Capillary Isoelectric Focusing: Mass Spectrometry Method for the Separation and Online Characterization of Monoclonal Antibody Charge Variants at Intact and Subunit Levels.

Monoclonal antibodies (mAbs) are one of the most widely used types of protein therapeutics. Charge variants are important quality attributes for evaluating developability, activity, and safety for mAb therapeutics. Here, we report a novel online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for mAb charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization.

Factors that determine a Patient's willingness to physician selection in online healthcare communities: A trust theory perspective.

Health care users and patients are increasingly using online health communities to seek medical service, especially during the COVID-19 epidemic. The factors that determine the online trust between physicians and patients perplex the stakeholders for a long time. Based on the trust theory, this study explored the influence of physicians' personal quality and online reputation on patients' selection.

Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Ribosomal Proteins and Protein Complexes.

With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the 70S ribosome.

Online capillary electrophoresis - mass spectrometry analysis of histatin-5 and its degradation products.

Histatin-5 (Hst-5) is a human salivary peptide with antibacterial and antifungal activities. Thorough characterization and reliable quantification of Hst-5 and its degradation products are essential for understanding the Hst-5 degradation pathway. Due to the highly basic and strong cationic nature of the Hst-5 peptide, the quantitative analysis of Hst-5 and its degradation forms by online mass spectrometry remains challenging.

Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry.

Membranous nephropathy (MN) is one of the most common causes of primary glomerular diseases worldwide. The M-type phospholipase A2 receptor (PLA2R), an antigen expressed in more than 70% of cases of idiopathic membranous nephropathy (IMN), is a biomarker which is now used by physicians for clinical diagnosis. Despite the prevalence of PLA2R in the cases of MN, it is not always effective to use PLA2R for differentiating primary or secondary MNs.

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray.

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g.

Investigation of electrospray for a capillary electrophoresis-mass spectrometry interface in reverse polarity and negative ion mode.

Capillary zone electrophoresis (CZE) paired with mass spectrometry (MS) is a powerful analytical technique for examining mixtures of ionic analytes such as glycosaminoglycans. This study examines the mechanics of the electrospray process for a sheath flow CZE-MS interface under reverse polarity negative ionization conditions. Liquid flow in a sheath flow nano-electrospray CZE-MS interface is driven by two mechanisms, electroosmotic flow and electrospray nebulization.

Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides.

Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points.

On-line capillary electrophoresis/laser-induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump-based nanospray ion source.

Rationale: N-linked glycan analysis of recombinant therapeutic proteins, such as monoclonal antibodies, Fc-fusion proteins, and antibody-drug conjugates, provides valuable information regarding protein therapeutics glycosylation profile. Both qualitative identification and quantitative analysis of N-linked glycans on recombinant therapeutic proteins are critical analytical tasks in the biopharma industry during the development of a biotherapeutic.

Methods: Currently, such analyses are mainly carried out using capillary electrophoresis/laser-induced fluorescence (CE/LIF), liquid chromatography/fluorescence (LC/FLR), and liquid chromatography/fluorescence/mass spectrometry (LC/FLR/MS) technologies.

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12.

Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants.

We report a new online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for monoclonal antibody (mAb) charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source and a time-of-flight MS with pressure-assisted chemical mobilization. To develop a successful, reliable CIEF-MS method for mAb, we have selected and optimized many critical, interrelating reagents and parameters that include (1) MS-friendly anolyte and catholyte; (2) a glycerol enhanced sample mixture that reduced non-CIEF electrophoretic mobility and band broadening; (3) ampholyte selected for balancing resolution and MS sensitivity; (4) sheath liquid composition optimized for efficient focusing, mobilization, and electrospray ionization; (5) judiciously selected CIEF running parameters including injection amount, field strength, and applied pressure. The fundamental premise of CIEF was well maintained as verified by the linear correlation (R = 0.

Analysis of heparin oligosaccharides by capillary electrophoresis-negative-ion electrospray ionization mass spectrometry.

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid.

Capillary Electrophoresis-Mass Spectrometry for the Analysis of Heparin Oligosaccharides and Low Molecular Weight Heparin.

Heparins, highly sulfated, linear polysaccharides also known as glycosaminoglycans, are among the most challenging biopolymers to analyze. Hyphenated techniques in conjunction with mass spectrometry (MS) offer rapid analysis of complex glycosaminoglycan mixtures, providing detailed structural and quantitative data. Previous analytical approaches have often relied on liquid chromatography (LC)-MS, and some have limitations including long separation times, low resolution of oligosaccharide mixtures, incompatibility of eluents, and often require oligosaccharide derivatization.

Paired single residue-transposed Lys-N and Lys-C digestions for label-free identification of N-terminal and C-terminal MS/MS peptide product ions: ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo sequencing.

Rationale: Paired Lys-N and Lys-C proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Data from these parallel experiments provide constraints that are applied before data analysis. With this approach, we can find matched spectra before analysis, distinguish ion type, and determine residue level confidence.

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated.

Analysis of a membrane-enriched proteome from postmortem human brain tissue in Alzheimer's disease.

Purpose: The present study is a discovery mode proteomics analysis of the membrane-enriched fraction of postmortem brain tissue from Alzheimer's disease (AD) and control cases. This study aims to validate a method to identify new proteins that could be involved in the pathogenesis of AD and potentially serve as disease biomarkers.

Experimental Design: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the membrane-enriched fraction of human postmortem brain tissue from five AD and five control cases of similar age.

Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards.

A hallmark of neurodegeneration is the aggregation of disease related proteins that are resistant to detergent extraction. In the major pathological subtype of frontotemporal lobar degeneration (FTLD), modified TAR-DNA binding protein 43 (TDP-43), including phosphorylated, ubiquitinated, and proteolytically cleaved forms, is enriched in detergent-insoluble fractions from post-mortem brain tissue. Additional proteins that accumulate in the detergent-insoluble FTLD brain proteome remain largely unknown.

Aberrant septin 11 is associated with sporadic frontotemporal lobar degeneration.

Background: Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches.

Results: We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U.

Characterization and diagnostic value of amino acid side chain neutral losses following electron-transfer dissociation.

Using a large set of high mass accuracy and resolution ETD tandem mass spectra, we characterized ETD-induced neutral losses. From these data we deduced the chemical formula for 20 of these losses. Many of them have been previously observed in electron-capture dissociation (ECD) spectra, such as losses of the side chains of arginine, aspartic acid, glutamic acid, glutamine, asparagine, leucine, histidine, and carbamidomethylated cysteine residues.

Phosphoproteomic analysis reveals site-specific changes in GFAP and NDRG2 phosphorylation in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported.

Sub-part-per-million precursor and product mass accuracy for high-throughput proteomics on an electron transfer dissociation-enabled orbitrap mass spectrometer.

We demonstrate a new approach for internal mass calibration on an electron transfer dissociation-enabled linear ion trap-orbitrap hybrid mass spectrometer. Fluoranthene cations, a byproduct of the reaction used for generation of electron transfer dissociation reagent anions, are co-injected with the analyte cations in all orbitrap mass analysis events. The fluoranthene cations serve as a robust internal calibrant with minimal impact on scan time (<20 ms) or spectral quality.

Multiplex SILAC analysis of a cellular TDP-43 proteinopathy model reveals protein inclusions associated with SUMOylation and diverse polyubiquitin chains.

Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons.

Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database.

Background: Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P.