cRPLC) was integrated as the second separation dimension. Fractions from SCX were collected offline and loaded onto cRPLC. Effluents from cRPLC were directly deposited onto the MALDI target plates and further digested by using a rapid on-probe tryptic digestion technique. This approach minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and transfer. MALDI-TOF/TOF was used for subsequent analyses. The sensitivity of on-target digestion is showed by analyzing 0.07 ng of myoglobin, 0.07 ng of cytochrome c and 0.7 ng BSA. The high efficiency of the overall system was demonstrated by the analysis of intact proteins extracted from normal human liver tissue. In total, 458 proteins were identified, which proved the system's promising potential for analysis and application in proteomics.     

鼠肝蛋白质组的纳升级多维液相色谱分离

搭建了一个纳升级的二维液相色谱分离平台(nano-2D-LC),该平台可以自动完成进样、除盐、分离及鉴定。以离子交换色谱(SCX)为第一维,反相液相色谱(RPLC)为第二维,对鼠肝组织的蛋白质组进行了研究。SCX采用阶梯式洗脱,RPLC运用线性梯度洗脱,以200 nL/min的速度进行分离,峰容量可达620。     

Multidimensional liquid chromatography system with an innovative solvent evaporation interface

An orthogonal two-dimensional liquid chromatographic (2D-LC) system was developed by using a vacuum-evaporation loop-type valve interface. Normal-phase liquid chromatography (NPLC) with a bonded CN phase column was used as the first dimension, and reversed-phase liquid chromatography (RPLC) with a C(18) column was used as the second dimension. All the solvents in the loop of the interface were evaporated at 90 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loop. The mobile phase of the second dimension dissolved the analytes in the loop and injected them onto the secondary column, allowing an on-line solvent exchange of a selected fraction from the first dimension to the second dimension. The chromatographic resolution of analytes on the two dimensions was maintained at their optimal condition. Sample loss due to evaporation in the interface was observed that depended on the boiling point of the compound. Separation of sixteen polycyclic aromatic hydrocarbon mixtures and a traditional Chinese medicine Angelica dahurica was demonstrated.     

Ultra rapid liquid chromatography as second dimension in a comprehensive two-dimensional method for the screening of pharmaceutical samples in stability and stress studies

Separation orthogonality has been explored with respect to comprehensive two-dimensional liquid chromatography (2D-LC) for different reversed-phase stationary phases. The outcome of this study points out an SB-CN x BEH-C18 combination, used in the first and the second dimension, respectively, as the most orthogonal chromatographic system for the samples assayed. The present investigation reports the employment of an ultra high pressure liquid chromatography system (UPLC) as the second chromatographic dimension, increasing the sensitivity and the speed, completing the whole chromatographic separation in a reasonable time frame. Finally, an off-line 2D-LC method with diode array detection based on the UPLC has been optimized, allowing the separation and minimizing the run time. SB-CN and BEH-C18 were employed as first and second dimension, respectively, with gradient elution applied in each dimension. Alprazolam degraded tablets were studied as a proof of concept of the utility of this type of setups for impurity profiling of complex samples.     

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography x liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ((2)D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ((1)D) column can be derived. Theory clearly indicates that the choice of the (1)D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8mm for SEC and 4.6mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.     

全二维微柱液相色谱-质谱鉴定人胃癌组织中的差异蛋白质

构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱-质谱分离平台.采用了醋酸铵缓冲液梯度洗脱,实现了第一维肽段的分步洗脱,洗脱的肽段经富集除盐后通过接口进入反相色谱微柱,通过线性梯度实现第二维进一步分离,最后进入质谱进行检测.采用此平台分析了人胃癌组织与正常组织提取蛋白质信息,其中正常胃组织鉴定蛋白质数为537个,而癌症组织鉴定蛋白质数目为506个.对胃癌和正常组织两种提取蛋白质酶解产物的蛋白质检索结果进行比较分析,将鉴定的蛋白质按照物理性质进行分布,找出正常组织与癌症组织间蛋白质差异,筛选出一种可能发生变异的癌症特有蛋白.     

