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Therapeutic antibodies are a major class of pharmaceutical drugs used to treat a wide variety of diseases. They have several advantages including the high specificity and binding affinity to their molecular targets, and generally low immunotoxicity and mild adverse effects. The characterization of therapeutic antibodies is crucial to ensure drug efficacy and safety. Charge variant analysis can be used to examine the charge variant forms of therapeutic antibodies, which may reflect modifications that impact the drug quality. Native capillary electrophoresis-mass spectrometry (nCE-MS) analysis by an integrated ZipChip CE-MS system is an alternative and complementary method to cation-exchange chromatography and imaged capillary isoelectric focusing to support the characterization of charge variants. In this study, we performed nCE-MS analysis to evaluate the charge variants and impurities in therapeutic antibodies including immunoglobin G (IgG) monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), and alternative formats such as therapeutic antibodies with addition or removal of antigen-binding domain. With the ZipChip CE-MS system, high-resolution charge variant separation was achieved for different types of therapeutic antibodies. Moreover, ZipChip nCE-MS analysis enabled high-sensitivity detection and identification of species with low abundance, including proteolytic cleavage and fragmentation in mAb, monospecific mAb impurities in bsAb, and O-glycosylation in alternative formats to support biopharmaceutical development and investigations.

Objectives Oligoclonal band (OCB) determination in cerebrospinal fluid (CSF) is the gold standard to detect intrathecal inflammation. However, there is uncertainty about the significance of one isolated band in CSF. Free light chains kappa (FLC-k) are gaining interest as a complementary method to detect intrathecal inflammation. The aim of this study was to investigate the performance of an additive measurement of FLC-k in patients with one isolated band in CSF. Materials & Methods FLC-k were analyzed using the nephelometric Siemens FLC-k kit in paired samples of CSF and sera (n = 56) in patients with one isolated band in isoelectric focusing. According to medical diagnosis, samples were subdivided in inflammatory neurological disease, non-inflammatory neurological disease controls and symptomatic controls. Intrathecal fraction of FLC-k was plotted in a FLC-k quotient diagram. OCB interpretation was done blinded by three experienced raters. Results Of 6695 OCB analyses, 91 (1.4%) had one isolated band in CSF. After exclusion of patient samples due to unclear OCB pattern after reevaluation and sample availability, 56 patient samples were included in the study. All patients with an inflammatory origin of disease (n = 13) had FLC-k values above the upper discrimination line (Qlim) in the FLC-k quotient diagram, resulting in a sensitivity of 100% with a positive predictive value of 52% and a negative predictive value of 100%. Fourteen patients (36%) with a non-inflammatory origin of disease (n = 39) had FLC-k values above Qlim. Conclusions In patients with one isolated band in CSF, a lack of intrathecal fraction of FLC-k strongly favors a non-inflammatory orgin of disease. Implementation of FLC-k measurement can help the clinician in the diagnostic process of neurological diseases.

We report capacitively coupled contactless conductivity detection (C4D) of proteins separated by microfluidic capillary isoelectric focusing (μCIEF). To elucidate the evolution of negative conductivity peaks during focusing and seek IEF conditions for sensitive conductivity detection, numerical simulation was performed using a model protein GFP (green fluorescence protein) and hypothetical carrier ampholytes (CAs). C4D was successfully applied to the μCIEF by optimizing assay conditions using a simple and effective pressure-mobilization approach. The conductivity and fluorescence signals of a focused GFP band were co-detected, confirming that the obtained negative C4D peak could be attributed to the actual protein, not the non-uniform background conductivity profile of the focused CAs. GFP concentrations of 10 nM–30 μM was quantified with a detection limit of 10 nM. Finally, the resolving power was analyzed by separating a mixture of R-phycoerythrin (pI 5.01), GFP-F64L (pI 5.48), and RK-GFP (pI 6.02). The conductivities of the three separated fluorescence proteins were measured with average separation resolution of 2.06. We expect the newly developed label-free μCIEF-C4D technique to be widely adopted as a portable, electronics-only protein-analysis tool.

Background α1-Antitrypsin (A1AT) deficiency predisposes patients to pulmonary disease due to inadequate protection against human neutrophil elastase released during inflammatory responses. A1AT deficiency is caused by homozygosity or compound heterozygosity for A1AT variants; individuals with A1AT deficiency most commonly have at least one Z variant allele (c.1096G > A (Glu366Lys)). Null variants that result in complete absence of A1AT in the plasma are much rarer. With one recent exception, all reported A1AT variants are characterized by a single pathogenic variant. Case An 8 years old patient from Edmonton, Alberta, Canada, was investigated for A1AT deficiency. His A1AT phenotype was determined to be M (wild type)/Null by isoelectric focusing (IEF) but M/Z by targeted genotyping. Gene sequencing revealed two heterozygous variants: Z and Ile100Asn (c.299 T > A). The Ile100Asn substitution is predicted to disrupt the secondary structure of an α-helix in which it resides and the neighbouring tertiary structure, resulting in intracellular degradation of A1AT prior to hepatocyte secretion. Methods Family testing was conducted to verify potential inheritance of an A1AT allele carrying the two mutations in cis, as this arrangement of the mutations would explain “Z” detection by genotyping but not by IEF. Molecular modeling was used to assess the effect of the variants on A1AT structure and stability. Discussion Carrier status for a novel variant NullCanada with in cis mutations (c.[299 T > A;1096G > A], p.[(Ileu100Asn;Glu366Lys)]) was confirmed. A sibling was identified as having A1AT deficiency on the basis of compound heterozygosity for two alleles: NullCanada and the common Z allele. A separate pedigree from the Maritimes was subsequently recognized as carrying NullCanada. Conclusion In cis mutations such as NullCanada may be more common than previously described due to failure to detect such mutations using historical testing methods. Combined approaches that include gene sequencing and segregation studies allow recognition of rare A1AT variants, including in cis mutations.

