Capillary electrophoresis coupled to mass spectrometer for automated and robust polypeptide determination in body fluids for clinical use

We describe the application of capillary electrophoresis (CE) coupled on-line to an electrospray ionization-time of flight-mass spectrometer (ESI-TOF-MS) to the analysis of human urine and serum for the identification of biomarkers for clinical diagnostics. CE-MS led to display > 1000 polypeptides present in complex biological samples within 45-60 min in a single analysis run. To extract the information of the CE-MS spectra in a timely fashion, a software was designed to automatically deconvolute and normalize the spectra. Both urine and serum contain several hundred polypeptides in samples from healthy individuals. Hence, it is possible to establish typical "normal urine" or "normal serum" polypeptide patterns. Samples from patients with different diseases display polypeptide patterns that differ significantly from those obtained from healthy individuals. Examining series of patients with the same disease allowed the establishment of polypeptide patterns typical for specific diseases. This permits the search for marker peptides specific for diseases. The data indicate that a single polypeptide present in all patients with the same disease, but absent in all healthy control individuals does not exist. The combination of several polypeptides found in either urine or serum or both are forming a specific pattern, which is indicative not only for the particular disease, but also for the stage of disease. CE-MS detects many polypeptides in single samples and the application of the software to the search of identical polypeptides excreted in urine allows the unbiased diagnosis based on a pattern and does not rely on single disease markers.     

Determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers

The on-line coupling of capillary electrophoresis (CE) with electrospray-time-of-flight mass spectrometry (MS) has been used to obtain patterns of peptides and proteins present in the urine of healthy human individuals. This led to the establishment of a "normal urine polypeptide pattern", consisting of 247 polypeptides, each of which was found in more than 50% of healthy individuals. Applying CE-MS to the analysis of urine of patients with kidney disease revealed differences in polypeptide pattern. Twenty-seven polypeptides were exclusively found in samples of patients. Another 13, present in controls, were missing. These data indicate that CE-MS can be applied as powerful tool in clinical diagnostics.     

Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urine

We describe the use of capillary electrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47 patients who underwent prostate biopsy. Of this group, 21 patients had benign pathology and 26 with CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47 urine samples were compared to that of 41 young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474 samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP.     

Mass spectrometry for the detection of differentially expressed proteins: a comparison of surface-enhanced laser desorption/ionization and capillary electrophoresis/mass spectrometry

The discovery of biomarkers is currently attracting much interest as it harbors great potential for the diagnosis and monitoring of human diseases. Here we have used two advanced mass spectroscopy based technologies, surface enhanced laser desorption ionization (SELDI-MS) and capillary electrophoresis/mass spectrometry (CE/MS), to obtain proteomic patterns of urine samples from patients suffering from membranous glomerulonephritis (MGN) and healthy volunteers. The results indicate that CE/MS analysis is able to display a rich and complex pattern of polypeptides with high resolution and high mass accuracy. In order to analyze these patterns, the MosaiqueVisu software was developed for peak identification, deconvolution and the display of refined maps in a three-dimensional format. The polypeptide profiles obtained with SELDI-MS from the same samples are much sparser and show lower resolution and mass accuracy. The SELDI-MS profiles are further heavily dependent on analyte concentration. SELDI-MS analysis identified three differentially expressed polypeptides, which are potential biomarkers that can distinguish healthy donors from patients with MGN. In contrast, approximately 200 potential biomarkers could be identified by CE/MS. Thus, while SELDI-MS is easy to use and requires very little sample, CE/MS generates much richer data sets that enable an in-depth analysis.     

Capillary electrophoresis coupled to mass spectrometry to establish polypeptide patterns in dialysis fluids

Combination of capillary electrophoresis with mass spectrometry (CE-MS) allows generation of polypeptide patterns of body fluids. In a single CE-MS (45 min) run more than 600 polypeptides were analyzed in hemodialysis fluids obtained with different membranes (high-flux/low-flux). Larger polypeptides (M(r) > 10 000) were almost exclusively present in high-flux dialysates only, while in low-flux dialysates additional small polypeptides were detected. Comparison to the normal urine pattern yielded a surprisingly low consensus: a number of polypeptides present in urine were missing. We established a fast and sensitive technique, easily applicable to the monitoring of different modalities of dialyzers.     

