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Most conventional analytical tools for the assessment of protein-protein interactions yield information on the bulk sample. By employing the efficient separation of intact proteins, affinity capillary electrophoresis (ACE) can measure the interaction of components of heterogeneous proteins with a target protein. In this work, the hyphenation of ACE with mass spectrometry (MS) is presented as a novel, highly selective tool for the assessment of protein-protein interactions. The binding of the protease inhibitor aprotinin to trypsinogen was used as protein-protein affinity model. A trypsinogen sample comprising several modifications was analyzed using a background electrolyte of 25 mM ammonium acetate (pH 8.0) containing increasing concentrations of aprotinin (0-300 μM). A capillary coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was employed to prevent adsorption of the proteins to the capillary wall. The trypsinogen variants were separated and could be assigned based on detected molecular masses and relative migration. In presence of aprotinin, both free and aprotinin-bound trypsinogen were detected revealing a 1:1 binding stoichiometry. For most trypsinogen variants, shifts in electrophoretic mobility were observed upon raising the aprotinin concentration, allowing determination of their dissociation constants (Kd's). The interacting trypsinogen variants showed similar affinity toward aprotinin (Kd's of 3-9 μM), which were not significantly different from the values obtained with ACE-UV and were in agreement with an earlier reported value. The use of the ratio of obtained MS signal intensities of free and protein-protein complex for the determination of Kd's was also explored. Derived Kd values (20-104 μM) for the binding variants were similar to those obtained with direct-infusion MS, but higher and less precise as compared with values based on mobility shifts. The suitability of the ACE-MS methodology for the affinity profiling of heterogeneous protein samples was evaluated, and components with high, medium, or low affinity toward aprotinin could be successfully discriminated.

This review treats the coupling of capillary electrophoresis (CE) with fluorescence detection (Flu) for the analysis of natively fluorescent biomolecular and pharmaceutical compounds. CE-Flu combines the excellent separation efficiency of CE with the high selectivity and sensitivity of Flu. In CE-Flu, an appropriate design of the fluorescence detection cell is very important in order to achieve efficient analyte excitation in and emission light collection from the small cylindrically-shaped detection volume. Therefore, due attention is paid to the various optical detection designs used for CE-Flu, including the applied excitation sources and emission light detectors. Special attention is devoted to wavelength-resolved Flu and to sensitivity issues. Furthermore, he specific requirements for fluorescence detection in microfluidic systems (i.e. chip-based electrophoresis) are discussed. Subsequently, an overview of described applications of CE-Flu for the analysis of natively fluorescent biomolecules and drugs is presented in extensive tables, treating amino acids, peptides, proteins, bioactive compounds, flavins, pharmaceuticals and also single cell analysis. The tables provide information on analyte nature, sample matrix, optical detection aspects, CE mode and limits of detection. A selection of descriptive applications is discussed in detail to illustrate the potential of native fluorescence detection in CE. It is concluded that CE-Flu is a powerful tool for biomolecular and pharmaceutical analysis, and provides good opportunities for use in lab-on-chip devices., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Since its introduction in 1987, CE-MS has become an increasingly important technique for the analysis of biomolecules. Since our previous update on CE-MS methods within the field of intact protein analysis (Electrophoresis 2011, 32, 66-82), a variety of interesting methodological improvements and applications have been reported in literature. Therefore, this article presents an overview of the development and application of CE-MS for intact protein analysis as published between June 2010 and June 2012. The article is divided in sections that treat CE coupled to MS through ESI, MALDI, and ICP ionization, respectively. In the section about CE-ESI-MS, technological developments with respect to CE-MS interfacing, prevention of protein adsorption, and chip-based CE-MS are treated in more detail. Novel interfacing strategies and the development of improved capillary coating strategies appeared to be the major developments. Furthermore, in all sections, the applicability of CE-MS for intact protein analysis is demonstrated by representative examples, including important developments in the fields of biopharmaceutical characterization and the analysis of proteins in biological samples. Finally, some general conclusions and future perspectives are given., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.

