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Postcapillary affinity detection is developed to analyze structurally related compounds following capillary zone electrophoresis separation. In this work, biotin and biotin derivatives are resolved by capillary zone electrophoresis and selectively identified on the basis of their cross-reactivity with streptavidin−fluorescein isothiocyanate, added via a postcapillary reactor. Direct sensing of the affinity reaction is achieved by monitoring the resulting fluorescence enhancement of streptavidin−fluorescein isothiocyanate upon biotin binding. The effects of postcapillary reactor parameters, including capillary gap distance, dilution ratio, and reaction distance, on peak area and peak efficiency of the biotin−streptavidin complex are investigated. A reaction kinetic model is presented for evaluating the dynamic range and detection limits of postcapillary affinity detection as a function of streptavidin concentration in the postcapillary reactor. The detection limit for postcapillary affinity detection of bi...

Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-α1-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.

The combination of micellar electrokinetic chromatography (MEKC) with mass spectrometry (MS) is very attractive for the direct identification of analyte molecules, for the possibility of selectivity enhancement, and for the structure confirmation and analysis in a MS-MS mode. The direct coupling of MEKC with MS can be hazardous due to the effect of nonvolatile MEKC surfactants on MS performance, including the loss of analyte sensitivity and ion source contamination. The possibility of off-line coupling between MEKC and matrix-assisted laser desorption/ionization (MALDI)-MS remains to be investigated. Various approaches for on-line coupling MEKC with electrospray ionization (ESI)-MS, including the use of high-molecular-mass surfactant, an electrospray-chemical ionization (ES-CI) interface, a voltage switching and buffer renewal system, partial-filling micellar plug and anodically migrating micelles, are reviewed and evaluated. The use of an ES-CI interface is most promising for routine operation of on-line MEKC-MS under the influence of nonvolatile salts and surfactants. The use of a high-molecular-mass surfactant allows the formation of a micellar phase at very low surfactant concentrations and avoids the generation of a high level of background ions in the low m/z region. Alternatively, the application of a partial-filling micellar plug and anodically migrating micelles eliminate the introduction of MEKC micelles into the ESI-MS system. It is possible to directly transfer the conventional MEKC separations to partial-filling MEKC-ESI-MS and MEKC-ESI-MS using anodically migrating micelles without any instrument modifications.

On-line combination of capillary isoelectric focusing with electrospray ionization mass spectrometry is applied for a two-dimensional analysis of Escherichia coli proteins. The proteins are focused and cathodically mobilized in a polyacrylamide coated capillary. At the end of the capillary, various protein zones are analyzed by mass spectrometry coupled through an electrospray interface. Comparisons with silver-stained two-dimensional gel electrophoresis are made with regard to mass determination, resolution, speed, and sensitivity. Direct identification of a recombinant fusion protein of glutathione S-transferase and striped bass growth hormone is achieved without any prior protein isolation procedures.

Rapid identification of transient partially folded intermediates formed during protein refolding and aggregation has been difficult, particularly with separation methods relying on solid matrices. Capillary zone electrophoresis equipped with laser-induced fluorescence detection provides a fast sensitive means of identifying folding and aggregation intermediates using the intrinsic tryptophan fluorescence. The in vitro refolding of the trimeric P22 tailspike, a model system for the study of protein folding, misfolding and aggregation, has been monitored after dilution out of denaturant. Both monomeric and trimeric folding intermediates were resolved. The refolding kinetics and yields measured by capillary zone electrophoresis were in good agreement with those obtained via fluorescence spectrophotometry and polyacrylamide gel electrophoresis. In comparison with typical UV detection, laser-induced tryptophan fluorescence increased detection sensitivity. In addition, the fluorescence signal carries information on the packing of the tryptophan residues in the folding intermediates. For tailspike and many other proteins, the off pathway aggregation reactions proceed from a thermolabile intermediate at the junction with the productive pathway. By monitoring refolding intermediates after temperature shifts, the structured monomeric intermediate was identified as the thermolabile junctional intermediate between the productive and aggregation pathways.

On-line micellar electrokinetic chromatography (MEKC)-electrospray ionization mass spectrometry (ESIMS) is demonstrated for the analysis of chlorotriazine herbicides and barbiturates. In this study, the micellar velocity is directly manipulated by the adjustment of electroosmosis rather than the electrophoretic velocity of the micelle. The electroosmotic flow is adjusted against the electrophoretic velocity of the micelle by changing the solution pH in MEKC. The elimination of MEKC surfactant introduction into ESIMS is achieved with an anodically migrating micelle, moving away from the electrospray interface. The effects of moving surfactant boundary in the MEKC capillary on separation efficiency and resolution of triazine herbicides and barbiturates are investigated. The mass detection of herbicides and barbiturates sequentially eluted from the MEKC capillary is acquired using the positive and negative electrospray modes, respectively.

