Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/×/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-μm particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.     

在线二维液相色谱技术在中药研究中的应用进展

中药的组成复杂,其化学成分的表征和识别一直是中药研究的基础和关键。在线二维液相色谱是基于两种分离模式构建的色谱分析技术,主要包括中心切割二维液相色谱和全二维液相色谱两种模式,因二者具有更高的峰容量而在中药研究中备受青睐。该文对在线二维液相色谱技术的概念和特点进行了讨论,并对二维液相色谱在中药研究中的应用进行了综述,以期为该技术在中药质量控制、物质基础表征、活性成分筛选等研究方面提供一定参考。     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

A four-dimensional separation approach by offline 2D-LC/IM-TOF-MS in combination with database-driven computational peak annotation facilitating the in-depth characterization of the multicomponents from Atractylodis Macrocephalae Rhizoma (Atractylodes macrocephala)

Plant metabolites represent complex chemical system, which renders it difficult to clarify the chemical composition by conventional liquid chromatography/mass spectrometry (LC/MS) due to the limited selectivity and peak capacity. The rhizomes of Atractylodes macrocephala have been utilized as a traditional Chinese medicine Atractylodis Macrocephalae Rhizoma (Bai-Zhu), and have been reported containing multiple categories of plant metabolites. Targeting the multicomponents from A. macrocephala, an integral approach by offline two-dimensional liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) was established and validated. By configuring an XBridge Amide column of Hydrophilic Interaction Chromatography and an Atlantis Premier BEH C18AX column of mixed ion exchange and reversed-phase modes, the established 2D-LC/IM-QTOF-MS system showed high orthogonality up to 0.91. Dimension-enhanced, data-independent high-definition MS^E (HDMS^E) in the positive ESI mode was conducted on a Vion IM-QTOF mass spectrometer, and its hyphenation to offline 2D-LC could enable the four-dimensional separation (each dimension in 2D-LC, IM, and MS). Particularly, HDMS^E facilitated the acquisition of high-definition MS¹ and MS² spectra. In-house library-driven computational peak annotation by the bioinformatics platform UNIFI could efficiently process and annotate the HDMS^E data for the structural elucidation. By integrating reference compounds comparison, we could identify or tentatively characterize 251 components from A. macrocephala (including 115 sesquiterpenoids, 90 polyacetylenes, 11 flavonoids, 9 benzoquinones, 12 coumarins, and 14 others), which indicated large improvement in identifying those minor plant components, compared with the conventional LC/MS approach. Conclusively, offline 2D-LC/IM-QTOF-HDMS^E in combination with computational data interpretation proves to be powerful facilitating the in-depth multicomponent characterization of herbal medicine.     

IEX/RP二维液相色谱中弱保留组分收集模式的构建及系统评价

以十通阀和捕集柱接口形式,构建了弱阴离子交换/反相(WAX/RP)二维液相色谱分离系统.通过将第一维离子交换色谱分析中的前部集中洗脱出的弱保留组分收集后单独分析,剩余样品进一步采用二维液相色谱分析,可以有效避免第二维色谱柱对特殊样品局部集中流出导致的第二维分离超柱容量问题,提高了系统的整体分离能力.使用4种蛋白胰蛋白酶酶解后的多肽样品评价该系统,在不分流的系统中共检测到115个峰.对第一维分离的前15 min分流后得到的组分单独分析,共识别出58个峰,后续的二维分离中共得到78个峰.2种方法相比,第二种方法检测峰数增加了21个,一些低丰度的组分在弱保留组分收集后被识别.     

Bias reduction in the quantitative analysis of a target analyte present in a limited quantity in human plasma using dual-mode heart-cutting two-dimensional liquid chromatography coupled with isotope dilution mass spectrometry

Dual-mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS were performed with triple quadrupole mass spectrometry using folic acid standards and/or ¹³C₅-folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked ¹³C₅-folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma, eliminating matrix effects and saving analysis time.     

二维液相色谱接口技术

二维液相色谱具有峰容量大、分辨率高、分析速度快等优点,已经成为复杂样品分离分析的重要工具。两种分离模式的转换通常需要经过一个特殊接口来完成,接口是二维液相色谱系统的核心,也是限制二维液相色谱应用的瓶颈;两种流动相不互溶时,接口尤为重要。本文针对二维液相色谱接口技术近期的发展和应用进行总结。引用文献51篇。     

A green protocol for efficient discovery of novel natural compounds: Characterization of new ginsenosides from the stems and leaves of Panax ginseng as a case study

Exploration of new natural compounds is of vital significance for drug discovery and development. The conventional approaches by systematic phytochemical isolation are low-efficiency and consume masses of organic solvent. This study presents an integrated strategy that combines offline comprehensive two-dimensional liquid chromatography, hybrid linear ion-trap/Orbitrap mass spectrometry, and NMR analysis (2D LC/LTQ-Orbitrap-MS/NMR), aimed to establish a green protocol for the efficient discovery of new natural molecules. A comprehensive chemical analysis of the total ginsenosides of stems and leaves of Panax ginseng (SLP), a cardiovascular disease medicine, was performed following this strategy. An offline 2D LC system was constructed with an orthogonality of 0.79 and a practical peak capacity of 11,000. The much greener UHPLC separation and LTQ-Orbitrap-MS detection by data-dependent high-energy C-trap dissociation (HCD)/dynamic exclusion were employed for separation and characterization of ginsenosides from thirteen fractionated SLP samples. Consequently, a total of 646 ginsenosides were characterized, and 427 have not been isolated from the genus of Panax L. The ginsenosides identified from SLP exhibited distinct sapogenin diversity and molecular isomerism. NMR analysis was finally employed to verify and offer complementary structural information to MS-oriented characterization. The established 2D LC/LTQ-Orbitrap-MS/NMR approach outperforms the conventional approaches in respect of significantly improved efficiency, much less use of drug materials and organic solvent. The integrated strategy enables a deep investigation on the therapeutic basis of an herbal medicine, and facilitates new compounds discovery in an efficient and environmentally friendly manner as well.     

Novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography, an excellent orthogonal system for practical analysis

A novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography (2D-RPLC/HILIC) system is developed with the introduction of the click beta-cyclodextrin (beta-CD) stationary phase. The offline system shows excellent abilities for the separation of polar and medium-polarity components in traditional Chinese medicine (TCM). It facilitates us not only to separate components that cannot be resolved by uni-dimensional chromatography, but also to achieve much more efficient detection of components with low abundance. The orthogonality of this new system is excellent. A simple geometric approach is developed to characterize the practical orthogonality of 2D-LC in the analysis of complex unknown samples. The mathematical characterization results are in good accordance with the experimental findings, and both demonstrate that the proposed 2D-RPLC/HILIC system is a powerful tool for the separation of polar and medium-polarity complex samples. This system may have a great potential for the separation of peptides and metabolomic compounds with similar polarity.     

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。     

离线2D-LC-MS系统的建立及其用于人肝蛋白质组学的研究

在蛋白质组学研究中,近年来提出的多维色谱-质谱联用技术有望成为继2D-PAGE-MS技术之后又一项重要的高通量技术平台,Yates,Davis,Wagner和Hancock等报道了二维强阳离子交换色谱-反相色谱-质谱(SCX-RPLC-MS)的在线联用分析系统,在该系统中,各维色谱的分离条件相互制约,只能从整体上加以平衡。     

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。     

二维液相色谱-质谱联用分离中药复方葛根芩连汤中的有效成分

建立了二维液相色谱-质谱联用方法分离中药复方葛根芩连汤的成分。以CN柱作第一维色谱柱,水和甲醇梯度洗脱分离;以ODS柱作第二维色谱柱,20 mmol/L乙酸铵缓冲液和乙腈梯度洗脱分离;质谱检测采用电喷雾电离/大气压化学电离（ESI/APCI）复合离子源,正负离子扫描。实验结果表明搭建的二维液相色谱的峰容量显著高于一维色谱,分离效率得到了明显的提高。以第一维色谱的第3个流分为例,对其二维分离进行仔细分析,发现质谱比紫外光谱检测到的组分多,质谱中采用负离子模式比正离子模式检测到的组分多。表明搭建的二维液相色谱-质谱分离平台分离效果好,提高了液相色谱的峰容量和分离效率。该方法操作简便,可作为中药等复杂体系分离分析的有效手段。     

Large-scale identification of human biliary proteins from a cholesterol stone patient using a proteomic approach

Gallbladder bile, one of the most important body fluids, is composed of water, inorganic ions, conjugated bile salts, phospholipids, cholesterol, bilirubin, mucin and proteins. The separation and identification of bile proteins remain difficult due to the complexity of this matrix. In the present study, human gallbladder bile was obtained from a cholesterol stone patient, and the proteins were isolated and purified by dialysis, precipitation and delipidation procedures. The resulting proteins were divided into several aliquots. One aliquot was subjected to two-dimensional gel electrophoresis (2DE). The protein spots were then in-gel digested and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). Another aliquot was directly digested and analyzed by a combination of strong cation-exchange (SCX) and reversed-phase (RP) chromatography prior to tandem mass spectrometry (2D-LC/MS/MS). Eventually, 48 and 218 unique proteins were identified from 2DE/MS and 2D-LC/MS/MS, respectively, resulting in a total of 222 unique identified proteins. Of the 218 proteins identified by 2D-LC/MS/MS, 92 were identified based on more than one unique tryptic peptide, and, of the total 222 proteins, 98 were identified based on more than one unique tryptic peptide.     

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.     

Technologies to improve the sensitivity of existing chromatographic methods used for bioanalytical studies

Chromatographic method has long been recognized as the most widely used separation method in bioanalytical research. However, the relatively low sensitivity of existing chromatographic methods remains a significant challenge, as the requirements for experimental procedures become more demanding. This review discusses the main causes for the low sensitivity of chromatographic methods and aims to introduce different technologies for enhancing their sensitivity in the following aspects: (i) different pretreatment methods for improving clean-up efficiency and recovery; (ii) derivatization step for altering the chromatographic behavior of analytes and enhancing MS ionization efficiency; (iii) optimal LC–MS conditions and appropriate separation mechanism; and (iv) applications of other chromatographic methods, including miniaturized LC, 2D-LC, 2D-GC, and supercritical fluid chromatography. Altogether, this review is devoted to summarizing the recent technologies reported in the literature and providing new strategies for the detection of bioanalytes.     

Development and validation of an on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of prostaglandins E(2) and F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha) levels in human plasma

We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E(2) as well as PGF(2alpha) and its metabolite 13,14-dihydro-15-keto PGF(2alpha) (F(2alpha)-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE(2) and 2.5 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5-50 pg/mL for PGE(2) and 2.5-500 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE(2), PGF(2alpha), and F(2alpha)-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE(2), 5.70 pg/mL for PGF(2alpha) and 9.48 pg/mL for F(2alpha)-M.     

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。     

Separation and analysis of peptides and proteins from Vipera lebetina snake venom

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences.The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α₂-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.