Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.     

A comparison of LC and SFC for cellulose- and amylose-derived chiral stationary phases

This paper presents a systematic comparison of liquid chromatography (LC) and supercritical fluid chromatography (SFC) for Chiralcel OD and Chiralpak AD chiral stationary phases (CSPs), performed using various chiral compounds having a known or potential pharmaceutical activity. The chiral recognition mechanisms involved in LC and SFC for the enantiomeric separation of β-blockers have been studied more particularly. As a general rule, it appears that the presence of polar functions, like primary or secondary hydroxyl or amine functions, may result in marked discrepancies in selectivity between LC and SFC. This result is peculiar to cellulose- and amylose-derived CSPs, for which the interactions involved in chiral recognition mechanism are not always well balanced, contrary to what happens for independent CSPs. In the case of chiral resolution of polar solutes or polymer-type CSPs, the analyst should try both the LC and SFC techniques to be able to choose the more stereoselective one. © 1995 Wiley-Liss, Inc.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2‐D peptide separations employing mixed mode reversed phase anion exchange (MM (RP‐AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP‐AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP‐AX) compared with the traditional SCX hyphenated to RP‐LC‐MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP‐AX) for 2‐D LC proteomic studies was also extended in the analysis of iTRAQ‐labelled HeLa and cyanobacterial proteomes using nano‐flow chromatography interfaced to the MS/MS. We demonstrate MM (RP‐AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.     

Ultrahigh pressure fast size exclusion chromatography for top‐down proteomics

Top‐down MS‐based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top‐down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size‐based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high‐resolution separation of intact proteins for top‐down proteomics. Fast separation of intact proteins (6–669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP‐SEC provides high‐resolution separation of intact proteins using a MS‐friendly volatile solvent system, allowing the direct top‐down MS analysis of SEC‐eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP‐SEC is an attractive LC strategy for the size separation of proteins with great potential for top‐down proteomics.     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.     

Application of Box-Behnken Design to Hybrid Electrokinetic-Adsorption Removal of Mercury from Contaminated Saline-Sodic Clay Soil

A hybrid electrokinetic-adsorption (HEKA) technique using uniform electric field and granular activated carbon (GAC) produced from date palm pits was investigated for the removal of mercury from natural saline-sodic clay heavily contaminated with heavy metals, phenol, and kerosene. Response surface methodology (RSM) was employed to model, optimize, and interpret the results obtained with the aid of Design Expert software. According to the Box-Behnken experimental design, 15 experiments were conducted each with residence time of three weeks. The effects of voltage gradient (0.2–1 V/cm), initial Hg concentration (mg/Kg), and polarity reversal interval (0-48 hours) on Hg removal efficiency and energy consumed for Hg removal were investigated. Respectively, the responses fitted reduced cubic (R² = 99.3%) and quadratic models (R² = 92.3%) with the overall relative contributions of the investigated parameters on the responses following the order: voltage gradient > initial Hg concentration > polarity reversal interval based on analysis of variance (ANOVA). The optimal conditions obtained with desirability of 90% aimed at maximizing Hg removal were 24 hours polarity reversal interval, 0.2 V/cm voltage gradient, and 100 mg/kg initial Hg concentration. This optimum operating condition yielded good removal of Hg (99.5%) at reduced energy consumption of 50.1kWh.m^?3mg^?1. Experimental validation of the models showed good prediction of Hg removal efficiency (0.0368% prediction error). The results presented herein suggest that HEKA technology could be utilized effectively for the removal of Hg from contaminated, low permeable soils under extreme soil and contamination conditions.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.     

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).     

Two dimensional liquid phase separations of proteins using online fractionation and concentration between chromatographic dimensions

Multi-dimensional liquid chromatography is often presented as an alternative to two-dimensional (2-D) gel electrophoresis for separating complex protein mixtures. The vast majority of analytical-scale 2-D LC systems have employed either off-line fractionation or stepped gradients in the first dimension separation. The latter severely restrict flexibility in setting up the first dimension gradient. We propose a novel two-dimensional LC system that employs online fractionation of proteins into a series of small reversed phase trapping columns. These traps effectively decouple the two separation dimensions and avoid problems associated with off-line fraction collection. Flexibility in determining the gradient programs for the two separations is thus enhanced. The reduced diameter of the trapping columns concentrates analyte between chromatographic dimensions. The apparatus is coupled with online electrospray time-of-flight mass spectrometry to characterize ribosomal proteins of Caulobacter crescentus.     

Spontaneous reversal of polarity of the voltage across LLC-PK1 renal epithelial cell sheets