Post-capillary affinity detection is developed to selectively evaluate protein microheterogeneity in complex sample matrices. In this scheme, separation of protein variants is accomplished prior to affinity detection. A post-capillary reactor is constructed to allow the transfer of separated variants as well as the addition of a fluorescein-labeled analyte-binding protein into the reaction capillary. Affinity complexes are formed within the reaction capillary and monitored by laser-induced fluorescence detection. Fluorescence enhancement, upon affinity complex formation, allows selective detection of protein variants. Successful resolution of IgG Fc variants is presented and compared with affinity capillary electrophoresis techniques reported in the literature. Additionally, the effects of analyte transfer and affinity complex formation on the resolution and detection limits of IgG Fc variants are discussed.

A novel sheathless capillary isotachophoresis (CITP/CZE)-mass spectrometry (MS) interface featuring a large inner diameter (i.d.) separation capillary, and a detachable small i.d. porous electrospray ionization (ESI) emitter was developed in this study to simultaneously achieve large sample loading capacity and stable nanoESI operation. Crucial operating parameters, including sample loading volume, flow rate, and separation window, were systematically investigated to attain optimum CITP/CZE separation efficiency and MS detection sensitivity. The performance of CITP/CZE-nanoESI-MS using the new sheathless interface was evaluated for its achievable low limit of quantification (LOQ) by analyzing targeted peptides, leu-enkephalin and angiotensin II, spiked in a BSA tryptic digest matrix at different concentrations. A linear dynamic range spanning 4.5 orders of magnitude and a 10 pM LOQ with measurement reproducibility of the CV22% were obtained experimentally for both targeted peptides, representing a 5-fold sensitivity improvement as compared to using the sheath liquid interface developed previously.1.

The inherent disadvantage of biomarker dilution in complex biological fluids such as serum/plasma, urine, and saliva necessitates highly sensitive analytical approaches. Furthermore, in addition to sample complexity, the large variation of protein-relative abundances having potential biological significance in mammalian systems presents a major analytical challenge for achieving comprehensive proteomic studies of clinical specimens. Because the sizes of human tissue biopsies are becoming significantly smaller due to the advent of minimally invasive methods and early detection and treatment of lesions, more effective proteomic and metabolomic technologies are critically needed to enable investigative studies of proteins and metabolites that will have diagnostic and therapeutic relevance. This chapter focuses on the most recent advances in capillary electrophoresis-based single and multidimensional separations coupled with mass spectrometry for performing comparative analysis of protein expression levels and metabolite profiles within normal and diseased tissues, including biological fluids.

Due to the inherent disadvantage of biomarker dilution in complex biological fluids such as serum/plasma, urine, and saliva, investigative studies directed at tissues obtained from the primary site of pathology probably afford the best opportunity for the discovery of disease biomarkers. Still, the large variation of protein relative abundances with clinical specimens often exceeds the dynamic range of currently available proteomic techniques. Furthermore, since the sizes of human tissue biopsies are becoming significantly smaller due to the advent of minimally invasive methods and early detection and treatment of lesions, a more effective discovery-based proteomic technology is critically needed to enable comprehensive and comparative studies of protein profiles that will have diagnostic and therapeutic relevance.This review therefore focuses on the most recent advances in capillary electrophoresis-based single and multidimensional separations coupled with mass spectrometry for performing comprehensive proteomic analysis of clinical specimens. In addition to protein identification, monitoring quantitative changes in protein expression is essential for the discovery of disease-associated biomarkers. Comparative proteomics involving measurements in changes of biological pathways or functional processes are further expected to provide relevant markers and networks, molecular relationships among different stages of disease, and molecular mechanisms that drive the progression of disease.