Electrophoretic methods for analysis of urinary polypeptides in IgA-associated renal diseases

We evaluated the utility of SDS-PAGE/Western blot and CE coupled with MS (CE-MS) for detection of urinary polypeptide biomarkers of renal disease in patients with IgA-associated glomerulonephritides. In a reference cohort of 402 patients with various renal disorders and 207 healthy controls, we defined CE-MS patterns of renal damage and IgA nephropathy (IgAN). In a blinded analysis of a separate cohort of patients with IgAN (n = 10), Henoch-Schoenlein purpura (HSP) with nephritis (n = 10), and IgA-associated glomerulonephritis due to hepatitis C virus (HCV)-induced cirrhosis (n = 9), and healthy controls (n = 12), we compared SDS-PAGE/Western blot and CE-MS against clinical urinalysis for detection of urinary proteins/polypeptides. Urinalysis indicated proteinuria for 50, 90, and 33% of patients, respectively, and for none of the healthy controls. SDS-PAGE/Western blot showed urinary polypeptides abnormality for 90, 80, and 67% of patients, respectively, and for none of the healthy controls. CE-MS indicated a Renal Damage Pattern in 80, 80, and 100 of patients, respectively, and in 17% of healthy controls, with the more specific IgAN Pattern in 90, 90, and 1%, respectively, and in none of the healthy controls. Based on differences in CE-MS patterns, the disease mechanisms may differ among various IgA-associated glomerulonephritides. These exploratory findings should be evaluated in a prospective study with contemporaneous renal biopsy and urinary testing. If validated, it may be feasible to adapt the CE-MS methodology to develop novel tests to detect renal injury at earlier stages, assess clinical manifestations, and monitor responses to therapy in patients with IgA-associated renal diseases.     

Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.     

Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.     

Capillary electrophoresis coupled to mass spectrometry for clinical diagnostic purposes

The introduction of fast, sensitive, and robust techniques for proteomic analysis into clinical practice represents a major step toward a new diagnostic approach of body fluids. In addition, proteomics emerges as a key technology for the discovery of disease biomarkers in various body fluids. However, even in relatively protein-deprived body fluids such as urine, the complexity and wide dynamic range of protein expression pose a considerable challenge to both separation and identification technologies. In the present review we discuss from a clinical point-of-view recent advances of the use of proteomics in clinical diagnosis as well as therapy evaluation. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS) and discuss CE-MS from an application point of view evaluating its merits and vices with regard to biomarker discovery. This review further presents examples of clinical applications of CE-MS for detection and identification of biomarkers in urine.     

Capillary electrophoresis-mass spectrometry for glycoscreening in biomedical research

Application of capillary electrophoresis (CE) in combination with mass spectrometry (MS) and tandem MS to glycoscreening in biomedical projects is highlighted. In the first part recent CE-MS experiments by sheath liquid CE and multiple stage MS are reported. Neutral and negatively charged N-glycan mixtures from ribonuclease B and fetuin, high-mannose type N-glycoforms, oligosaccharides from lipopolysaccharides (LPS) of Haemophilus influenzae, polysaccharides of Pseudomonas aeruginosa and Staphylococcus aureus were analyzed. A particular emphasis is devoted to the applicability of novel off- and on-line CE-MS and tandem MS methods for screening of proteoglycan-derived oligosaccharides, glycosaminoglycans (GAGs), such as hyaluronates from Streptococcus agalactiae, chondroitin/dermatan sulfates (CS/DS) from bovine aorta and human skin fibroblast decorin, and heparin/heparan sulfate (HS) from porcine and bovine mucosa. The performance of CE-MS/MS for identification of glycoforms in glycopeptides and glycoproteins is illustrated by experiments performed on complex mixtures from urine of patients suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease) and urine of patients suffering from cancer cachexia. For determination of glycosylation patterns in glycoproteins like enzymes and antibodies by CE/MS, both CE-matrix assisted laser desorption/ionization (MALDI) and CE-electrospray ionization (ESI)-MS were functional. Finally, the potential of CE-ESI-MS strategy in glycolipid analysis is demonstrated for gangliosides from bovine brain for which particular CE buffer conditions are required.     