Freeze-drying is commonly used to improve stability of liquid formulations of labile biopharmaceuticals. Lyo- and cryoprotectants such as sucrose are traditionally utilized as excipients, but have low glass transition (Tg′) and collapse temperatures (Tc). Consequently, these formulations require low primary drying temperatures making the lyophilization cycle time-consuming and costly. We investigated different dextrans (1, 40, 150, and 500 kDa) and mixtures of dextran with sucrose as alternative excipients. The influence of dextran on thermal properties, cake appearance, and other quality attributes in the solid state was studied using bovine serum albumin as model protein. Especially at higher weight ratios of dextran to sucrose, dextrans of medium to high molecular weight (MW) of 40–500 kDa showed up to 20 °C higher Tc compared to sucrose, which was reflected in elegant lyophilisates. However, this resulted in slower reconstitution times. Addition of dextran led to lower residual moisture levels and higher Tg values compared to sucrose. We confirmed the thermal properties for two monoclonal antibodies (mAb) at two weight ratios of sucrose and dextran with different MW, and tested for stability at 40 °C for 14 days. While no loss in relative potency of the antibodies was observed after storage, size exclusion chromatography and isoelectric focusing revealed a strong increase in high molecular weight species (HMWs) and acidic species, which were dependent on the MW of the dextrans. With further characterization of selected formulations (dextran 1 kDa) by boronate affinity chromatography and mass spectrometry analysis, we demonstrated that HMWs were a result of glycation by free terminal glucose of the dextran. This chemical modification was strongly reduced when adding sucrose, which protects the protein possibly by shielding its surface. Our results demonstrate that formulation scientists need to use dextrans as excipients in freeze-dried mAb formulations with caution. A binary mixture of sucrose and dextran in adequate ratio however might potentially be superior to pure sucrose formulations allowing for faster freeze-drying cycles resulting in elegant lyophilisates and good protein stability.

In this study, we used the commercial soybean protein hydrolysate Hinute-DC6 as a novel starting material from which to purify and identify multifunctional cationic peptides. After fractionation, Hinute-DC6 was separated into 20 fractions with varying isoelectric points (pI) by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we purified and identified the cationic peptides from fractions 19 and 20, which had pI values greater than 12, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectrometry. Of the 83 cationic peptides identified, 14 had high pI values and net charges greater than +2, and were chemically synthesized and assayed for various bioactivities, including hemolytic, antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. None of the 14 cationic peptides tested exhibited hemolytic activity toward mammalian red blood cells at concentrations up to 1000 μM. Five of the cationic peptides exhibited antimicrobial activities against at least one of four human-pathogenic microorganisms tested. In addition, in chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, the 50% effective concentrations of these 14 peptides were between 0.069 and 5.2 μM. Tube-formation assays in human umbilical vein endothelial cells showed that each of the 14 cationic peptides exhibited significant angiogenic activities at 10 μM, with values similar to those of the positive control LL-37. Our results demonstrate that the 14 identified cationic peptides have multiple functions with negligible hemolytic activity. These data indicate that the cationic peptides isolated from Hinute-DC6 and fractions containing these cationic peptides have the potential to be used as multifunctional ingredients for healthcare applications.

Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

We present a set of novel low-molecular-mass (LMM) compounds possessing ampholytic properties. The compounds were designed to perform as markers of isoelectric point (pI) in different isoelectric focusing (IEF) formats and feature direct detectability in UV and visible wavelength regions. Capillary isoelectric focusing (cIEF) was used to determine the purity of the focusing species and the compounds' pI values. Nitrophenol-based pI markers (NPIMs) published previously were used as standards for the pI value calibration. The presented compounds focused very well, but small portion of them contained focusing impurities, thus, we recommend them for use in other IEF formats like gel IEF and preparative IEF. Moreover, multi-wavelength detection enabled determination of individual markers based on their specific spectral profiles and different absorption at selected detection wavelengths in the electropherogram. The presented compounds compose a group of chemicals featuring excellent shelf stability and isoelectric focusing properties, inexpensive synthesis, universal/multimode detectability, and good solubility at pI. The presented results provide a solid ground for their use as reference standards in various isoelectric focusing methods.

Charge heterogeneity is an important critical quality attribute for the analysis of monoclonal antibodies (mAbs). For this, (imaged) capillary isoelectric focusing ((i)cIEF), ion exchange chromatography (IEC) and, recently, capillary zone electrophoresis (CZE) are the predominantly used techniques. In order to investigate which one is most suitable to answer a specific analytical question, here, the four aforementioned separation techniques were systematically evaluated using NISTmAb and Infliximab as test molecules. The performance parameters (precision, separation efficiency, linearity and sensitivity) were determined under comparable conditions. Moreover, important aspects for daily routine such as speed and ease of use were considered. Each technique has its own pros and cons. The (i)cIEF methodology is distinguished by its excellent separation efficiency. In addition, the native fluorescence mode in icIEF is a good tool to analyze small sample amounts (LOQ: 2.8 mg/l for Infliximab). Nevertheless, high performance liquid chromatography (HPLC) still has superior precision. CZE, and also micellar electrokinetic chromatography (MEKC), have emerged as further interesting alternatives. For all techniques, variations connected to the sample preparation strongly influence precision. Looking at the relative standard deviation (RSD) values of the relative peak areas, all techniques provide acceptable performance (RSD: 0.6–1.6%).

