Capillary electrochromatography of proteins and peptides

This review summarizes applications of CEC for the analysis of proteins and peptides. This "hybrid" technique is useful for the analysis of a broad spectrum of proteins and peptides and is a complementary approach to liquid chromatographic and capillary electrophoretic analysis. All modes of CEC are described--granular packed columns, monolithic stationary phases as well as open-tubular CEC. Attention is also paid to pressurized CEC and the chip-based platform.     

Capillary electrochromatography of peptides and proteins

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.e., design of new stationary phases and development of new column configurations, we describe here a variety of column architectures along with their advantages and disadvantages. Newly emerged column technologies in CEC for high speed and high efficiency separation are also discussed. Different analytical platforms of CEC such as pressure-assisted CEC or voltage-assisted micro- high-performance liquid chromatography (HPLC), CEC with different detection techniques, CEC on microchip platforms and multidimensional electrochromatography with their applications in peptide and protein analysis are presented.     

Capillary electrochromatography of peptides and proteins

This review surveys the accomplishments in the separation of peptides and proteins by capillary electrochromatography (CEC) over the last decade. A significant number of research articles have been published on this topic since the last review. Peptide and proteins separations have been carried out in all three formats of CEC, i.e., packed bed, continuous bed and open-tubular (OT) format. In addition to electrophoresis, different chromatographic modes have been successfully exploited with the most prevalent being reversed-phase mode followed by ion-exchange. Although many researchers continue to use model proteins and peptides primarily to evaluate the performance of novel stationary phases some researchers have also applied CEC to the analysis of real-life samples. The potential of CEC to yield complementary information and sometimes a superior separation with respect to established techniques, i.e., microbore HPLC and capillary electrophoresis has been demonstrated. Instrumental modifications in order to facilitate coupling of CEC to mass spectrometry have further upgraded the value of CEC for proteomic analysis. Capillaries are still the separation vehicle of choice for most researchers yet the microfluidic platform is gaining momentum, propelled particularly by its potential for multitasking, e.g., performing different chromatographic modes in series.     

Folded emitters for nanoelectrospray ionization mass spectrometry

Electrospray ionization (ESI) has revolutionized mass spectrometry (MS), providing a facile method for the ionization of macromolecules for analysis by mass. The development of nanoESI-MS has further extended the utility of ESI-MS, permitting the analysis of small-volume samples with enhanced sensitivity over conventional ESI-MS. Traditional nanoESI-MS experiments use pulled-glass capillary emitters, which are expensive to purchase and require specialized instruments and training to fabricate in-house. Furthermore, these emitters suffer from problems including clogging, sample contamination, and irreproducible spray stability. Here, we report a new emitter for nanoESI-MS, made by folding small pieces of polyimide tape. In comparison with conventional pulled-glass capillary emitters, the new emitters are inexpensive and simple to make. Their low cost makes them disposable after a single use, such that sample contamination or clogging is never a problem. Emitter performance has been evaluated for diverse analytes encompassing a large mass range, including small molecules, peptides, proteins, and synthetic polymers. In all cases, the performance is similar to that of pulled-glass capillary emitters, with the advantages of low cost, ease of use, and disposability.     

Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection.

On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-microns I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15-20 mM) of ammonium acetate or ammonium formate, and high organic modifier content (5-50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0-0.04 microliter/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous 'infusion' of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.     

Top-down characterization of proteins and drug-protein complexes using nanoelectrospray tandem mass spectrometry

We report a 'top-down' approach for characterization of proteins, and identification of binding sites in protein-drug complexes using nanoelectrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry (nanoESI-MS/MS). The efficiency of direct fragmentation of intact protein ions and the feasibility of this method were initially demonstrated using several well-characterized proteins with different molecular weights including metallothionein (6126 Da), cytochrome c (horse, 12360 Da), myoglobin (horse, 16592 Da), and hemoglobin (human, 64453 Da). Simply varying collision energy without enzyme digestion and gel or LC separation generated a range of peptide fragments of these proteins. Over 80% of these peptide ions matched those in the SWISS-PROT database with mass accuracy of 8 to 32 ppm with external calibration. This technique was further applied to fragment a cisplatin-metallothionein complex to identify the binding sites, demonstrating a potential application in the study of drug-protein binding.     

Induced nanoelectrospray ionization for matrix-tolerant and high-throughput mass spectrometry

No-contact rule: The title method is ultra-sensitive, high-throughput (4 samples per second), easily multiplexed, and is compatible with serum, urine, and concentrated salt solutions. Other features of this method, which avoids physical contact between the electrode and the solvent, include sample economy and the ability to produce both positive and negative-ion spectra in one cycle.     