CE coupled to MS has proven to be a powerful analytical tool for the characterization of intact proteins, as it combines the high separation efficiency of CE with the selectivity of MS. This review provides an overview of the development and application of CE-MS methods within the field of intact protein analysis as published between January 2007 and June 2010. Ongoing technological developments with respect to CE-MS interfacing, capillary coatings for CE-MS, coupling of CIEF with MS and chip-based CE-MS are treated. Furthermore, CE-MS of intact proteins involving ESI, MALDI and ICP ionization is outlined and overviews of the use of the various CE-MS methods are provided by tables. Representative examples illustrate the applicability of CE-MS for the characterization of proteins, including glycoproteins, biopharmaceuticals, protein-ligand complexes, biomarkers and dietary proteins. It is concluded that CE-MS is a valuable technique with high potential for intact protein analysis, providing useful information on protein identity and purity, including modifications and degradation products., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A capillary electrophoresis-mass spectrometry (CE-MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol-water-acetic acid (75:25:0.1, v/v/v) at 2 μL min(-1) resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE-MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE-MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE-MS system, determination coefficients (R(2)) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A lamp-based fluorescence detection (Flu) system for CE was extended with a wavelength-resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe-Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut-off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal-intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE-WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged-polymer coated capillary. Using the 3-D data set, signal averaging over time and emission-wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.

Complex Regional Pain Syndrome (CRPS) is characterized by various combinations of sensory, autonomic and motor disturbances. Pain disproportionate to the severity and duration of the inciting event is the most devastating symptom. Diagnosis of CRPS is difficult as the underlying mechanisms remain unclear. To try to derive metabolic indicators potentially characteristic for CRPS, we applied capillary electrophoresis time-of-flight mass spectrometry (CE-ToF-MS) to the explorative analysis of urine. The CE-ToF-MS method provided fast and stable metabolic profiles of urine samples. The mean intraday and interday CVs were <2% and <9% for migration times and peak areas, respectively, demonstrating robustness of the method. With the use of multivariate chemometric analysis, discrimination between urine samples from CRPS patients and controls was obtained, emphasizing differences in metabolic signatures between CRPS-diseased patients and controls. Several compounds, such as 3-methylhistidine, were responsible for discriminating the samples. The biological relevance of these compounds with regard to CRPS is discussed. Thus, CE-ToF-MS-based metabolic profiling of urine from CRPS patients and controls revealed metabolites that differentiate between diseased and control, illustrating the usefulness of this approach to get more insight into the pathology underlying CRPS.

This paper describes the hyphenation of cIEF and MALDI-TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al., Electrophoresis, 2008, 29, 1985-1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF-MALDI-TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH4OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under- or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm(-2) trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol microL(-1) horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm x 50 microm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.

Developments in the fields of protein chemistry, proteomics and biotechnology have increased the demand for suitable analytical techniques for the analysis of intact proteins. In 1989, capillary electrophoresis (CE) was combined with mass spectrometry (MS) for the first time and its potential usefulness for the analysis of intact (i.e. non-digested) proteins was shown. This article provides an overview of the applications of CE-MS within the field of intact protein analysis. The principles of the applied CE modes and ionization techniques used for CE-MS of intact proteins are shortly described. It is shown that separations are predominantly carried out by capillary zone electrophoresis and capillary isoelectric focusing, whereas electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the most popular ionization techniques used for interfacing. The combination of CE with inductively coupled plasma (ICP) MS for the analysis of metalloproteins is also discussed. The various CE-MS combinations are systematically outlined and tables provide extensive overviews of the applications of each technique for intact protein analysis. Selected examples are given to illustrate the usefulness of the CE-MS techniques. Examples include protein isoform assignment, single cell analysis, metalloprotein characterization, proteomics and biomarker screening. Finally, chip-based electrophoresis combined with MS is shortly treated and some of its applications are described. It is concluded that CE-MS represents a powerful tool for the analysis of intact proteins yielding unique separations and information.

Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC-UV-mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification.

The feasibility of using noncovalently bilayer-coated capillaries for capillary electrophoresis-mass spectrometry (CE-MS) of acidic proteins was investigated using background electrolytes (BGEs) of medium pH. The capillary was coated by successively rinsing the capillary with solutions of the oppositely charged polymers polybrene (PB) and poly(vinyl sulfonic acid) (PVS). Volatile BGEs containing ammonium formate and/or N-methyl morpholine were tested at pH 7.5 and 8.5. Overall, these BGEs provided relatively fast protein separations (analysis times of ca. 12 min) and showed high efficiencies (70,000-300,000 plates) when the ionic strength was sufficiently high. Migration-time reproducibilities were very favorable with RSDs of less than 1.0%. Infusion experiments showed satisfactory MS responses for studied proteins dissolved in ammonium formate (pH 8.5), however, high concentrations of N-methyl morpholine appeared to seriously suppress the MS protein signals. Evaluation of the CE-MS system was performed by analyzing a mixture of intact proteins yielding efficient separations and good-quality mass spectra. CE-MS analysis of a reconstituted formulation of the biopharmaceutical recombinant human growth hormone (rhGH) which was stored for a prolonged time, revealed one degradation product which was provisionally identified as desamido rhGH. Based on the MS responses the amount of degradation was estimated to be ca. 25%.

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.

The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.

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.

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.