Fatty acids are fundamental as energy and structural source to the human cells. They are not usually found free in human circulation. Alteration in fatty acids metabolism is linked to diseases such as diabetes, preeclampsia, heart disease, and some infectious diseases. Increased levels of non-esterified fatty acids (NEFA) may cause cell dysfunction and lipotoxicity. Since physiologically fatty acids are transported bound to albumin, we propose here a simple and cheap test that consists of albumin isoelectric focusing determination to measure the potential systemic NEFA cytotoxicity. For validation of this method, albumin isoelectric focusing in 51 serum samples from 40 critically ill patients and 11 controls was compared with NEFA/albumin ratios measured by HPLC. We called this approach an albumin saturation test. This test may indicate to physicians the potential NEFA lipotoxicity guiding them throughout better patient management. The albumin saturation test can point out serum albumin-NEFA saturation through a cheap assay that could be performed by any care facility.

International audience; Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.

Mass spectrometry has become a powerful tool in the field of bioanalysis. The combination of capillary electrophoresis with mass spectrometry (CE-MS) offers fast and high-resolution separation, sensitive and selective detection with small sample volumes, which is suitable for cell analysis. Here, we review the recent technical development of CE, MS and hyphenated strategies as well as their potential in cell analysis. In particular, we discuss the multiple MS compatible CE modes including capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic capillary chromatography and capillary electrochromatography for achieving better separation efficiency and selectivity before MS detection. For enhancing the sensitivity of CE-MS, several online preconcentration methods including dynamic pH junction, transient isotachophoresis and solid phase extraction are also reviewed and discussed. We also summarize the existing applications of CE-MS in cell analysis including cell biomacromolecule analysis, cell metabolism and single cell analysis.

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.

Focusing on microtubule heterogeneity and brain specificity allowed for initial discoveries of multiple tubulin isotypes four decades ago. Methods evolved from using radioactive labelling and single cell cultures to monoclonal antibodies recognizing discrete forms of tubulin in single neurons. With the advantage of molecular cloning and fluorescent protein tagging, essential components for microtubule dynamics/stability and function were identified, including activity-dependent neuroprotective protein, ADNP and its peptide snippet, NAP (drug candidate, davunetide/CP201). ADNP/NAP through the SxIP motif interact with microtubule end binding proteins EB1 and EB3 to increase microtubule dynamics, axonal transport and dendritic spine formation. Recent transcriptomic analysis of the young mouse brain at the single cell level enabled characterization of cell-type specific cytoskeleton related gene signatures (e.g., tubulin transcripts, microtubule-associated protein Tau, Mapt and microtubule end binding protein, EB3, Mapre3) at unprecedented detail. Here, we review these findings with a methodological perspective to highlight how cutting-edge techniques have allowed us to disentangle cytoskeleton dynamics in health and disease.

In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme’s specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme’s isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 μg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.

Proteome changes derived from animals that have suffered pre-slaughter stress are a fact. In this study, Proteomic analysis was carried out on 20 bovine loin samples from Asturiana de los Valles and crossbreds cattle previously classified as normal and DFD meat at 24 h post-mortem using pH measurements. Sarcoplasmic sub-proteome of Longissimus thoracis at 24 h post-mortem was fractionated by the use of liquid isoelectric focusing (OFFGEL) in the pH range 3–10, followed by SDS-PAGE analysis of each retrieved fraction. The protein fractionation profile showed high reproducibility along the different sample groups. Five protein bands showed significant differences (p, This work was funded by the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2014-00034-C04).

In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6-5.5 μM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23-31% in the presence of peptides at 10 μM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 μM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.

The present study proposed the cheese differentiation, according to geographical production area and with respect to species (cow, sheep) of two traditional cheese specialties, (salty and ripened), produced in Transylvania, Romania. For this purpose, the elemental profile and carbon isotopic ratios (13C/12C) of cheese and extracted casein were corroborated through statistic supervised techniques to get the best discrimination markers. The manganese content, along with Rare Earth Elements (REE) concentrations, proved to be very powerful predictors, for the traditional salted cheese mainly, due to the direct influence of the local salted water. Despite that proposed techniques are not acknowledged methods for species differentiation, this approach allowed a successful discrimination of the animal species that produced the raw materials for cheese manufacturing (milk). The results generated by the developed chemometric model, for species differentiation, were compared with those obtained using Isoelectric focusing (IEF) and DNA tests. The proposed association of isotopic and elemental markers allowed a differentiation better than 92% for geographical provenance, of each investigated cheese specialties while, for species discrimination (cow vs. sheep) a percentage of 100% was obtained.

In this study, we prepared fractions containing multifunctional cationic peptides by separating the commercial soybean protein hydrolysate Hinute-AM into 20 fractions. These fractions contained peptides with various isoelectric points (pI), as indicated by ampholyte-free isoelectric focusing (autofocusing). Thus, we purified and identified the cationic peptides from fractions 19 and 20, which had pH values greater than 10, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among 19 identified cationic peptides, NKNAKPPSPR, PGKKNAIV, KSGPGMSPR, NVSKPPRVV, RKVGAGGRKPLG, and LPCVIGGVPKRV had high pI values and were included as chemically synthesized peptides in assays of various functions, including lipopolysaccharide (LPS)-neutralizing and angiogenic activities. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysates showed that 50% effective concentrations of these six peptides were between 1.63 and 2.65 μM, and were higher than that (0.12 μM) of polymyxin B. Moreover, in tube-formation assays in human umbilical vein endothelial cells, all of the six cationic peptides except LPCVIGGVPKRV exhibited angiogenic activities similar to those of the positive control LL-37. In addition, the six identified cationic peptides had no hemolytic activity at concentrations up to 500 μM in mammalian red blood cells. Our results demonstrate that five of the identified cationic peptides, excluding LPCVIGGVPKRV, have multiple functions and little or no hemolytic activity. These data indicate that fractions containing cationic peptides from Hinute-AM have the potential to be used as dietary supplements and functional ingredients in food products.

The purpose is to determine the isoelectric point (IEP) pIμ of cytochrome c (cytC, a globular haemoproteid) adsorbed on montmorillonite (MM, plate-like colloid particles) by microelectrophoresis and to compare the pIμ value with pIf9.44 measured by isoelectric focusing in gel with covalently linked ampholytes, and with pInz10.0-10.6 of free cytC globule calculated by three programs for pH-dependent net charge nz using the crystallographic structure of cytC. The pH-dependence of the electrophoretic mobility μ(pH) in the range pH 6-11 shows out that IEP of cytC-MM particles appears at pH 9.35. The near courses of μ(pH) and nz(pH) reveal that the pH-independent negative charge of the MM substrate is hydrodynamically shielded by the adsorbed protein globules. The nearness of IEP and pIf allows attributing IEP value of cytC-MM particles to the isoelectric point pIμ of cytC. A short survey for pI of cytC reported in the literature since 1941 shows out that pI is dispersed in the range pH 9.0-10.65 although cytC is used now as pIf marker with well known IEP; the reason for that and the imperfections of the employed methods are discussed.

Charge heterogeneity is an important aspect of research into the development of monoclonal antibody drugs. In the present study, charge variants were separated into four fractions using weak cation exchange chromatography and were thoroughly analyzed using liquid chromatography-mass spectrometry at multiple levels. Molecular weight analysis of intact antibody and subunits confirmed the presence of heavy-chain leader sequences, light-chain leader sequences, dehydration, and cysteinylation. Peptide mapping of the fractions using different enzymes further localized the modified sites. Modified proportions identified at peptide level were compared with the purity detected by imaged capillary isoelectric focusing, the results showed that basic variant 1 consisted of cysteinylation and dehydration of asparagine, and basic variant 2 fully accounted for the N-terminal leader sequence of the heavy chain. About 14.8% of the acidic variant can be explained by N-terminal leader sequences in the light chain, and 18% of the acidic variant was demonstrated to be deamidation of asparagine in the heavy chain. There was approximately 54.2% of the acidic variant still cannot be explained. It was hypothesized that those acidic variants that have not yet been identified are an ensemble of molecules with slight molecular weight differences or the same molecular weight but different structures.

Background There has been long-term interest in cladribine as a drug for the treatment of MS. The current study focused on the effect of cladribine on oligoclonal bands (OCB) expression in the CSF in relapsing-remitting MS (RRMS) patients observed over 10 years. Methods 29 treatment-naive subjects with RRMS were prospectively enrolled and received induction therapy with subcutaneous parenteral cladribine (at a cumulative dose of 1.8 mg/kg; divided into 6 courses every 5 weeks given for 4–6 days, depending on patients’ body weight). Selected patients received maintenance doses in the follow-up period. Results Isoelectric focusing revealed that 55% of patients did not have OCB in CSF after cladribine treatment as compared to baseline testing when 100% of patients were positive for OCB. There were no significant differences in Expanded Disability Status Scale scores at baseline and at the end of treatment cycle between OCB-positive vs. OCB-negative subgroups. At the last follow-up, OCB-negative patients had lower disability compared to OCB-positive patients (p = 0.03). Conclusion Cladribine-induced immune reconstitution leads to long lasting suppression of intrathecal humoral response, which might be an additional mechanism that enhances the therapeutic effect on disease progression in RRMS patients.

Olive is one of the most important fruit crop trees in the history of Mediterranean because of the high quality oil. Olive oil has a well-balanced fatty acid composition along with biophenols, which make it exceptional in human diet and provide an exceptional value to the olive oil. Leaf non-glandular peltate trichomes are specialized cell types representing a protective barrier against acute environmental conditions. To characterize the proteome of this highly differentiated cell type, we performed a comparative proteomic analysis among isolated trichomes and trichome-less leaves. Proteins were separated and identified using the 2-DE MALDI-TOF/MS method. A number of enzymes involved in abiotic and biotic stress responses are present and may be responsible for the adaptation to prolonged adverse environmental conditions. The results show that this highly differentiated cell type is physiologically active fulfilling the demands of the trichomes in furnishing the leaf with a highly protective mechanism.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment. The exposure of living organisms to TCDD may result in numerous disorders, including reproductive pathologies. By employing two-dimensional fluorescence difference gel electrophoresis we aimed to identify proteins potentially involved in the mechanism of TCDD action and toxicity in porcine granulosa cells. The porcine granulosa cells were treated with TCDD (100 nM) for 3, 12 or 24 h, and afterwards, cytoplasmic proteins were isolated and labeled with cyanines. Next, samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF MS analysis. A total of 75 differentially expressed protein spots (p 0.05 and fold change ≥2.0) were found in granulosa cells treated with TCDD. After 3, 12 and 24 h of TCDD treatment, we were able to identify 29, 34 and 12 spots, respectively. Functional analysis showed that cytoskeletal proteins formed the largest class of proteins significantly affected by TCDD in all time points. We also demonstrated that most of the identified proteins were associated with the "structural constituent of cytoskeleton" and "chaperone binding" Gene Ontology categories. Based on the analysis of the porcine granulosa cell proteome, we demonstrated that TCDD may affect the ovarian follicle fate by the rearrangement of the cytoskeleton and extracellular matrix as well as the modulation of proteins important for the cellular response to stress. The results of the current study present an extended insight into the TCDD mechanism of action in porcine granulosa cells, providing new directions for future functional studies.

Modern analytical ion-exchange chromatography is one of the conventional tools used for assessment of product-related quality attributes in bio-therapeutic monoclonal antibodies (mAbs). Here, we present an approach to resolve, identify, and quantify product-related substances of therapeutic mAb at its intact molecular level by cation exchange (CIEX) HPLC coupled directly to electrospray ionization - quadrupole time of flight mass spectrometry (ESI-QTOF-MS). This method utilizes pH gradient elution mode comprised of ammonium formate buffer components, and a weak cation exchange column as stationary phase. Furthermore, ion-mobility mass spectrometry (IM-MS) provided additional insights on its higher order structure. Also, orthogonal assays such as conventional CIEX-HPLC, high resolution capillary isoelectric focusing, peptide mapping, spectroscopic, and fluorescence methods were used considerably to support the findings. Additionally, an in vitro assay was included to assess the associated impact on Fc mediated function. Overall, the developed method with simultaneous detection of UV peak area percentage at 280 nm and native ESI-MS is found to be a rapid and robust analytical tool for direct assessment of structural and purity attributes, process optimization, product development, and to decipher the relevant role of micro-variants on quality, stability, and clinical outcomes.

Objectives: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. Methods: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. Results: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (similar to 20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. Conclusion: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. Clinical significance: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.

Product identity is one of the release testing requirements that needs to be established to ensure that there is no misidentification of drugs. Here, we demonstrated the challenges that can come across while establishing a product identity method for monoclonal antibody (mAb) and mAb-related products using icIEF method. A unique three-point identity criteria tool (visual comparison, pI of individual peaks and ΔpIs) was applied to distinguish mAb1 from the other in-house mAbs. A reduction approach followed by icIEF showed higher potential for establishing identity for mAb1 product as compared to native and enzymatic digestion approach. In general, icIEF method lacks specificity required to unequivocally establish the identity for mAbs, therefore, risk analysis is recommended before implementing icIEF as a stand-alone identity method for monoclonal antibodies.

Background IgG concentrations in cerebrospinal fluid generally range from 20 to 45 mg/L. In multiple sclerosis immune reactions lead to intrathecal synthesis of specific IgGs that can be detected in biological fluid samples both quantitatively and qualitatively by isoelectric focusing of supplementary oligoclonal IgG bands. Method A simple tool, using the MATLAB application, to facilitate and improve isoelectric focusing profile analysis is presented and evaluated in terms of its sensitivity, repeatability and reproducibility. A comparison between human readers and semi-automatic method has also been performed. Results Results from the semi-automatic method were found to be equivalent or superior to generally employed laboratory methods. Repeatability analysis for semi-automatic processing yielded coefficients of variation (CVs) in the 3–7% range, and using a sample with an estimated IgG concentration of 200 mg/L, four bands were still visible after dilution to 5 mg/L, corresponding to band concentrations of 1.1–1.6 mg/L. Discordances between visual inspection and automatic analysis only appear at threshold levels for interpretation (the gray zone). Conclusion The semi-automatic method has acceptable performance for routine implementation.

In this study, single-step magnetic-beads based immunoassays was developed and for the first time adapted to microfluidic droplet operation for sequential determination of well-established amyloid-β peptide biomarkers ( i.e . monomeric Aβ 1–42 and Aβ 1–40) for molecular diagnosis of Alzheimer’s disease (AD). With the developed sandwich assay protocol, the capture antibodies ( i.e. monoclonal anti-Aβ antibodies that are specific for N-terminus of Aβ 1–42 and Aβ 1–40) grafted onto magnetic beads and the detection antibodies ( i.e. beta amyloid 1–16 monoclonal antibody labeled with horseradish peroxidase) can simultaneously bind to monomeric β peptides in a single step. Aβ 1–42 and Aβ 1–40 in cerebrospinal fluid (CSF) samples were successfully detected using the developed batchwise immunoassay approach. With the aim at expanding the spectrum of traced Aβ peptides beyond Aβ 1–42 and Aβ 1–40 for more precise diagnostics of AD, the developed sandwich assay was for the first time coupled as a downstream module to peptide fractionation and collection using capillary isoelectric focusing. Demonstration of this methodological combination was carried out with Aβ 1–40, Aβ 1–40 and Aβ 5–40. The immunoassay in batchwise format was downscaled into a purpose-made microfluidic droplet platform allowing significant sample volume reduction and higher throughput. Using a series of 4 programmable magnetic tweezers to manipulate a train of nano-scale confined droplets containing magnetic beads, sample, washing and detection solutions, a sequence of 8 analyses could be realized within 45 min. This droplet based immunoassay was realized in a dedicated platform integrating an in-house-made light emitting diode (LED)-based fluorescent detector, replacing conventional microscopic setup so as to significantly reduce the construction cost and simplify the detection protocol. Using this microfluidic configuration coupled with LED-based detection, information on both Aβ 1–42 and Aβ 1–40 concentrations can be collected in a single sequence with less than 1 μL of sample.