用于人血浆中蛋白质分离的在线阵列式二维常规柱液相色谱系统的建立

构建了一种在线阵列式二维常规柱液相色谱系统,并将其应用于分离血浆中的完整蛋白质。该系统以1根强阴离子交换柱作为第一维分离柱,8根阵列式反相色谱柱作为第二维分离柱。强阴离子交换柱分离的馏分通过十通阀被依次转移到第二维预柱上并得到保留富集,随后第二维流动相通过分流器同时将预柱上的蛋白质反冲至分析柱上进行分离。二维之间以及第二维阵列色谱柱之间均相互独立,从而可以提高系统分离的通量和总峰容量。采用该系统对血浆中的蛋白质进行了完整蛋白质水平上的分离。该系统具有高通量和高分辨率的特点,为血浆样品中高丰度蛋白质的去除以及血浆样品的深入研究提供了一种有效的手段。     

全二维液相色谱(NPLC×RPLC)接口及其应用

用内径为0.53 mm的填充毛细管正相液相色谱为第一维, 用4.6 mm(i.d.)×50 mm RP-18e整体柱反相色谱为第二维, 建立了定量环-阀切换接口的全二维液相色谱系统(NPLC×RPLC). 第一维色谱分离洗脱出的组分交替存储在十通阀上的两个定量环中, 同时定量环中前一个组分被转移到第二维进行反相分离. 因为第一维的流动相流量仅是第二维的1/500, 自然解决了流动相兼容问题. 采用芳香族化合物的混合物和中药丹参正己烷提取液对该全二维液相系统的分离能力进行了评价.     

Comprehensive on-line RPLC-CZE-MS of peptides

Peptide standards and tryptic digests of ribonuclease B are separated by comprehensive two-dimensional reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) and detected on-line by electrospray mass spectrometry. The RPLC column is coupled to the CZE column by a transverse flow gating interface. A new rugged microelectrospray needle is described that combines high ionization efficiency, low flow rates, and a sheath flow. The result is a system combining the separation capabilities of both RPLC and CZE with on-line mass spectrometric detection, all in about 15 min.     

Development of a Parallel-Tandem Column Interface in a Two-Dimensional Liquid Chromatography System

An ion exchange-reversed-phase two-dimensional liquid chromatography (2D-LC) system was developed based on a newly designed interface. The interface was constructed by alternately connecting tandem columns to one of the parallel separation columns to increase the separation efficiency. Separation on the tandem column does not affect the switching frequency; the valve switching could be maintained at high frequency. The constructed interface could be used in both ambient and elevated temperature modes. Under high-temperature mode, only the tandem column was heated, which simplified the operation, decreased the backpressure, improved the solvent compatibility, and speeded up the separation. Tryptic digestion of four proteins was used to evaluate the performance of the developed system. The peak capacity of the 2D-LC system reached 650 under ambient conditions and 890 under high temperature conditions. When compared with the traditional parallel column interface constructed in this paper, the new interface produced a 60% higher peak capacity.     

Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in "trapping" and separating neurotransmitters

Quantification of neurotransmitters as biologically active analytes in neurological samples is of high interest for studying their effect on multiple targets. This work is part of a strategy involving two-dimensional liquid chromatography (2D LC) system with mass spectrometry (MS) detection. The concept of the on-line LC system is the coupling of reversed phase liquid chromatography (RPLC, the second separation dimension) to ion-exchange chromatography (IEC, the first dimension). Our objective in this study is to find the appropriate second dimension column, ensuring that samples of neurotransmitters are refocused and separated on it. Silica-based columns designed specifically to retain polar compounds were tested in LC conditions and compared with results obtained with a porous graphitic carbon (PGC, Hypercarb) column. These polar embedded, polar endcapped, and high-density alkyl chain columns successfully separated analytes in question using mobile phase systems with high percentage of water, or even pure water. Only Hypercarb column provided efficient retention of the most polar neurotransmitters and could be used for trapping and preconcentrating the compounds without rapid breakthrough.     