A practical interface for microfluidics and nanoelectrospray mass spectrometry

We report a new method for fabricating nanospray ionization tips for MS, formed from glass substrates and the inert polymer, parylene-C. Using a single photolithography step, the emitters are formed contiguously with microchannels, such that no dead volumes are observed. In addition, because the devices are very thin (approximately 0.3 mm) and the tips are formed at rectangular corners, the Taylor cone volumes are small, which makes the method attractive for future integration with microfluidic separations. Device performance was demonstrated by evaluating diverse analytes, ranging from synthetic polymers, to peptides, to nucleic acids. For all analytes, performance was similar to that of conventional emitters (pulled-glass capillaries and the Agilent HPLC Chip) with the advantage of rapid, batch fabrication of identical devices.     

Capillary electrophoresis tandem mass spectrometry of bromine-containing charged derivatives of peptides

Novel bromine-containing positively charged labels 5-bromo-1-ethyl-thiazolium (BET+) and 5-bromo-1-ethyl-pyridinium (BEP+) ions were studied for improving the interpretation of MS/MS spectra of peptides. 2,5-Dibromo-1-ethyl-thiazolium tetrafluoroborate (DBET) reacts in the order: varepsilon->alpha-amino group>hydroxyl group of Tyr while 2,5-dibromo-1-ethyl-pyridinium tetrafluoroborate (DBEP) reacts preferably with thiol group of Cys>hydroxyl group of Tyr. In this study a simple and fast CE/MS/MS method is presented for investigating the labeling reaction with these new reagents, where the difference in migration times of labeled and unlabeled peptides also gives us information about the position of labeling. These bromine-containing reagents simplify the MS/MS spectra of peptides: the charge of the derivatives increases the intensity of the corresponding ions, thus enhancing the sensitivity of the detection and the characteristic distribution of the bromine isotope (the 79Br and 81Br ratio is nearly one) facilitating the recognition. By eliminating the non-doubled peaks, clear and easily interpretable MS/MS spectra can be produced that contain only the labeled fragments.     

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.     

Capillary electrochromatography of therapeutic peptides on mixed-mode butylmethacrylate monoliths

In this study, a porous mixed-mode n-alkyl methacrylate-based monolith has been used in the separation of therapeutic peptides. While the sulfonic acid (SCX) moiety derived from 2-acrylamido-2-methyl-1-propanesulfonic acid supports the generation of a stable electroosmotic flow (EOF) at both acidic and basic pH values, the butyl ligands provide the nonpolar sites for chromatographic resolution. The performance of the monolith was evaluated regarding the influence of pH on chromatographic resolution of peptides. The suitability of the butylmethacrylate/SCX monolith for the analysis of therapeutic peptides containing basic centres, for example arginine, at moderately high pH 9.5 and the stability to repeat injections of a mixture of peptides was demonstrated. Separations with efficiencies as high as 5.0 x 10(5) plates/m were obtained and the migration behaviour of the peptides at both low (2.8) and high (9.5) pH values could be rationalised based on their charge, molecular mass/shape and relative hydrophobicities.     

Characterization of micromachined hollow tips for two-dimensional nanoelectrospray mass spectrometry

In this work an improved design of chip-based nanoelectrospray nozzles is reported. Two-dimensional matrices of out-of-plane 10 microm i.d. silicon dioxide tips with a tapered shape were manufactured using deep reactive ion etching technology. Using a peptide sample, six micromachined tips and six commercially pulled silica capillary tips were compared employing an ion trap mass spectrometer. At a flow rate of 100 nL/min, the detectability obtained was approximately the same for the two types of tips. The relative standard deviation of the signal-to-noise ratio for the peptides between six different tips was on average 22% for the micromachined tips and 45% for the pulled capillary tips. The usefulness of the micromachined tips for analysis of non-covalent protein-ligand complexes was demonstrated by the analysis of a sample of RNase A and cytidine 2'-monophosphate. In another test, analyzing a tryptic digest of 1 pmol/microL cytochrome C, 18 peptides corresponding to a 82% sequence coverage were detected. Using MS/MS, the whole sequence of an 11 amino acid cytochrome C fragment was obtained. Computer simulations were performed on the shape and magnitude of the electrical field around micromachined and pulled capillary tips. To reach the threshold electric field density at the tip apex required to initiate an electrospray, a higher electrospray voltage was needed for the chip-based tips compared with pulled capillary tips. This is due to the influence of the chip base.     

Off-line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time-of-flight mass spectrometry and tandem mass spectrometry for glycoconjugate analysis

Implementation and optimization of an off-line capillary electrophoresis (CE)/(−)nanoESIchip-quadrupole time-of-flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping and structural investigation of components in complex carbohydrate mixtures is described. The approach was developed for glycoscreening and applied to O-glycosylated peptides from urine of a patient suffering from α-N-acetylhexosaminidase deficiency, known as Schindler's disease. The fundamental issue of sensitivity, previously representing a serious drawback of the off-line CE/MS analysis, could be positively addressed by the off-line conjunction of CE with automated chip-based ESI-QTOF-MS to provide flexibility for CE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures. Copyright © 2004 John Wiley & Sons, Ltd.     

Direct monitoring of protein-chemical reactions utilising nanoelectrospray mass spectrometry

The feasibility of nanoelectrospray mass spectrometry (nanoESI) for the direct analysis of protein chemical reactions and structural changes of proteins has been evaluated. Taking advantage of the long spraying time and the capability of nanoESI for employing a wide range of solvent conditions such as buffers and detergents, applications of monitoring reaction pathways, and dynamics have been carried out with several peptides and proteins. The time course of proteolytic digestions with trypsin and pepsin was investigated for several model polypeptides, and nanoESI showed to provide an efficient tool for optimising digestion conditions for the mass spectrometric peptide mapping analysis. Examples of specific protein chemical modification reactions at arginine and tyrosine residues illustrate the feasibility of nanoESI to monitoring reaction yields and modification sites for more than 180 min. Furthermore, changes of the pattern of protonated molecules caused by temperature effects and by protein unfolding due to disulfide bond reduction have been studied with the model proteins cytochrome c and hen eggwhite lysozyme. The results indicate that nanoESI is an efficient technique for the direct, molecular characterisation of protein-chemical reactions in solution.     

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.     

Micro-Electrospray: Zeptomole/attomole per microliter sensitivity for peptides

The micro-electrospray ionization source has been optimized for the specific analysis of neuropeptides such as neurotensin and methionine enkephalin. The source has the option of integrating nanoliter flow-rate desalting and preconcentration techniques into the micro-electrospray spray needle, eliminating post-column dead volumes. For neurotensin, the most sensitive neuropeptide analyzed thus far in this work, the injection of 10 μL of a solution containing 320 zeptomolesy/gmL gave an [M + 3H]⁺³ ion at m/z 558.4 with S/N of > 8∶1. The MS/MS analysis of this peptide for the fragment ion at m/z 578.9 gave a S/N > 20∶1 for a solution containing 32 attomoles/μL.     

Capillary electrochromatography of proteins and peptides with a cationic acrylic monolith

For the separation of proteins and peptides by capillary electrochromatography (CEC), columns with a monolithic stationary phase were prepared from silanized fused-silica capillaries of 50 microm I.D. by in situ copolymerization of glycidyl methacrylate, methyl methacrylate and ethylene glycol dimethacrylate in the presence of propanol and formamide as porogens. The epoxide groups at the surface of the porous monolith were reacted with N-ethylbutylamine to form fixed tertiary amino functions with ethyl- and butyl-chains. A mixture of ribonuclease A, insulin, alpha-lactalbumin and myoglobin was separated isocratically by counterdirectional CEC with hydro-organic mobile phases containing acetonitrile and sodium phosphate buffer, pH 2.5. The separation of four angiotensin type peptides by CEC was also achieved under similar conditions. The elution order of proteins was similar to that obtained in reversed-phase chromatography. Plots of the migration factors for proteins and peptides against the acetonitrile concentration exhibit opposite trends. This is most likely due to the greater chromatographic retention and lower electrophoretic migration velocity of proteins than that of peptides in the counterdirectional CEC system. From this it is concluded that the separation is governed by a dual mechanism that involves the complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Determination of protein-ligand association thermochemistry using variable-temperature nanoelectrospray mass spectrometry

A novel temperature-controlled nanoelectrospray (nanoES) device, interfaced with a Fourier transform ion cyclotron resonance mass spectrometer, is used to measure the association constants, Kassoc, for a series of protein-carbohydrate complexes at solution temperatures ranging from 5 to 40 degrees C. From a van't Hoff analysis of the Kassoc values, the enthalpies and entropies of association (DeltaHassoc, DeltaSassoc) at 25 degrees C are determined. The nanoES/mass spectrometry-derived thermodynamic parameters are in agreement with values previously determined by isothermal titration calorimetry.     

Review coupling of capillary electrochromatography to mass spectrometry

This review discusses the development of capillary electrochromatography (CEC) coupled to mass spectrometric (MS) detection over the last few years. Major topics addressed are instrumental setups employed and applications of this technology published in the recent literature. The instrumental section includes a discussion of the most commonly used interfaces for the hyphenation of CEC and MS as well as ionization techniques. Applications reviewed in this paper come from a variety of different fields such as the analysis of biomolecules like proteins, peptides, amino acids or carbohydrates, chiral separations or the analysis of pharmaceutical an their metabolites in a series of matrices.