An isoelectric focusing method (IEF) has been used to assess the charge heterogeneity profile of a monoclonal antibody during the early stages of product development. A more precise and sensitive ion exchange chromatography (IEC/CEX) method was developed and implemented as development progressed and was used concurrently with IEF for lot release and to monitor charge heterogeneity. Charge variants resolved by both methods (IEC and IEF) were purified and characterized. Tryptic peptide mapping and N- linked oligosaccharide profile analyses of the IEC and IEF fractions indicated a structural correlation between the charge variants separated by these two methods. The major sources of molecular heterogeneity were due to the variation in the sialyated carbohydrate structure and heavy chain C-terminal lysine truncation. By monitoring the rates of change in the charge heterogeneity profiles of the monoclonal antibody stored at elevated temperatures by the IEC and IEF methods, a positive correlation between the two methods was established. This approach enabled replacement of the IEF method with the more precise IEC method.

This study describes a new method for simultaneous identification of uropathogens in the case of polybacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is ≥104 cells mL−1. Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF.

Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster.

Capillary electrophoresis (CE) has emerged as a powerful technique for comprehensive physicochemical characterization of monoclonal antibodies (mAbs) as well as other therapeutic modalities. The method provides high resolution separation and high sensitivity characterization for analysis of therapeutic biomolecules. CE based techniques such as sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE, also referred to as CE SDS), capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF) have been increasingly adopted to assess size heterogeneity, glycosylation heterogeneity and charge heterogeneity in mAbs and related therapeutic modalities. This paper reviews the latest application developments of CE based methods for routine release testing, stability testing and in-depth characterization. In addition, advantages and disadvantages of each of these techniques are critically discussed.

In bacteria transcription is coupled to translation, and while it is broadly accepted that transcription–translation complexes (TTCs) are formed in growing bacterial cells, the exact spatial organization of these macromolecular assemblies is not known with certainty. Recent studies indicated the formation of orderly cytosolic superstructures in growing E. coli cells. The bacterial nucleic acid (NA)-binding protein Hfq has been shown to function at the interface of RNA synthesis–degradation machinery; multiple, independent studies link Hfq to orderly cytosolic assemblies. In this work, using fast cell lysis/2D-PAGE and in vitro reconstitution analyses we studied the Hfq modifications and small protein-associated molecules (SPAM). We demonstrate that native Hfq carries stable modifications and simulate 2D patterns of native Hfq–SPAM complexes in reconstitution experiments with purified Hfq and synthetic NA probes. We also demonstrate that genetically engineered Hfq lacking the conserved arginine residues positioned near the rim of the disc formed by the subunits’ N-terminal domains binds DNA with a reduced affinity in comparison with wild-type Hfq. These results are consistent with the proposed Hfq-mediated DNA remodeling and point to the involvement of this patch of conserved arginines in interactions with DNA.

In this study, we hydrolyzed rice endosperm protein (REP) with pepsin and generated 20 fractions containing multifunctional cationic peptides with varying isoelectric point (pI) values using ampholyte-free isoelectric focusing (autofocusing). Subsequently, we determined antimicrobial activities of each fraction against the pathogens Prophyromonas gingivalis, Propionibacterium acnes, Streptocossus mutans, and Candida albicans. Fractions 18, 19, and 20 had pI values greater than 12 and exhibited antimicrobial activity against P. gingivalis, P. acnes, and C. albicans, but not against S. mutans. In further experiments, we purified and identified cationic peptides from fractions 18, 19, and 20 using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. We also chemically synthesized five identified peptides (RSVSKSR, RRVIEPR, ERFQPMFRRPG, RVRQNIDNPNRADTYNPRAG, and VVRRVIEPRGLL) with pI values greater than 10.5 and evaluated antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. Among these synthetic peptides, only VVRRVIEPRGLL exhibited antimicrobial activity against P. gingivalis, with an IC50 value of 87μM. However, all five cationic peptides exhibited LPS-neutralizing and angiogenic activities with little or no hemolytic activity against mammalian red blood cells at functional concentrations. These present data show dual or multiple functions of the five identified cationic peptides with little or no hemolytic activity. Therefore, fractions containing cationic peptides from REP hydrolysates have the potential to be used as dietary supplements and functional ingredients in food products.

Imaged capillary isoelectric focusing (icIEF) separates ampholytic components of biomolecules in an electric field according to their isoelectric points and has been used for protein charge variants quantification and characterization. Denaturants are ordinarily incorporated into icIEF to stabilize charge species in solution. In certain circumstances, however, denaturants are detrimental to stable isoelectric separation of proteins due to their unique structural and biophysical features, such as an aggregation-prone antibody we encountered recently. Here we report our novel matrix formula non-detergent sulfobetaine and taurine (NDSB-T). It is deprived of denaturants that notably ameliorates the assay robustness and peak resolution for this antibody. NDSB-T is a combination of non-detergent sulfobetaine (NDSB) and taurine possessing the stabilization and separation power while maintaining protein integrity. As a result, assay throughputs are tremendously increased for more than 10 folds along with extraordinarily improved assay accuracy. Furthermore, NDSB-T can separate and quantify protein charge species in native state and therefore avoid partial denaturation derived peaks which are often misleading and hard to characterize. NDSB-T may be a valuable tool for proteins incompatible with conventional icIEF matrices and potentially opens a new window for icIEF assay in native conditions.

