Phenotypes and Genotypes of Patients with Pantothenate Kinase-Associated Neurodegeneration in Asian and Caucasian Populations: 2 Cases and Literature Review

Objectives. Pantothenate kinase-associated neurodegeneration (PKAN) is a rare disease caused by pantothenate kinase 2 (PANK2, OMIM 606157) mutations. This study is aimed to investigate clinical presentations, pathologies, and genetics in patients with PKAN. Methods. Two patients with PKAN were reported. We reviewed the literature to include additional 19 patients with PKAN in Eastern Asia. These patients were divided into classic and atypical groups by the age of onset. We compared the data on PKAN patients of Asian and Caucasian populations. Results. We found iron deposits in the globus pallidus in our Patient 1 and a heterozygous truncating mutation (c.1408insT) in Patient 2. Literature review shows that generalized dystonia and bulbar signs are more common in classic PKAN patients, whereas segmental dystonia and tremors are more specific to atypical ones. Asian patients have less complex presentations-lower prevalence of pyramidal signs, mental impairment, and parkinsonism-than Caucasians. D378G in exon 3 is the most frequent mutation (28%) in Asians. Conclusions. Our study demonstrates that the distribution of dystonia is the major distinction between subgroups of PKAN. Caucasian patients have more complex presentations than Asians. Exon 3 and 4 are hot spots for screening PANK2 mutations in Asian patients

CD5L is a canonical component of circulatory IgM

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L

CD5L is a canonical component of circulatory IgM

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L

Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry

Standard methods for mass analysis measure ensembles of thousand to millions of molecules. This approach enables analysis of monodisperse recombinant proteins, whereas some heterogeneous protein assemblies pose a significant challenge, whereby co-occurring stoichiometries, sub-complexes, and modifications hamper analysis using native mass spectrometry. To tackle the challenges posed by mass heterogeneity, single-particle methods may come to the rescue. Recently, two such approaches have been introduced, namely, mass photometry (MP) and Orbitrap-based charge detection mass spectrometry (CDMS). Both methods assess masses of individual molecules, albeit adhering to distinct physical principles. To evaluate these methods side by side, we analyzed a set of ribosomal particles, representing polydisperse ribonucleoprotein assemblies in the MDa range. MP and CDMS provide accurate masses for intact ribosomes and enable quantitative analysis of concomitant distinct particles within each ribosome sample. Here, we discuss pros and cons of these single-molecule techniques, also in the context of other techniques used for mass analysis

Biomolecular Clusters Distribution up to Mega Dalton Region Using MALDI-Quadrupole Ion Trap Mass Spectrometer

We present the first report on complete cluster distributions of cytochrome c (molecular weight of 12.4 kDa) and bovine serum albumin ((BSA), molecular weight of 66.4 kDa) with mass-to-charge ratio (m/z) reaching 350,000 and 1,400,000, respectively, by matrix-assisted laser desorption/ionization (MALDI). Large cluster distributions of the analytes were measured by our homemade frequency-scanned quadrupole ion trap (QIT) mass spectrometer with a charge detector. To our knowledge, we report the highest m/z clusters of these two biomolecules. The quantitative results indicate that large clusters ions of cytochrome c and BSA follow the power law (r2 > 0.99) with cluster size distribution, which provides experimental evidence for the laser ablation studies of MALDI

Comparative Analysis of Antibodies and Heavily Glycosylated Macromolecular Immune Complexes by Size-Exclusion Chromatography Multi-Angle Light Scattering, Native Charge Detection Mass Spectrometry, and Mass Photometry

Qualitative and quantitative mass analysis of antibodies and related macromolecular immune complexes is a prerequisite for determining their identity, binding partners, stoichiometries, and affinities. A plethora of bioanalytical technologies exist to determine such characteristics, typically based on size, interaction with functionalized surfaces, light scattering, or direct mass measurements. While these methods are highly complementary, they also exhibit unique strengths and weaknesses. Here, we benchmark mass photometry (MP), a recently introduced technology for mass measurement, against native mass spectrometry (MS) and size exclusion chromatography multi-angle light scattering (SEC-MALS). We examine samples of variable complexity, namely, IgG4Δhinge dimerizing half-bodies, IgG-RGY hexamers, heterogeneously glycosylated IgG:sEGFR antibody–antigen complexes, and finally megadalton assemblies involved in complement activation. We thereby assess the ability to determine (1) binding affinities and stoichiometries, (2) accurate masses, for extensively glycosylated species, and (3) assembly pathways of large heterogeneous immune complexes. We find that MP provides a sensitive approach for characterizing antibodies and stable assemblies, with dissociation correction enabling us to expand the measurable affinity range. In terms of mass resolution and accuracy, native MS performs the best but is occasionally hampered by artifacts induced by electrospray ionization, and its resolving power diminishes when analyzing extensively glycosylated proteins. In the latter cases, MP performs well, but single-particle charge detection MS can also be useful in this respect, measuring masses of heterogeneous assemblies even more accurately. Both methods perform well compared to SEC-MALS, still being the most established method in biopharma. Together, our data highlight the complementarity of these approaches, each having its unique strengths and weaknesses

Comparative Analysis of Antibodies and Heavily Glycosylated Macromolecular Immune Complexes by Size-Exclusion Chromatography Multi-Angle Light Scattering, Native Charge Detection Mass Spectrometry, and Mass Photometry

Qualitative and quantitative mass analysis of antibodies and related macromolecular immune complexes is a prerequisite for determining their identity, binding partners, stoichiometries, and affinities. A plethora of bioanalytical technologies exist to determine such characteristics, typically based on size, interaction with functionalized surfaces, light scattering, or direct mass measurements. While these methods are highly complementary, they also exhibit unique strengths and weaknesses. Here, we benchmark mass photometry (MP), a recently introduced technology for mass measurement, against native mass spectrometry (MS) and size exclusion chromatography multi-angle light scattering (SEC-MALS). We examine samples of variable complexity, namely, IgG4Δhinge dimerizing half-bodies, IgG-RGY hexamers, heterogeneously glycosylated IgG:sEGFR antibody–antigen complexes, and finally megadalton assemblies involved in complement activation. We thereby assess the ability to determine (1) binding affinities and stoichiometries, (2) accurate masses, for extensively glycosylated species, and (3) assembly pathways of large heterogeneous immune complexes. We find that MP provides a sensitive approach for characterizing antibodies and stable assemblies, with dissociation correction enabling us to expand the measurable affinity range. In terms of mass resolution and accuracy, native MS performs the best but is occasionally hampered by artifacts induced by electrospray ionization, and its resolving power diminishes when analyzing extensively glycosylated proteins. In the latter cases, MP performs well, but single-particle charge detection MS can also be useful in this respect, measuring masses of heterogeneous assemblies even more accurately. Both methods perform well compared to SEC-MALS, still being the most established method in biopharma. Together, our data highlight the complementarity of these approaches, each having its unique strengths and weaknesses

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions

Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The ∼895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the ∼935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric "pentamer" stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria