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A straightforward capillary isoelectric focusing (cIEF) method is established with a isoelectric point (pI) range spanning from 3.5 to 7.0, exhibiting excellent linearity and repeatability, with an R 2 value of 0.9937 and migration time RSDs for all standard proteins below 0.3%. Subsequently, this method was applied to model exosomes derived from cell lines and healthy human serum, and the peak attributions of exosomes were identified using DiI labeled exosomes and lysed exosomes. The reproducibility of this method in exosome detection was also validated, as the RSDs of all pI values were less than 1%. Then, we observed a significant increase in the pI of exosomes with higher cholesterol content, irrespective of whether they originated from cell culture medium or mouse plasma. Notably, serum exosomes from healthy volunteers exhibited higher pI values compared to those from hepatocellular carcinoma patients, suggesting a potential diagnostic perspective for cancer. These findings underscore the significance of pI measurement in reflecting modifications in exosomal lipid membrane composition and their implications in biological functions mediated by exosomes. Graphical Abstract

High-resolution isoelectric focusing (HiRIEF) of peptides followed by mass spectrometry analysis, combined with accurate peptide pI prediction, allows a reduction of protein database search space, enabling deep proteome coverage and the discovery of protein-coding loci in human and mouse. We present a liquid chromatography–mass spectrometry (LC-MS)-based method permitting unbiased (gene prediction–independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7–5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST’s overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST’s charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user’s laboratory.Graphical AbstractRepresentative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein–protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.

Human dentin is not only a composite material of a collagenous matrix and mineral to provide strength and elasticity to teeth, but also a precious reservoir full of bioactive proteins. They are released after demineralization caused by bacterial acids in carious lesions, by decalcifying irrigants or dental materials and they modulate tissue responses in the underlying dental pulp. This work describes a first-time analysis of the proteome of human dentin using a shotgun proteomic approach that combines three different protein fractionation methods. Dentin matrix proteins were extracted by EDTA and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), OFFGEL isoelectric focusing (IEF) or strong cation exchange chromatography (SCX). Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 813 human proteins with high confidence, however, isoelectric focusing turned out to be the most beneficial prefractionation method. All Proteins were categorized based on the PANTHER system and representation analysis revealed 31 classes and subclasses to be overrepresented. The acquired knowledge provides a comprehensive insight into the number of proteins in human dentin as well as their physiological and pathological functions. Thus, the data presented paves the way to the analysis of specific functions of dentin matrix proteins in vivo and their potential in tissue engineering approaches to regenerate dental pulp.

Separation and determination of proteins by capillary isoelectric focusing (CIEF) and mass spectrometry (MS) are essential and complementary techniques in the field of bioanalysis. The hyphenation of these two techniques is challenging due to the nonvolatile substances required for the CIEF separation. An additional separation step prior to MS enables the removal of the nonvolatile substances. However, it is complicated due to the small transfer volume and the required high voltages in the CIEF process. In order to remove nonvolatile substances and transfer the analytes toward the mass spectrometer, we applied a four-port valve to couple CIEF online to capillary electrophoresis-mass spectrometry. To demonstrate the power of this concept, hemoglobin and glycated hemoglobin with an isoelectric point difference of 0.037 were separated via isoelectric focusing and characterized by MS. In general, this setup guaranties interference-free mass spectra and will provide an information-rich and sensitive top down protein characterization. Graphical abstract Interference free coupling of capillary isoelectric focusing to mass spectrometry by applying a mechanical valve. The focused proteins were tranferred from the isoelectric focusing to capillary electrophoresis by a mechanical valve. Afterwards, the transferred protein was sepearated from ionization interfering substances in the capillary electrophoresis prior to the mass spectrometry detection.

We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

In this paper, a novel nanocomposite based on magnetic nanoparticles decorated by dopamine were reported. Three modified magnetic nanocomposites by dopamine were offered with different type of linkers. The mentioned magnetic nanocomposites were applied to separate α-amylase protein from fresh bovine milk. All of the magnetic nanocomposites were characterized and investigated by using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning microscope, X-ray diffraction pattern, and vibrating-sample magnetometer analyses. To investigate the purifying application, sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis, and alpha-amylase activity assay were employed. With paying attention to factors such as yield of purification and concentration of separated protein by each of magnetic nanocomposite, it could be concluded that the length of linkers played an important role in α-amylase protein separation. According to the results, the best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Also, docking calculation has shown that the binding energy is − 1.697 kcal/mol and ΔG = − 6.844 kcal/mol which result that the interaction process between dopamine and α-amylase protein is spontaneous.

Oligoclonal bands (OCBs) represent the presence of intrathecal immunoglobulin G (IgG) as detected by isoelectric focusing and immunofixation. Cerebrospinal fluid (CSF) analysed alongside a paired serum sample gives five different immunofixation patterns. These are: type 1—the normal physiological state with no intrathecal IgG synthesis; type 2—evidence for intrathecal IgG synthesis, with CSF-restricted OCBs; type 3—evidence for intrathecal IgG synthesis, with CSF-restricted OCBs, but with additional, identical bands in the CSF and serum; type 4—absence of intrathecal IgG synthesis, but with identical OCBs in CSF and serum; and type 5—absence of intrathecal IgG synthesis, with a monoclonal band in CSF and serum. Analysis of these patterns can help to diagnose a range of neurological conditions, including multiple sclerosis. However, it is important to interpret OCB results alongside other CSF tests and their clinical context.