CE - a multifunctional application for clinical diagnosis

CE has been used widely as an analytical tool with high separation power taking advantage of size, charge-to-size ratio, or isoelectric point of various analytes. In combination with detection methods, such as UV absorption, electrochemical detection, fluorescence, or mass spectrometry (MS), it allows the separation and detection of inorganic and organic ions, as well as complex compounds, such as polypeptides, nucleic acids, including PCR amplicons from viruses or bacteria. Recent interest in identification of biomarkers of diseases using body fluids leads to development of CE-MS techniques. These applications allowed identification of new potential biomarkers for clinical diagnosis and monitoring of therapeutic interventions. In this report, we present a technical overview of various CE techniques and discuss their applications in clinical medicine.     

Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS

The need for sensitive analytical technologies applicable to metabolic profiling of volume-restricted biological samples is high. Here, we demonstrate feasibility of capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (MS) with sheathless nano-electrospray interface for non-targeted profiling of ionogenic metabolites in body fluids of experimental animals. A representative mixture of the metabolites and body fluids of mice such as cerebrospinal fluid (CSF), urine and plasma were used as examples of low-volume biological samples for method evaluation. An injection volume of only 9?nL resulted in limits of detection between 0.7 and 12?nM for the metabolite mixture. The method allowed the detection of ～350 molecular features in mouse CSF (an injection volume of ca. 45?nL), while ～400 features were observed in mouse plasma and ～3,500 features in mouse urine (an injection volume of ca. 9?nL). The low-volume body fluid samples were analyzed directly after only 1:1 dilution with water, thereby fully retaining sample integrity, which is of crucial importance for non-targeted metabolic profiling. As little is known about the metabolic composition of mouse CSF, we identified a fraction of the molecular features in mouse CSF using accurate mass information, migration times, MS/MS data, and comparison with authentic standards. We conclude that sheathless CE-MS can be used for sensitive metabolic profiling of volume-restricted biological samples.     

<Superscript>0,2</Superscript>A<Subscript>n</Subscript> cross-ring cleavage as a general diagnostic tool for glycan assignment in glycoconjugate mixtures

In contrast to proteomics significantly less efficient analytical tools are presently available for high throughput glycomics using mass spectrometry. In this article, a strategy to use the (0,2)A(n) ring cleavage ion at the reducing end of free glycans as a diagnostic ion for assignment of free glycans, in presence of glycopeptides containing similar glycosylation patterns, is presented for rapid distinction in complex mixtures by mass spectrometry. The MS to MS/MS automatic switching, already previously introduced for the on-line LC-MS and CE-MS analysis, is shown in this contribution to be highly functional to obtain diagnostic fragmentation patterns of free glycan precursors in rapid screening of highly complex glycoconjugate mixtures obtained from clinical samples, namely from the urine of patients suffering from congenital disorders of glycosylation. Congenital disorders of glycosylation (CDG) are inherited metabolic diseases based on defects in the glycosylation pathways of glycoconjugates. The urine of CDG patients was reported to contain O-glycans and glycosylated amino acids at concentrations two to three orders of magnitude higher in comparison with the healthy control, characterized by a high degree of heterogeneity concerning the type, number, and values of molecular ions. Using the (0,2)A(n) ring cleavage ion approach by tandem MS, it was possible to sort out free glycans and get them assigned.     

Confirmation testing of amphetamines and designer drugs in human urine by capillary electrophoresis-ion trap mass spectrometry

Monitoring of amphetamines and designer drugs in human urine is a timely topic in clinical toxicology, surveillance of drug substitution, forensic science, drug testing at the workplace, and doping control. Confirmation testing of urinary amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) by capillary electrophoresis (CE) combined with atmospheric pressure electrospray ionization and ion trap mass spectrometry (MS) is described. Using an aqueous pH 4.6 buffer composed of ammonium acetate/acetic acid, CE-MS and CE-MS2 provided data that permitted the unambiguous confirmation of these drugs in external quality control urines. Furthermore, other drugs of abuse present in alkaline urinary extracts, including methadone and morphine, could also be monitored. The data presented illustrate that the sensitivity achieved with the benchtop MS is comparable to that observed by CE with UV absorption detection. CE-MS2 is further shown to be capable of identifying comigrating compounds, including the comigration of amphetamine with nicotine.     

