Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.     

Gangliosides as Biomarkers of Human Brain Diseases: Trends in Discovery and Characterization by High-Performance Mass Spectrometry

Gangliosides are effective biochemical markers of brain pathologies, being also in the focus of research as potential therapeutic targets. Accurate brain ganglioside mapping is an essential requirement for correlating the specificity of their composition with a certain pathological state and establishing a well-defined set of biomarkers. Among all bioanalytical methods conceived for this purpose, mass spectrometry (MS) has developed into one of the most valuable, due to the wealth and consistency of structural information provided. In this context, the present article reviews the achievements of MS in discovery and structural analysis of gangliosides associated with severe brain pathologies. The first part is dedicated to the contributions of MS in the assessment of ganglioside composition and role in the specific neurodegenerative disorders: Alzheimer’s and Parkinson’s diseases. A large subsequent section is devoted to cephalic disorders (CD), with an emphasis on the MS of gangliosides in anencephaly, the most common and severe disease in the CD spectrum. The last part is focused on the major accomplishments of MS-based methods in the discovery of ganglioside species, which are associated with primary and secondary brain tumors and may either facilitate an early diagnosis or represent target molecules for immunotherapy oriented against brain cancers.     

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera^® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera^® and Reditux™ displayed differences at the molecular level. MabThera^® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera^®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.     

热脱附/气相色谱-质谱法测定模拟工业源挥发性有机物

文章研究了Tenax TA吸附管收集模拟工业源挥发性有机物(VOCs),利用热脱附/气相色谱-质谱联用仪(GC-MS)进行定性定量分析。采用制气装置将混合液体标样通过高纯氦气吹扫气化,经Tenax管收集,制得模拟工业源VOCs样品。样品置于热脱附仪二次解析后直接进GC-MS分析。结果表明,通过优化热脱附仪条件及色谱条件,能对57种模拟工业源VOCs实现准确、快速的定性定量分析。     

Blood Plasma Proteome: A Meta-Analysis of the Results of Protein Quantification in Human Blood by Targeted Mass Spectrometry

A meta-analysis of the results of targeted quantitative screening of human blood plasma was performed to generate a reference standard kit that can be used for health analytics. The panel included 53 of the 296 proteins that form a “stable” part of the proteome of a healthy individual; these proteins were found in at least 70% of samples and were characterized by an interindividual coefficient of variation <40%. The concentration range of the selected proteins was 10⁻¹⁰–10⁻³ M and enrichment analysis revealed their association with rare familial diseases. The concentration of ceruloplasmin was reduced by approximately three orders of magnitude in patients with neurological disorders compared to healthy volunteers, and those of gelsolin isoform 1 and complement factor H were abruptly reduced in patients with lung adenocarcinoma. Absolute quantitative data of the individual proteome of a healthy and diseased individual can be used as the basis for personalized medicine and health monitoring. Storage over time allows us to identify individual biomarkers in the molecular landscape and prevent pathological conditions.     

Protein-Mediated Transformations of Superparamagnetic Nanoparticles Evidenced by Single-Particle Inductively Coupled Plasma Tandem Mass Spectrometry: A Disaggregation Phenomenon

Progress toward translating superparamagnetic iron oxide nanoparticles (SPIONs) with specific diagnostic and therapeutic properties for clinical applications depends on developing and implementing appropriate methodologies that would allow in-depth characterizations of their behavior in a real biological environment. Herein, we report a versatile approach for studying interactions between SPIONs and proteins using single-particle inductively coupled plasma tandem mass spectrometry. By monitoring the changes in the size distribution upon exposure to human serum, the formation of stable protein corona is revealed, accompanied by particle disaggregation.     

电喷雾质谱法研究不同碳链长度的全氟羧酸与肌红蛋白的相互作用

采用电喷雾质谱法(ESI-MS)检测了全氟丁酸、全氟己酸、全氟辛酸和全氟癸酸分别与肌红蛋白的相互作用,通过直接计算法测定了4种全氟羧酸与肌红蛋白的结合常数,并研究了碳链长度对全氟羧酸类化合物与肌红蛋白相互作用的影响。结果表明,不同碳链长度的全氟羧酸类化合物与肌红蛋白相互作用的能力有差别,碳链越长,其结合能力越强,且与蛋白质结合的配体数目越多。     

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10^?9 m ), which is nearly identical to that of the full‐length enzyme (K_D=16×10^?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.