Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC and ZIC-cHILIC) provide high resolution separation and increase sensitivity in proteome analysis

The complexity of peptide mixtures that are analyzed in proteomics necessitates fractionation by multidimensional separation approaches prior to mass spectrometric analysis. In this work, we introduce and evaluate hydrophilic interaction liquid chromatography (HILIC) based strategies for the separation of complex peptide mixtures. The two zwitterionic HILIC materials (ZIC-HILIC and ZIC-cHILIC) chosen for this work differ in the spatial orientation of the positive and negative charged groups. Online experiments revealed a pH-independent resolving power for the ZIC-cHILIC resin while ZIC-HILIC showed a decrease in resolving power at an acidic pH. Subsequently, we extensively evaluated the performances of ZIC-HILIC and ZIC-cHILIC as first dimension in an off-line two-dimensional liquid chromatography (2D-LC) strategy in combination with reversed phase (RP), with respect to peptide separation efficiency and how the retention time correlates with a number of peptide physicochemical properties. Both resins allowed the identification of more than 20,000 unique peptides corresponding to over 3500 proteins in each experimental condition from a remarkably low (1.5 μg) amount of starting material of HeLa lysate digestion. The resulting data allows the drawing of a comprehensive picture regarding ZIC- and ZIC-cHILIC peptide separation characteristics. Furthermore, the extent of protein identifications observed from such a level of material demonstrates that HILIC can rival or surpass traditional multidimensional strategies employed in proteomics.     

HPLC separation of oligonucleotides in isocratic and temperature-programming mode

In HPLC, temperature programming and isocratic separation were investigated for resolving a mixture of nucleotide polymers. A mixture of short oligonucleotides pd(A)(12)(-)(18) was resolved in less than 4 min by utilizing isocratic separation. The method is sensitive to organic modifier concentration; a 0.5% change of organic modifier in the mobile phase leads to more than doubling of the total analysis time. Temperature programming was used to optimize the separation of larger oligonucleotides pd(A)(25)(-)(30) and pd(A)(40)(-)(60). When the column temperature was changed by programming during the separation, the analysis time was 75% less than for an isocratic/isothermal run. The low amounts of ion-pairing agent in the mobile phase (between 5 and 10 mM) make these methods suitable for electrospray ionization mass spectrometry.     

Isolation and characterization of glycosylphosphatidylinositol-anchored peptides by hydrophilic interaction chromatography and MALDI tandem mass spectrometry

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney membrane dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma membrane proteins.     

Approximate and exact equations for peak capacity in isocratic high-pressure liquid chromatography

In this letter, we examine an equation that had been published in Analytical Chemistry in a paper entitled "Comprehensive Study on the Optimization of Online Two-Dimensional Liquid Chromatographic Systems Considering Losses in Theoretical Peak Capacity in First- and Second-Dimensions: A Pareto-Optimality Approach" by Vivo-Truyols, G.; van der Wal, Sj.; Schoenmakers, P. J. Anal. Chem. 2010, 82, 8525-8536. In that paper, the authors considered, among many issues, the effects of extra-column and column broadening on isocratic peak capacity. They developed an equation to cover all possible conditions and offered a derivation based on two Taylor-series expansions and a regression. We have found an exact equation that covers all conditions and have compared the results using our equation to the results using their approximation in predicting ratios of peak widths. Their approximation works well, as we show, but we wish to offer the exact equation which is simpler in form than the approximate solution.     

Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides

We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH <5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.     

Chiral separations of polar compounds by hydrophilic interaction chromatography with evaporative light scattering detection

The chiral separations of drug substances and underivatized amino acids were demonstrated in this study through the use of hydrophilic interaction chromatography (HILIC). The polar character of the model compounds presented challenges for their analysis by traditional modes of chromatography, but through the employment of multimodal chromatography utilizing the HILIC mechanism and cyclodextrin- or teicoplanin-derivatized stationary phases, effective resolution was achieved. The analytes lacked sufficient ultraviolet chromophores, requiring their determination by evaporative light scattering detection. HILIC was demonstrated to represent a novel technique for the facilitation of chiral chromatography by providing an environment of solubility and retention that could not be achieved through the use of the traditional methods of reversed-phase, normal-phase, or polar organic mode.     

Hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes

A porous zwitterionic monolith was prepared by thermal copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine and ethylene dimethacrylate inside a 100-mum-i.d. capillary. The resulting monolith was evaluated as a hydrophilic liquid chromatography (HILIC) stationary phase. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical HILIC mechanism was observed at higher organic solvent content (ACN% > 60%). The poly(SPE-co-EDMA) monolith showed very good selectivity for neutral, basic, and acidic polar analytes. For charged analytes, both hydrophilic interactions and electrostatic interactions contributed to their retention, which provide chromatographers more choice to optimize the separations.     

Evaluation of automated isocratic and gradient nano-liquid chromatography and capillary electrochromatography

An automated liquid nano-separation system has been developed for nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) using both isocratic and gradient elution. One fused-silica nanocolumn, typically 75 μm i.d. × 39 cm (25 cm effective packed length), packed with Spherisorb ODS 1, 3 μm particle size, can be used with either technique without having to remove the column upon switching from one mode to the other. The mobile phase is delivered by two reciprocating micro-LC pumps at a flow rate of 30 μL/min to a postinjection splitter that houses the nanocolumn inlet. The splitter is directly connected to a micro-injection valve with a 0.5 μL injection volume. In the CEC mode, pressure is not applied (no restriction on splitter) to the column inlet or outlet and the voltage is continuously applied during sample injection and mobile phase delivery. In the nano-LC mode, the restrictor is coupled to the splitter. Using the same nanocolumn under isocratic conditions, the repeatabilities of retention time and peak area for nano-LC were better than 0.2% and 4%, respectively, and those for CEC were better than 0.6% and 6%, respectively. On average, column efficiency was 57% higher in CEC compared to nano-LC. Gradient elution separations of parabens and polynuclear aromatic hydrocarbons (PAHs) were accomplished by CEC.     

Atom-transfer radical graft polymerization initiated directly from silica applied to functionalization of stationary phases for high-performance liquid chromatography in the hydrophilic interaction chromatography mode

Initiation of atom-transfer radical polymerization of a number of monomers (styrene, methyl acrylate, 3-[N,N-dimethyl-N-(methacryloyloxyethyl)ammonium] propanesulfonate, butyl methacrylate, 2,3-epoxypropyl methacrylate) directly from chlorinated porous silica particles has been performed. The grafting has been confirmed and evaluated by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. This initiation technique results in a hydrolytically stable initial Si-C bond, tethering the polymer to the silica substrate. The resulting grafted particles have been used as separation materials for both reversed-phase and hydrophilic interaction chromatography.     

Archiving and absolute quantitation of solutes separated by single charged droplet coulomb explosion

The electrospray (ES) ion source relies on the transfer of low-volatility solutes to the gas phase as an outcome of coulomb explosion events of charged droplets generated by electrical atomization. Introduced here are two methods for archiving compounds separated by coulomb explosion of single droplets having net charge that had been levitated in an electrodynamic balance. We categorized compounds separated by the explosion as either material ejected, including progeny droplets, or the material retained in the main residue. The potential for this methodology is illustrated by (i) qualitative characterization of solute states, aqueous versus precipitated in the separated material, and (ii) absolute quantitation of solutes separated by such an event. For a droplet containing 5 x 10(7) 20-nm-diameter fluospheres, its first encountered coulomb explosion event resulted in the ejection of 1.70% of them. The capability to acquire such detailed information regarding the individual steps in the process of transferring low-volatility solutes to the gas phase in an ES ion source is essential to develop strategies for absolute quantitation in applications of ES mass spectrometry.     

Development of an on-line SPE-LC-ESI-MS method for urinary nucleosides: hyphenation of aprotic boronic acid chromatography with hydrophilic interaction LC-ESI-MS

The development of an on-line automated SPE-HPLC--ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside levels. The setup comprises a boronate affinity column as a trapping device, a hydrophilic interaction chromatography (HILIC) separation and information-dependent MS detection modes. The system was optimized using standards and tested on biological samples, detecting a number of modified nucleosides. Other urinary biomarkers could also be analyzed by the system developed: for example, the urinary nucleobases were also available for analysis. A simultaneous creatinine-monitoring experiment was also demonstrated to be viable when utilizing the method, which is of benefit as creatinine is a urinary normalizing factor.     

Evaluation of the deuterium isotope effect in zwitterionic hydrophilic interaction liquid chromatography separations for implementation in a quantitative proteomic approach

Quantitative methodologies for the global in-depth comparison of proteomes are frequently based on chemical derivatization of peptides with isotopically distinguishable labeling agents. In the present work, we set out to study the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC (zwitterionic hydrophilic interaction liquid chromatography) technology for quantitative proteomics. We first addressed the potential issue of isotope effects perturbing the essential coelution of differently labeled peptides under ZIC-cHILIC separation. The deuterium incorporation-induced effect can be largely eliminated by favoring the mixed-mode ZIC-cHILIC separation based on combined hydrophilic and ionic interactions. Then, we evaluated the performance and applicability of this strategy using a sample consisting of human cell lysate. We demonstrate that our approach is suitable to perform unbiased quantitative proteome analysis, still quantifying more than 2500 proteins when analyzing only a few micrograms of sample.     