We report on a Mexican mestizo with a multisystemic syndrome including neurological involvement and a type I serum transferrin isoelectric focusing (Tf IEF) pattern. Diagnosis of PMM2-CDG was obtained by clinical exome sequencing (CES) that revealed compound heterozygous variants in PMM2, the encoding gene for the phosphomannomutase 2 (PMM2). This enzyme catalyzes the conversion of mannose-6-P to mannose-1-P required for the synthesis of GDP-Man and Dol-P-Man, donor substrates for glycosylation reactions. The identified variants were c.422G>A (R141H) and c.178G>T, the former being the most frequent PMM2 pathogenic mutation and the latter a previously uncharacterized variant restricted to the Latino population with conflicting interpretations of pathogenicity and that we here report causes leaky non-functional alternative splicing (p.V60Cfs*3).

The identification and control of monoclonal antibody (mAb) critical quality attributes (CQAs) is a key component of quality by design (QbD). In this work, rapid peptide mapping and native intact charge variants analysis have been developed to comprehensively characterize and monitor mAb CQAs using a microfluidic capillary electrophoresis - mass spectrometry (CE-MS) platform. The ultrafast peptide mapping simultaneously analyzed multiple CQAs, including protein primary structure, oxidation, deamidation, succinimide, C-terminal lysine (Lys) clipping, N-terminal cyclization, and glycosylation. The microfluidic CE-MS based peptide mapping acquired results comparable to conventional but lengthy liquid chromatography - MS (LC-MS) based approach. The native intact analysis resolved mAb charge variants with a comparable resolution as commonly achieved using capillary isoelectric focusing (cIEF). Charge variants' identities were assigned based on characteristic mass shifts, knowledge learned from peptide mapping, and changes in electrophoretic mobility. Major mAb glycoforms of each charge variants were resolved and identified in the deconvoluted mass spectra. Furthermore, a model simulation was performed to reconstruct intact deconvoluted mass spectra using peptide mapping results. The reconstructed and experimentally determined intact deconvoluted mass spectra were highly correlated, suggesting that our data collected at the peptide level and intact level were consistent and highly comparable. Overall, the microfluidic CE-MS based peptide mapping and native intact charge variants analysis are high-throughput methods that have great potential to support biopharmaceutical development.

This review article describes the significant recent advances in Isoelectric Focusing from the period 2015-2020. The review highlights the principles and common challenges faced in Isoelectric Focusing as well as its applications. This review also details the recent advances in various modes of Isoelectric Focusing in various platforms and future directions for the technique.

Cationic ionic liquids-based surfactants (ILS), such as 4-methyl pyridinium bromide (CnPBr, where n=4,6,8), were used in preparation of polyacrylamide gels, sample buffer, and running buffer for cationic ILS polyacrylamide gel electrophoresis (ILS-PAGE). These ILS are liquids in the pure state and were selected for improved separation of ribonuclease b (Rib b) glycoforms in a single step and a protein mixture containing bovine serum albumin (BSA, pI-4.8, 66.5kDa), ovalbumin (Ova, pI-4.6, 44.3kDa), α-chymotrypsinogen (α-Chy, pI-8.8, 25.7kDa), myoglobin (Myo, pI-6.8, 16.9kDa), and cytochrome c (Cyt c, pI-10.0, 12.3kDa). Results acquired for Rib b glycoform separation by use of ILS were compared with conventional non-ILS surfactants-PAGE: sodium dodecylsulfate (SDS)-PAGE, cetyltrimethylammonium bromide (CTAB)-PAGE, and benzyldimethyl-n-hexadecylammonium chloride (16-BAC)-PAGE. A single protein band was observed with relatively short migration time in all the conventional PAGE techniques tested. In contrast, ILS-PAGE showed multiple bands with two distinct bands for Rib b protein. The two distinct bands of Rib b from ILS-PAGE were further analyzed using MALDI-MS. Examination of MALDI-MS spectral data revealed the presence of Rib b glycoforms. Furthermore, a two-dimensional isoelectric focusing (IEF)/SDS-PAGE map of Rib b protein revealed negative charge heterogeneity on the protein, which is a common observation for glycoproteins. This overall discovery greatly enhances the capability of using cationic ILS-PAGE for Rib b protein separation. Among all ILS tested, excellent protein separations were observed using C4PBr ILS at concentrations of 0.05% (w/v) in polyacrylamide gels, 0.01% (w/v) in protein sample buffer, and 0.1% (w/v) in running buffer. Under these optimum conditions, all other tested proteins were separated as sharp bands with good resolution.

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis.

In this study, to prepare the fraction containing multifunctional cationic peptides, we first hydrolyzed rice bran protein (RBP) with pepsin. We separated the enzymatic hydrolysate of RBP into 20 fractions containing peptides with different isoelectric point (pI) values by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we examined the antimicrobial activity of each fraction against four pathogens. In addition, we purified the cationic peptides from fractions exhibiting antimicrobial activity by reversed-phase high-performance liquid chromatography and identified them by matrix-assisted laser/desorption ionization–time-of-flight mass spectroscopy. Of five cationic peptides identified, we chemically synthesized three peptides with high pI values and evaluated their multiple functions, including antimicrobial, lipopolysaccharide-neutralizing and angiogenic activities. Our results demonstrated that the three identified cationic peptides exhibited multiple functions with little or no haemolytic activity. Fractions containing cationic peptides obtained from RBP hydrolysate have the potential to be used as dietary supplements and functional ingredients in food products.