二维液相色谱接口的改进及其在蛋白质组学研究中的应用

随着蛋白质组学、本草物质组学等组学概念的提出,所需分析的样品的成分越来越复杂,因此具有强大分离能力的多维液相色谱技术受到人们越来越多的关注。二维液相色谱中第二维的分离性能和速度是整个分离系统性能的关键。基于捕集柱模式,我们采用经特殊设计的流路系统,使得双捕集柱型接口具有预分离的功能。样品从第一维流出以后被富集在捕集柱1的柱头,经过脱盐后,正冲捕集柱,捕集柱1与第二维色谱柱联用对富集的样品进行分离,增加了第二维分离效率。当捕集柱上的样品全部被洗脱到第二维色谱柱上时,捕集柱2已经完成对第一维洗脱液中样品的捕集和脱盐,此时将阀进行切换,捕集柱2与第二维色谱柱直接相连进行洗脱。循环切换捕集柱1和捕集柱2,维持较高的阀切换频率,实现了第二维色谱柱的连续洗脱。因此保证了第二维分离具有较快速度,同时具有较高的分离效率。使用35 mm长捕集柱和十通阀为接口,以弱阴离子交换(WAX)色谱为第一维分离模式,以反相(RP)色谱为第二维分离模式,构建了WAX-RP二维液相色谱系统(2D-LC system)。以小鼠血清为样品对系统进行了初步评价。色谱流出曲线出现了明显的界面现象,这是由于捕集柱流动相中含有的较多盐分流出时的背景吸收造成的。同时,由于界面两侧的流动相黏度不同产生了黏性指进(VF)现象。当第二维色谱柱长度为50 mm时,理论上可将第二维分离效能提高70%。该接口可以应用于多种二维液相色谱模式,适用于蛋白质组学和本草物质组学研究中对于复杂样品的分离分析。     

强阳离子交换毛细管液相色谱-反相加压毛细管电色谱二维系统的构建及其在中药黄柏提取物分离中的应用

加压毛细管电色谱(pCEC)具有电泳和液相色谱的双重分离机理,其柱效高、选择性强、分辨率高和分离速度快并可进行梯度洗脱。我们在此基础上加入离子交换色谱模式,构建了强阳离子交换-反相加压毛细管液相色谱(micro strong cation exchange liquid chromatography/reversed phase pressurized capillary electrochromatography, μ-SCXLC/RP-pCEC)二维系统,并对中药黄柏的提取物进行了优化分离。第一维μ-SCXLC采用线性盐梯度分离,样品被切割成11个馏分洗脱收集后进入第二维,第二维脱盐后,采用RP-pCEC进行分离分析,梯度洗脱。以中药黄柏提取物为样品,此二维系统的分辨率和峰容量都较一维系统有很大提高,理论峰容量可达900左右,证明构建的二维体系非常适合复杂样品的分离分析。     

基于捕集柱阵列的制备型二维液相色谱纯化烟草中的4个组分

二维液相色谱(2D-LC)因具有较高的峰容量,在复杂样品的分离分析中获得了广泛的关注。然而,制备型2D-LC以纯化高纯单体为目标,在方法开发和设备构成等方面与分析型2D-LC有较大的不同,目前尚未得到充分的开发,在大规模的制备纯化中应用较少。本文以一套制备液相色谱模块为分离系统,以稀释泵、切换阀和捕集柱阵列为接口,构建了新型的制备型2D-LC系统,旨在规模化纯化多个活性成分。以烟叶中可以用作医药原料的烟碱、绿原酸、芦丁和茄尼醇等组分为目标物,考察了不同类型填料对样品的捕集效率、过载条件下的色谱保留行为等,优化了制备色谱条件。进而利用在线2D-LC系统实现了烟叶提取物的纯化,通过一次运行获得了4个高纯化合物。该系统具有中压色谱纯化成本低、系统在线运行自动化程度高、稳定性好及容易放大等优点。烟叶中活性化学成分的回收利用对促进烟草行业的发展及带动地方农业经济开发具有重大的意义。     

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。     

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

In this study, two mixed‐mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two‐dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed‐mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed‐mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off‐line 2D‐LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine—Uncaria rhynchophylla. The two‐dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed‐mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.