Capillary electrophoresis-mass spectrometry of proteins at medium pH using bilayer-coated capillaries

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%.     

Analyses of quaternary ammonium drugs in horse urine by capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry (CE-MS) method for the analysis of quaternary ammonium drugs in equine urine was developed. Quaternary ammonium drugs were first extracted from equine urine by ion-pair extraction and then analysed by CE-MS in the positive electrospray ionization (ESI) mode. Within 12 min, eight quaternary ammonium drugs, each at 1 ng/mL in horse urine, could be detected. The confirmation of these drugs in urine samples was achieved by capillary electrophoresis tandem mass spectrometry (CE-MS/MS). A direct comparison of this method was made with existing liquid chromatography/mass spectrometry (LC-MS) methods in the detection and confirmation of glycopyrrolate and ipratropium bromide in horse urine. While the two drugs could be detected within the same CE-MS run at 1 ng/mL in urine, they could only be detected in separate LC-MS runs at 5 ng/mL in urine. In addition, CE-MS consumed a much smaller volume of extract; the analyte peak widths, in some cases, were much narrower; and as the quaternary ammonium ions were well separated electrophoretically from the mainly neutral urine matrix, a much cleaner background in the CE-MS total ion trace was observed.     

Simplified 2-D CE-MS mapping: analysis of proteolytic digests

It has been demonstrated that CE-MS is a very useful hyphenated technique for proteomic studies. However, the huge amount of data stored in a single CE-MS run makes it necessary to account with procedures able to extract all the relevant information made available by CE-MS. In this work, we present a new and easy approach capable of generating a simplified 2-D map from CE-MS raw data. This new approach provides the automatic detection and characterization of the most abundant ions from the CE-MS data including their mass-to-charge (m/z) values, ion intensities and analysis times. It is demonstrated that visualization of CE-MS data in this simplified 2-D format allows: (i) an easy and simultaneous visual inspection of large datasets, (ii) an immediate perception of relevant differences in closely related samples, (iii) a rapid monitoring of data quality levels in different samples, and (iv) a fast discrimination between comigrating polypeptides and ESI-MS fragmentation ions. The strategy proposed in this work does not rely on an excellent mass accuracy for peak detection and filtering, since MS values obtained from an IT analyzer are used. Moreover, the methodology developed works directly with the CE-MS raw data, without interference by the user, giving simultaneously a simplified 2-D map and a much easier and more complete data evaluation. Besides, this procedure can easily be implemented in any CE-MS laboratory. The usefulness of this approach is validated by studying the very similar trypsin digests from bovine, rabbit and horse cytochrome c. It is demonstrated that this simplified 2-D approach allows specific markers for each species to be obtained in a fast and simple way.     

Liquid chromatography/mass spectrometry for metabonomics investigation of the biochemical effects induced by aristolochic acid in rats: the use of information-dependent acquisition for biomarker identification

The toxic effects of oral administrations of nephrotoxic and carcinogenic aristolochic acid (AA) to male Sprague-Dawley rats were investigated by using high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Analysis of the urine and plasma samples revealed distinct changes in the biochemical patterns in the AA-dosed rats. After peak finding and alignment, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for multivariate data analysis. Potential biomarkers were studied by high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses. The MS/MS spectra of all endogenous metabolites satisfying the pre-defined criteria were acquired in a single information-dependent acquisition (IDA) analysis, demonstrating that IDA was an efficient approach for structural elucidation in metabonomic studies. Citric acid and a glucuronide-containing metabolite were observed as potential biomarkers in rat urine. A significant increase in plasma creatinine concentration was also observed in the AA-dosed rats, which indicated that AA induced an adverse effect on the renal clearance function.     

Capillary electrophoresis-mass spectrometry for the analysis of intact proteins 2007-2010

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.