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 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.     

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.     

Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates

This paper describes a procedure for quantitative proteomics that selects peptides containing both cysteine and histidine residues from tryptic digests of cell lysates. Cysteine-containing peptides were selected first by covalent chromatography using thiol disulfide exchange. Following the release of cysteine-containing peptides from the covalent chromatography column with reductive cleavage, histidine-containing peptides were captured by passage through an immobilized metal affinity chromatography column loaded with copper. Quantification was achieved in a four-step process involving (i) differential labeling of control and experimental samples with isotopically differing forms of succinic anhydride, (ii) mixing the two globally labeled samples, (iii) fractionating the labeled peptides by reversed-phase liquid chromatography, and (iv) determining the isotope ratio in individual peptides by mass spectrometry. The results of these studies indicate that by selecting peptides containing both cysteine and histidine, the complexity of protein digests could be substantially reduced. Up-regulated proteins from plasmid bearing Escherichia coli that had been induced with isopropyl beta-thiogalacto-pyranoside were identified and quantified by the global internal standard technology (GIST) described above. Database searches were greatly simplified because the number of possible peptide candidates was reduced more than 95%.     

Signature-peptide approach to detecting proteins in complex mixtures

The objective of the work presented in this paper was to test the concept that tryptic peptides may be used as analytical surrogates of the protein from which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatography, lectin columns were used in this case. Affinity selected peptide mixtures were directly transferred to a high-resolution reversed-phase chromatography column and further resolved into fractions that were collected and subjected to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The presence of specific proteins was determined by identification of signature peptides in the mass spectra. Data are also presented that suggest proteins may be quantified as their signature peptides by using isotopically labeled internal standards. Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a peptide relative to that of the labeled internal standard. Peptides in tryptic digests were labeled by acetylation with acetyl N-hydroxysuccinimide while internal standard peptides were labeled with the trideuteroacetylated analogue. Advantages of this approach are that (i) it is easier to separate peptides than proteins, (ii) native structure of the protein does not have to be maintained during the analysis, (iii) structural variants do not interfere and (iv) putative proteins suggested from DNA databases can be recognized by using a signature peptide probe.     

反相/亲水色谱法分析糖苷类化合物

根据皂苷、黄酮苷等糖苷类化合物的结构特点,采用亲水色谱模式分析该类化合物,以弥补反相色谱模式分离结构类似糖苷类化合物选择性的不足。首先选择14个糖苷类化合物,比较了反相色谱柱(XAqua C18)和亲水色谱柱(Click XIon)的分离效果,评价了反相/亲水色谱的正交性,并构建了反相/亲水二维体系用于西洋参样品的分离。结果表明,糖苷类化合物在反相及亲水色谱柱上均有很好的保留,但两者具有不同的分离选择性,14种物质在反相和亲水柱的出峰顺序有较大差异,在反相色谱中不易分离的人参皂苷Rg1和Re在亲水色谱柱可获得很好的分离,反相/亲水模式分离糖苷化合物具有很好的正交性。以构建的RPLC/HILIC二维色谱体系分离西洋参样品,有效地提高了分离能力及峰容量,有利于后续更多极性及微量化合物的制备、结构表征与活性研究,且该方法操作简便、流动相兼容性好,可作为糖苷分离分析、制备的有效手段,也可以为其他中药复杂体系的分析提供参考。     

Off-line comprehensive two-dimensional high-performance liquid chromatography system with size exclusion column and reverse phase column for separation of complex traditional Chinese medicine Qingkailing injection

A comprehensive two-dimensional liquid chromatography system was constructed on the combination of size exclusion chromatography (SEC) and reverse phase liquid chromatography (RPLC). The first dimension used a SEC column with 300 mm x 8mm i.d., packed with Toyopearl HW-40S. The column was eluted with 0.05 mol/l Tris-HCl (pH 6.9) at a flow rate of 0.4 ml/min. The second dimension used a RPLC column with 100 mm x 4.6mm i.d., which was operated in gradient form at a flow rate of 1.0 ml/min. An automatic switching valve was used to collect the effluent of the SEC column every 3 min, and the effluent was automatically injected into the RPLC column. Mass spectrometer was used for peak identification. This system was used to separate Qingkailing injection, a traditional Chinese medicine (TCM). The result showed that the total peak capacity of this system could reach 1134 and the qualitative analysis of seven chemical components of the Qingkailing injection was accomplished by this system. The results show that comprehensive two-dimensional liquid chromatography system is of great importance and high value in the separation of complex TCM.     

Capillary electrochromatography of peptides in a microfabricated system.

Reversed-phase liquid chromatography of tryptic peptides is shown in the capillary electrochromatography mode using microfabricated columns. Although selectivity is different, a mixture of tryptic peptides from ovalbumin appears to be as easily separated in the CEC as HPLC mode. The major difference between a separation in the macrofabricated CEC column and conventional separations in the HPLC mode is that separations are more readily achieved in the isocratic mode in the lower surface area microfabricated CEC columns.     

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.     

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.     

质谱法分析大肠癌组织中踝蛋白的磷酸化修饰

踝蛋白的磷酸化修饰,特别是肿瘤等病理条件下的踝蛋白磷酸化状态,与肿瘤的发病、转移机理密切相关。本研究采用盐析、离子交换层析和电泳分离并纯化了人大肠癌组织中的踝蛋白(Talin),经胰酶水解获得其肽段混合物,进一步分别利用固定化Fe3+亲和层析和TiO2亲和层析在酸性条件下对其中磷酸化修饰肽段进行吸附,并以1%氨水进行洗脱。在Michrom Magic C18色谱柱上,以A:99%水+1%乙腈+0.1%甲酸和B:99%乙腈+1%水+0.1%甲酸两种流动相进行梯度洗脱分离,采用ESI质谱进行依赖数据的二级子离子扫描。结果显示,固定化Fe3+富集到8个磷酸化肽段而TiO2富集到9个磷酸化肽段。本研究提供了一种快速、准确地鉴定从人大肠癌组织中分离表征踝蛋白的方法。     

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.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

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.     

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.     

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.     

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.     

Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.     

Study on the degeneracy of antisense peptides using affinity chromatography

The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. Similar result was obtained from another experiment that Arg-substituted antisense peptide of fusion peptide (1-11) of influenza virus A was also shown the highest affinity binding to immobilized fusion peptide.     

Selectivity differences for C18 and C8 reversed-phase columns as a function of temperature and gradient steepness. II. Minimizing column reproducibility problems

The choice of T and tG as variables for controlling selectivity and resolution during reversed-phase liquid chromatography (RPLC) method development can be used to minimize problems caused by column batch-to-batch irreproducibility. When a new column fails to provide adequate separation of the sample, altered values of T and tG can be predicted that will restore the separation obtained with the previous column. Alternatively, columns from different manufacturers can be tested during method development, in order to find a common set of conditions (T and tG) that provide acceptable separation with two or more of these columns. In this way, any of several columns from different sources become usable for the method. Examples are shown of these different computer-assisted procedures for minimizing problems due to column variability.