Chitosan-based membrane chromatography for protein adsorption and separation

A chitosan-based membrane chromatography was set up by using natural chitosan/carboxymethylchitosan (CS/CMCS) blend membrane as the matrix. The dynamic adsorption property for protein (lysozyme as model protein) was detailed discussed with the change in pore size of the membrane, the flow rate and the initial concentration of the feed solution, and the layer of membrane in membrane stack. The best dynamic adsorption capacity of lysozyme on the CS/CMCS membrane chromatography was found to be 15.3 mg/mL under the optimal flow conditions. Moreover, the CS/CMCS membrane chromatography exhibited good repeatability and reusability with the desorption efficiency of ~ 90%. As an application, lysozyme and ovalbumin were successfully separated from their binary mixture through the CS/CMCS membrane chromatography. This implies that such a natural chitosan-based membrane chromatography may have great potential on the bioseparation field in the future.     

Speciation analysis of gadolinium-based MRI contrast agents in blood plasma by hydrophilic interaction chromatography/electrospray mass spectrometry

The first analytical method for simultaneous speciation analysis of five of the most important gadolinium-based magnetic resonance imaging (MRI) contrast agents in blood plasma samples was developed. Gd-DTPA (Magnevist), Gd-BT-DO3A (Gadovist), Gd-DOTA (Dotarem), Gd-DTPA-BMA (Omniscan), and Gd-BOPTA (Multihance) were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospray mass spectrometry (ESI-MS). Spiking experiments of blank plasma with Magnevist and Gadovist were performed to determine the analytical figures of merit and the recovery rates. The limits of detection ranged from 1 x 10 (-7) to 1 x 10 (-6) mol/L depending on the ionization properties of the individual compounds, and limits of quantification ranged from 5 x 10 (-7) to 5 x 10 (-6) mol/L. The linear concentration range comprised 2 orders of magnitude. With application of this method, blood plasma samples of 10 healthy volunteers, with Magnevist or Gadovist medication, were analyzed for Gd-DTPA and Gd-BT-DO3A, respectively. The obtained results were successfully validated with inductively coupled plasma-optical emission spectroscopy (ICP-OES).     

Centrifugation assisted microreactor enables facile integration of trypsin digestion, hydrophilic interaction chromatography enrichment, and on-column deglycosylation for rapid and sensitive N-glycoproteome analysis

Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.     

Aptamer-modified monolithic capillary chromatography for protein separation and detection

A capillary chromatography technique was developed for the separation and detection of proteins, taking advantage of the specific affinity of aptamers and the porous property of the monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully immobilized on a streptavidin-modified polymer monolithic capillary column. The aptamer, having a G-quartet structure, could bind to both cytochrome c and thrombin, enabling the separation of these proteins from each other and from the unretained proteins. Elution of strongly bound proteins was achieved by increasing the ionic strength of the mobile phase. The following proteins were tested using the aptamer affinity monolithic columns: human immunoglobulin G (IgG), hemoglobin, transferrin, human serum albumin, cytochrome c, and thrombin. Determination of cytochrome c and thrombin spiked into dilute serum samples showed no interference from the serum matrix. The benefit of porous properties of the affinity monolithic column was demonstrated by selective capture and preconcentration of thrombin at low ionic strength and subsequent rapid elution at high ionic strength. The combination of the polymer monolithic column and the aptamer affinities makes the aptamer-modified monolithic columns useful for protein detection and separation.     

Prediction of analyte retention for ion chromatography separations performed using elution profiles comprising multiple isocratic and gradient steps

This study addresses the simulation of ion chromatographic (IC) separations performed under conditions where the elution profile consists of a sequence of isocratic and gradient elution steps (referred to as "complex elution profiles"). First, models for prediction of retention under gradient elution conditions in IC were evaluated using an extensive database of gradient elution retention data. It is shown that one such model is preferred on the basis that it can be used to predict gradient retention times on the basis of isocratic input data. A method is then proposed for using this model for complex elution profiles whereby each step of the elution profile is treated separately and analyte movement through the column is mapped. An empirically based algorithm for predicting peak width under complex elution conditions is also proposed. Evaluation of the suggested approaches was undertaken on a set of 24 analyte anions and 13 analyte cations on 5 different Dionex columns using a range of 5-step complex elution profiles that gave R2 values for correlations between predicted and observed retention times of 0.987 for anions and 0.997 for cations. The simulation of separations of anions and cations using a 3-step complex elution profile is demonstrated, with good correlation between observed and predicted chromatograms. The proposed approach is useful for the rapid development of separations when complex elution profiles are used in IC.