Peptide Nucleic Acid (PNA) is a nucleic acid analogue, whose neutrally charged and hydrophobic backbone makes it more stable in vivo, so it might act as a potentially better protein probe as compared to aptamer. Currently the investigation of PNA and protein interaction is scarce. In this research, multiple modes of capillary electrophoresis were established and applied for PNA characterization and its interaction with ssDNA and protein. A 15-mer PNA having the same nucleobase sequence as 15-mer anti-thrombin DNA aptamer was chosen as PNA model for this study, its pI (7.71) was estimated by capillary isoelectric focusing (cIEF). Due to its neutral charge and strong hydrophobicity, three micellar electrokinetic chromatography (MEKC) modes containing (a) SDS, (b) Triton X-100 and (c) CTAB were compared for PNA related analysis. CTAB was not applicable for PNA analysis, while in 4mM SDS or 2mM Triton X-100, PNA and PNA-ssDNA complex can be identified directly. The significant peak of PNA-ssDNA complex helped in validating the two MEKC modes for PNA and target interaction study. Furthermore, the effect of SDS and Triton X-100 concentrations in the two MEKC modes on the protein target thrombin analysis was investigated by capillary zone electrophoresis (CZE). 4mM SDS caused thrombin denaturation. So in 2mM Triton X-100, interactions of PNA with thrombin, PNA with RNase A and a non-aptameric PNA (n-PNA) with thrombin were compared. PNA with thrombin exhibited strongest binding. In summary, cIEF mode for PNA pI determination, MECK mode for direct PNA, PNA-ssDNA and PNA-protein complex identification and CZE mode for the effect of surfactant in MEKC modes on protein target thrombin analysis were applied. Above results showed that multiple modes of CE provide rapid and very low-sample cost methods for PNA related studies.

Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]. However some proteins and antibodies are exceptions, we found urea and sucrose are not sufficient for a typical fusion protein (Fusion protein A) in icIEF assay and high variability is observed. We report a novel reagent, formamide, significantly improved reproducibility of protein charge profiles. Our results show formamide is a good supplementary reagent to reduce aggregation and stabilize proteins in isoelectric focusing. We further confirmed the method robustness, linearity, accuracy and precision after introducing the new reagent; extremely tight pI values, significantly improved method precision and sample on-board stability are achieved by formamide. Formamide is also proven to be equally functional to multiple antibodies as urea, which makes it an extra tool in icIEF method development.

The concentration gradient detection method based on the Schlieren optics employed for electrophoresis analyses by extending the technology to a multi-channel system using a prototyped microfluidic chip (thinXXS Micro-technology, Germany). The results prove that coupling a chip-based microfluidic device with Schlieren detection is an appropriate approach to improve the electrophoretic separations. The effects of channel's geometry and dimension were investigated by conducting the experiments in channels with different cross sectional areas. Fast kinetic data acquisition of the charge-coupled device (CCD) camera facilitated recording of a time sequence of optical images, demonstrating the potential of the CCD camera as a powerful tool for studying dynamic processes such as diffusion. Diffusion coefficients of sample proteins were measured under static and dynamic conditions, where the static mode demonstrated more accurate results. Furthermore, the Fourier transformation was employed to improve the Schlieren images for quantitative analysis of the diffusion coefficient measurement.

Haemoglobinopathies are among the most common inherited monogenic disorders worldwide. Thalassaemia screening for carrier status is recommended for adults of reproductive age if suspected of being at risk. Conventional laboratory methods for screening include the assessment of haematological indices, and high-performance liquid chromatography, capillary electrophoresis or isoelectric focusing to measure the levels of HbA2 and HbF, and to identify haemoglobin variants. Each screening method has its advantages and disadvantages, the main disadvantage being that none can fully resolve all variants. The complex nature of the genetics of haemoglobinopathies necessitates expertise in the interpretation of screening results to evaluate the most likely genotypes, which must then be confirmed using the DNA diagnosis. This review highlights the limits and pitfalls of each screening technique, and outlines a rational combination of different methods to overcome issues in thalassaemia carrier detection.

Described as intrinsically powerful building blocks for two-dimensional separations by Giddings [Anal. Chem., 56 (1984) 1258A-1270A], the coupling of chromatography and electrophoresis has been proven to enhance the resolution of a wide array of molecules in complex biological, environmental and food samples. This review provides a comprehensive overview of multidimensional chromato-electrophoretic (LC - E) and electrophero-chromatographic (E - LC) separation systems from inception to the most recent published examples. LC separation modes include reversed phase, ion exchange, and size exclusion. Electromigration separation modes include capillary, microchip or free flow electrophoresis; micellar electrokinetic chromatography; electrochromatography; and isoelectric focusing. The advantages and disadvantages of various non-gel based off-line and on-line hyphenation technologies of LC - E and E - LC are discussed, with conditions and system characteristics also provided.

More than seventy-five isotypes of α-1-antitrypsin (AAT) have been described. To assess risks associated with AAT deficiency, isotype identification is necessary. Isoelectric focusing (IEF) is traditionally used for isotype differentiation, however, IEF has limited scope since it is a manual procedure that is not suitable for automation, and antitrypsin variants must differ in net charge in order to be resolved. In comparison, mass spectrometric assays are easily automated and offer a more complete solution for characterization of proteins. To capitalize on these advantages, we have developed a qualitative top-down liquid chromatography–mass spectrometry (LC–MS) method for selective phenotyping of AAT. This technique requires no sample pretreatment, and has the potential for use in routine clinical diagnostics. We have validated our LC–MS results against both DNA sequencing and IEF. Thus far, this method has identified the AAT variants PLowell, S and Z, as well as unique fragments shared by different M alleles. Its high selectivity is indirectly illustrated by the detection of a variant carrying the amino acid substitution p.Ala308Ser, which cannot be visualized by IEF.