On-line capillary zone electrophoresis/electrospray ionization mass spectrometry (CZE-ESMS) and capillary isoelectric focusing/electrospray ionization mass spectrometry (CIEF/ESMS) were employed for protein analysis. The separation mechanisms and the detection limits of CZE/ESMS and CIEF/ESMS were compared by using model proteins including cytochrome c (horse heart), ribonuclease A (bovine pancreas), myoglobin (horse heart), carbonic anhydrase I (human erythrocytes) and β-lactoglobulin A (bovine milk). The effect of a moving ionic boundary inside the electrophoresis capillary on the separation resolution of model proteins by CZE/ESMS and CIEF/ESMS is described. In CZE/ESMS, the formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be eliminated by using a common counterion in both the background electrolyte and the sheath liquid. Additionally, the moving ionic boundary in CIEF/ESMS is minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. The concentration detection limits of model proteins in a full-scan CIEF/ESMS analysis are in the region of 10 -7 M, ∼20-50 times lower than that achievable using CZE/ESMS. Sample preconcentration taking place during the focusing step in CIEF is responsible for the improved detection limits.

On-line combination of partial filling micellar electrokinetic chromatography (PF-MEKC) and electrospray ionization mass spectrometry (ESI-MS) is demonstrated for the analysis of triazine herbicides including atrazine, propazine, ametryne and prometryne. In comparison with conventional micellar electrokinetic chromatography (MEKC), PF-MEKC involves filling a small portion of the capillary with a sodium dodecyl sulfate (SDS) micellar solution for achieving the separation. In PF-MEKC, the triazine analytes first migrate into the micellar plug where the separation occurs and then into the electrophoresis buffer which is free of surfactant. Consequently, the electroosmotic transfer of neutral triazine herbicides to ESI-MS at the end of PF-MEKC capillary is comparable to conventional capillary zone electrophoresis ESI-MS. Therefore, PF-MEKC-ESI-MS provides a mechanism for the separation and mass detection of neutral molecules without the interference of surfactant.

On-line capillary isoelectric focusing (CIEF)-electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of hemoglobin variants A, C, S, and F. The effects of moving ionic boundary inside the CIEF capillary and MS scan rate on the separation resolution and mass detection of hemoglobin variants are investigated. The formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. Hemoglobin variants F and A, with a pI difference of 0.05 pH unit, are almost baseline resolved and identified in CIEF-ESIMS. The concentration detection limit for each hemoglobin variant is in the range of 10(-)(8) M, comparable to that obtained in two-dimensional gel electrophoresis using silver staining. Initial preconcentration during the focusing step and the use of single-ion monitoring scan mode are responsible for improving detection limits.

On-line combination of capillary isoelectric focusing (CIEF) with electrospray mass spectrometry (ESMS) as a two-dimensional separation system is demonstrated. Mixtures of model proteins including cytochrome c (horse heart), myoglobin (horse heart), and carbonic anhydrase II (bovine erythrocyte) are focused and cathodically mobilized in a polyacrylamide-coated capillary. At the end of CIEF capillary, the mobilized protein zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. The effects of carrier ampholyte concentration on the CIEF separation and the protein electrospray ionization mass spectra are presented and discussed. In this study, the focusing effect of CIEF permits analysis of very dilute protein samples. A typical concentration factor of 50-100 times is observed. The concentration detection limit of myoglobin for a full-scan CIEF-ESMS analysis is in the range of 10{sup -7} M, 2 orders of magnitude over that possible with normal capillary zone electrophoresis ESMS. 35 refs., 5 figs., 2 tabs.

The resolution in capillary zone electrophoresis (CZE), with the assumption of diffusion control only, is strongly dependent on the direction and magnitude of electroosmotic flow. In fact, excellent separation resolution will be obtained if the electroosmotic flow is in the opposite direction of the electrophoretic migration. By applying various radial electric potential gradients across the capillary wall, the direct control of the zeta potential and the electroosmotic flow results in a great enhancement of chiral resolution in cyclodextrin-modified CZE. All 12 basic chiral drugs, including (+/-)-ephedrine, (+/-)-pseudoephedrine, (+/-)-norephedrine, (+/-)-epinephrine, (+/-)-norepinephrine, and (+/-)-isoproterenol, are separated and resolved within 22 min of the analysis time. There is no additional band broadening and dispersion introduced by the direct control of electroosmosis with the application of various radial electric potential gradients.

We demonstrate the direct coupling of transient capillary isotachophoresis/ capillary zone electrophoresis (CITP/CZE) with a high sensitivity triple quadrupole mass spectrometer operating in selected reaction monitoring (SRM) mode for sample quantitation. The capability of CITP/CZE for in situ sample enrichment and separation has been shown to significantly improve the analytical figures of merit. A linear dynamic range spanning 4 orders of magnitude was observed. An average signal-to-noise ratio (S/N) of 49.6 was observed for 50 attomoles of targeted peptide in the presence of a complex and much more abundant bovine serum albumin (BSA) digest product. A correlation of variation (CV) less than 10 % for peak area was measured from triplicate sample analyses at 50 pM peptide concentration, showing good reproducibility of this online CITP/CZE-SRM mass spectrometry (MS) platform, and with limit of quantitation (LOQ) demonstrated to be well below 50 pM.

Besides proteome complexity, the greatest bioanalytical challenge facing comprehensive proteomic analysis, particularly in the identification of low abundance proteins, is related to the large variation of protein relative abundances. In contrast to universally enriching all analytes by a similar degree, the result of the capillary isotachophoresis (CITP) stacking process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins drastically reduces the range of relative protein abundances within complex proteomes and greatly enhances the resulting proteome coverage. Furthermore, CITP offers seamless combination with nano-reversed phase liquid chromatography (nano-RPLC) as two highly resolving and completely orthogonal separation techniques critically needed for analyzing complex proteomes.

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.

Besides proteome complexity, the greatest bioanalytical challenge facing comprehensive proteomic analysis, particularly in the identification of low abundance proteins, is related to the large variation of protein relative abundances. In contrast to universally enriching all analytes by a similar degree, the result of the capillary isotachophoresis (CITP) stacking process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins drastically reduces the range of relative protein abundances within complex proteomes and greatly enhances the resulting proteome coverage. Furthermore, CITP offers seamless combination with nano-reversed phase liquid chromatography (nano-RPLC) as two highly resolving and completely orthogonal separation techniques critically needed for analyzing complex proteomes.

Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.

Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new "multidimensional" capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional in vitro studies utilizing the U87 glioma cell line, we found that expression of NNAT is associated with significant increases in cellular proliferation. Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P = 0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents.

The electrokinetic phenomena at the silica-solution interface under the influence of applied radial electric potential gradient were analyzed by a theory based on the Gouy-Chapman-Stern-Grahame (GCSG) model and the induced effect across the capillary wall. The effect of adsorbed ions at the silica-solution interface on the direct control of electroosmosis was studied with the application of lithium ions, tin(IV) ions, and dodecyltrimethylammonium bromide (DTAB). In addition, various organic coatings, including butyl phase, amino phase, and (glycidoxypropyl)trimethoxysilane-ethylene glycol diglycidyl ether (GOX-EGD), were employed for investigating the effect of surface deactivation on the direct control of electroosmosis

The leakage current phenomenon at the capillary-solution interface was investigated with the application of high radial electric potential gradients across the capillary wall for the direct control of electroosmosis. The leakage current traveling through the capillary wall gave rise to a different surface potential between the controlled and uncontrolled regions, and resulted in a linear electroosmotic velocity profile from the controlled region to the ends of the capillary. The mismatch between the elctroosmotic velocities was then balanced by the additional laminar flow over the entire length of the capillary. The additional dispersion induced by the laminar flow under the leakage current situation was demonstrated and compared with theoretical prediction.

The ζ potential and the electroosmotic florw in micellar electrokinetic capillary chromatography (MEKC) can be directly controlled by using a radial electric potential gradient across the capillary wall. In this study, the separations of neutral phenylthiohydantoin amino acids as the model system in MEKC with direct control of electroosmosis and retention window are presented. Significant enhancement in the separation resolution of phenylthiohydantoin amino acids under the direct control of electroosmosis is demonstrated

The ξ potential and the electroosmotic flow in capillary electrophoresis can be directly controlled by using a radial electric potential gradient across the capillary wall. flow profiles and dispersion in the capillary with the direct control of electroosmosis are studied. The values of total spatial variance measured from the experiment are in good agreement with the predictions based on only molecular diffusion. There is no measurable additional dispersion and band broadening induced by the direct control of electroosmosis. A dc short circuit phenomenon at the capillary/solution interface is proposed for explaining our experimental observations

Wireless technology offers flexibility and mobility that enables users to access to any service including voice call, anytime, anywhere with any device. Transferring call or session across devices is also made possible with today's session management protocol such as SIP, provided the user knows the contact information of the target device. In this paper, we propose a method which simplifies the transfer of session across devices whereby the target device is automatically selected based on its proximity to the active source device. Such automation is enabled with the use of a customised Location Server that performs location estimation of all registered devices using the RSSI-based triangulation method. Our proposed method successfully eliminated the error-prone manual intervention on the selection of target device during the transfer, and achieved a small average error of 0.21 meter.

Background Ovarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients. Methods and Findings We compared the proteomes of paired primary and recurrent post-chemotherapy ovarian high-grade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-κB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-κB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in naïve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells. Conclusions Proteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-κB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer.

The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.

A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)-based multidimensional separations and multidimensional liquid chromatography system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second dimension separation (reversed phase liquid chromatography) and mass spectrometry conditions, and consistent search parameters and cutoff established by the target-decoy determined false discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications, analytical reproducibility of the CITP proteome platform coupled with the spectral counting approach is determined by a Pearson R2 value of 0.98 and a coefficient of variation of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional liquid chromatography separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.

Mitochondria are responsible for several essential functions, including apoptosis, iron-sulfur cluster assembly, and several important catabolic pathways in mammalian cells. Characterization of the mitochondrial proteome may therefore be extremely useful for identification and validation of targets for therapeutic treatment of human diseases, including aging and age-related disorders, diabetes, and obesity. While capillary isotachophoresis (CITP) has been introduced for on-capillary analyte preconcentration prior to electrophoretic separation, the application of transient CITP/capillary zone electrophoresis (CZE) to selectively enrich and resolve trace amounts of proteins/peptide is employed in an effort to expand mitochondrial proteome coverage in mammals. In this study, a total of 2095 distinct mouse SwissProt protein entries are identified from synaptic mitochondria isolated from mouse brain, corresponding to 50% and 76% coverage of the Maestro and MitoP2 mitochondrial reference sets, respectively. The increased coverage of mouse mitochondrial proteome further reveals the top 17 biosynthetic and metabolic pathways in synaptic mitochondria using the Ingenuity Pathway Analysis.

The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining capillary isoelectric focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.

A two-dimensional microfluidic system is presented for intact protein separations combining isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) employing in situ photopolymerized polyacrylamide (PAAm) gels. The PAAm gels are used for multiple functions. In addition to serving as a highly-resolving separation medium for gel electrophoresis, discrete polyacrylamide gel plugs are used to enable the efficient isolation of different on-chip media including anolyte, catholyte, and sample/ampholyte solutions for IEF. The gel plugs are demonstrated as on-chip reagent containers, holding defined quantities of SDS for on-chip SDS–protein complexation, and enabling the use of a discontinuous buffer system for sample band sharpening during SDS-PAGE. The 2-D chip also employs several unique design features including an angled isoelectric focusing channel to minimize sample tailing, and backbiasing channels designed to achieve uniform interdimensional sample transfer. Separation results using E. coli cell lysate are presented using a 10-channel chip with and without the discontinuous buffer system, with resolving power more than doubled in the former case. Further improvements in separation resolution are demonstrated using a 20-channel chip design.

The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining capillary isoelectric focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.

Mitochondria are responsible for several essential functions, including apoptosis, iron-sulfur cluster assembly, and several important catabolic pathways in mammalian cells. Characterization of the mitochondrial proteome may therefore be extremely useful for identification and validation of targets for therapeutic treatment of human diseases, including aging and age-related disorders, diabetes, and obesity. While capillary isotachophoresis (CITP) has been introduced for on-capillary analyte preconcentration prior to electrophoretic separation, the application of transient CITP/capillary zone electrophoresis (CZE) to selectively enrich and resolve trace amounts of proteins/peptide is employed in an effort to expand mitochondrial proteome coverage in mammals. In this study, a total of 2095 distinct mouse SwissProt protein entries are identified from synaptic mitochondria isolated from mouse brain, corresponding to 50% and 76% coverage of the Maestro and MitoP2 mitochondrial reference sets, respectively. The increased coverage of mouse mitochondrial proteome further reveals the top 17 biosynthetic and metabolic pathways in synaptic mitochondria using the Ingenuity Pathway Analysis.

Direct control of electroosmosis in capillary electrophoresis with the application of an external electric field is demonstrated by the UV marker method. When the zeta potential at the capillary/aqueous solution interface is small, the capacitor model qualitatively and quantitatively predicts the effectiveness of the external electric field for controlling electroosmosis at different electrolyte concentrations and capillary dimensions. To investigate the separation efficiency of capillary electrophoresis with the direct control of electroosmosis, frontal analysis of dimethyl sulfoxide as a UV marker are examined. There is no measurable additional band broadening induced by the application of an external electric field.

Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.

Dynamic changes in the expression of multiple genes appear to be common features that distinguish transformed cells from their normal counterparts. We compared the proteomic profiles of four glioblastoma multiforme (GBM) tissue samples and four normal brain cortex samples to examine the molecular basis of gliomagenesis. Trypsin-digested protein samples were separated by capillary isoelectric focusing with nano-reversed-phase liquid chromatography and were profiled by mass spectrometric sequencing. Wolf-Hirschhorn syndrome candidate 1 (WHSC1), along with 103 other proteins, was found only in the GBM proteomes. Western blot and immunohistochemistry verified our proteomic findings and demonstrated that 30-kDa WHSC1 expression increases with ascending tumor proliferation activity. RNA interference could suppress glioma cell growth by blocking WHSC1 expression. Our novel findings encourage the application of proteomic techniques in cancer research.

Contains fulltext : 70228.pdf (Publisher’s version ) (Closed access) Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 +/- 0.094 vs. 0.018 +/- 0.009 day(-1)), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease.

Peptide identification of tandem mass spectra by a variety of available search algorithms forms the foundation for much of modern day mass spectrometry-based proteomics. Despite the critical importance of proper evaluation and interpretation of the results generated by these algorithms there is still little consistency in their application or understanding of their similarities and differences. A survey was conducted of four tandem mass spectrometry peptide identification search algorithms, including Mascot, Open Mass Spectrometry Search Algorithm, Sequest, and X! Tandem. The same input data, search parameters, and sequence library were used for the searches. Comparisons were based on commonly used scoring methodologies for each algorithm and on the results of a target-decoy approach to sequence library searching. The results indicated that there is little difference in the output of the algorithms so long as consistent scoring procedures are applied. The results showed that some commonly used scoring procedures may lead to excessive false discovery rates. Finally an alternative method for the determination of an optimal cutoff threshold is proposed.

Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.

Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and "leaked" proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry (MS), a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins. A search of bacterial protein sequences also identified many peptides unique to several organisms and unique to the NCBI nonredundant database. To the best of our knowledge, this proteome study represents the largest catalog of proteins measured from a single saliva sample to date. Data analysis was performed on individual MS/MS spectra using the highly specific peptide identification algorithm, OMSSA. Searches were conducted against a decoyed SwissProt human database to control the false-positive rate at 1%. Furthermore, the well-curated SwissProt sequences represent perhaps the least redundant human protein sequence database (12,484 records versus the 50,009 records found in the International Protein Index human database), therefore minimizing multiple protein inferences from single peptides. This combined bioanalytical and bioinformatic approach has established a solid foundation for building up the human salivary proteome for the realization of the diagnostic potential of saliva.

In this report, we take a heuristic approach to studying the effects of mass tolerance settings and database size on the sensitivity and specificity of MASCOT. We also examine the efficacy of the MASCOT Identity Threshold as a discriminator when applied to QqTOF data with an average mass accuracy of 10 ppm or better. As predicted, arbitrarily large mass tolerance settings negatively affect MASCOT's specificity, and to a lesser degree, sensitivity. Increased mass tolerances also render the generation of a significance threshold less effective. To study these effects, we used Bayes' Law to calculate MASCOT's predictive values. With a relatively small search database (Human IPI), MASCOT had a mean positive predictive value of 0.993 when combined with MASCOT's Identity Threshold. However, the corresponding average negative predictive value, or the probability that an ion was not present given no score or a score below threshold, was reduced as mass tolerances were tightened, and had an average value of 0.717. This value was improved upon by extrapolating an empirical threshold using a reversed database search and a new algorithm to rapidly identify false positive identifications. Using the empirical threshold reduced false negative identifications on the average 17% while limiting the false positive rate to below 5%; even larger reductions were obtained using mass tolerances approaching two times the actual error of the experimental data. A simple application of this strategy to the analysis of a microdissected glioblastoma multiforme sample analyzed by IEF/LC-MS/MS is reported, as is a description of the tools required to implement a large scale analysis using this alternative approach.

A simple method for fabricating nanoscale channels based on thermomechanical deformation of rigid polymer substrates is described. The process uses thermoplastic preforms containing microchannels with cross-sectional dimensions on the order of tens of microns, which are controllably deformed to produce sub-micron dimensions. Nanochannels with circular or elliptical cross-sections are defined in polycarbonate by varying the channel position and preform geometry prior to deformation. Arrays of parallel nanochannels with critical dimensions down to 400 nm are described. Using the fabrication method, a nanochannel network is fabricated for the detection of single protein molecules via confocal fluorescence microscopy, with channel dimensions matched to the confocal volume dimensions of the detection objective. Single molecule measurements of fluorescently-labeled bovine serum albumin at 15 nM and 150 nM concentrations are presented.