While sterilely monitoring transepithelial voltage (potential difference) across LLC-PK cell sheets over a 24-hr period, we noted that the apical-negative, transepithelial voltage, a key property of the LLC-PK1 renal epithelial cell line, reverses polarity to become apical-positive. This spontaneous change of polarity of electrical potential difference (PD) across LLC-PK1 cell sheets cultured on permeable filters was observed to occur approximately 12 hr after refeeding. Unlike the apical (luminal)-negative PD, the apical-positive PD was insensitive to phlorizin and ouabain. Both were insensitive to the diuretics amiloride, furosemide, and 4-acetamido-4-isothiocynato-stilbene-2,2-disulfonic acid (SITS). A pH gradient existed across apical-positive cell sheets (apical medium more acidic by 0.3 units) but an osmotic gradient did not. Unlike the temperature-sensitive apical-negative PD, the apical positive PD was unaffected by brief exposure to 4 degrees C temperature. Junctional disruptive agents such as the tumor promotor, TPA, dissipated both types of PD with similar time courses. The formation of the apical-positive PD correlated in time with apical glucose levels falling below the reported Km of the Na+-sugar contransporter. A high glycolytic rate per se may not be essential for this PD polarity reversal since the reversal could occur in glucose-free medium with a normal time course and magnitude. The lysis with time of floating cells with consequent release of KCl into the apical compartment was also considered as a possible cause of the polarity reversal, but the turnover of even 2 X 10(6) cells in 12 hr was found not to raise apical KCl sufficiently to produce the polarity shift. Although a significant K+ gradient did not exist across cell sheets with apical-positive PD values, a sizable gradient of Cl- did exist, directed apical to basolateral. This gradient, coupled with anion-selective tight junctions, should contribute to the observed apical positive voltage. The voltage polarity shift seen in these cell cultures with time is not unlike the polarity shift occurring in the renal proximal convoluted tubule, with distance from the glomerulus.     

Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection

An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C₁₈ column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a flow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofluids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipids in biological samples of different matrices.     

Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS

Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.     

Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca²⁺ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca²⁺ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.     

Recovery of small bioparticles by interfacial partitioning

In this article, a qualitative study of the recovery of small bioparticles by interfacial partitioning in liquid-liquid biphasic systems is presented. A range of crystallised biomolecules with varying polarities have been chosen such as glycine, phenylglycine and ampicillin. Liquid-liquid biphasic systems in a range of polarity differences were selected such as an aqueous two-phase system (ATPS), water-butanol and water-hexanol. The results indicate that interfacial partitioning of crystals occurs even when their density exceeds that of the individual liquid phases. Yet, not all crystals partition to the same extent to the interface to form a stable and thick interphase layer. This indicates some degree of selectivity. From the analysis of these results in relation to the physicochemical properties of the crystals and the liquid phases, a hypothetical mechanism for the interfacial partitioning is deduced. Overall these results support the potential of interfacial partitioning as a large scale separation technology.     

Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels

When linear or supercoiled DNA molecules are imbedded in agarose gels and subjected to electric fields, they become oriented in the gel matrix and give rise to an electric birefringence signal. The sign of the birefringence is negative, indicating that the DNA molecules are oriented parallel to the electric field lines. If the DNA molecules are larger than about 1.5 kilobase pairs, a delay is observed before the birefringence signal appears. This time lag, which is roughly independent of DNA molecular weight, decreases with increasing electric field strength. The field-free decay of the birefringence is much slower for the DNA molecules imbedded in agarose gels than observed in free solution, indicating that orientation in the gel is accompanied by stretching. Both linear and supercoiled molecules become stretched, although the apparent change in conformation is much less pronounced for supercoiled molecules. When the electric field is rapidly reversed in polarity, very little change in the birefringence signal is observed for linear or supercoiled DNAs if the equilibrium orientation (i.e., birefringence) had been reached before field reversal. Apparently, completely stretched, oriented DNA molecules are able to reverse their direction of migration with little or no loss of orientation. If the steady-state birefringence had not been reached before the field reversal, complicated orientation patterns are observed after field reversal. Very large, partially stretched DNA molecules exhibit a rapid decrease in orientation at field reversal. The rate of decrease of the birefringence signal in the reversing field is faster than the field-free decay of the birefringence and is approximately equal to the rate of orientation in the field (after the lag period).(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.     

Two-dimensional microcolumn separation platform for proteomics consisting of on-line coupled capillary isoelectric focusing and capillary electrochromatography. 1. Evaluation of the capillary-based two-dimensional platform with proteins, peptides, and human serum

In this report, an on-line coupling of capillary isoelectric focusing (CIEF) to capillary electrochromatography (CEC) is developed via a nanoinjector valve for performing two-dimensional (2D) proteomics separation. CIEF constitutes the first separation dimension, while CEC operates as the second separation dimension. Besides the orthogonal migration mechanisms of the two capillary-based separation modes, which lead to a 2D system whose overall peak capacity is the product of the peak capacity of the individual modes, the solvent of the CIEF mode is a weak eluent for the reversed-phase CEC (RP-CEC) mode, thus, allowing the transferring of focused fractions from CIEF to CEC without inducing band broadening, and instead zone sharpening would result. In fact, the transferred focused protein fraction from the CIEF column to the CEC column will stay tightly adsorbed to the inlet top of the CEC column until it will be eluted and separated into its protein components with a hydro-organic mobile phase. The theoretical peak capacity of the CIEF-CEC 2D platform is estimated at n(CIEF) (= 560) x n(CEC) (= 97) = 54 320. This peak capacity is more than needed for proteomics profiling. Also, only a fraction of this peak capacity is needed when looking at heart cuts for performing subproteomics. The 2D platform described here offers the convenience to generate the needed peak capacity to solve a given proteomic separation problem. This is facilitated by the RP-CEC dimension, which ensures rapid isocratic separation of proteins and peptides and rapid solvent change and column equilibration and avoids lengthy gradient elution. The RP-CEC column is based on neutral C17 monolith, which offers high separation efficiency and relatively high column permeability. To the best of our knowledge, the proposed 2D platform combining CIEF and CEC is reported for the first time for proteins and proteomics.