Charge detection mass spectrometry (CDMS) is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of their mass-to-charge ratio (m/z) and charge. Masses are determined for thousands of individual ions, and then the results are binned to give a mass spectrum. Using this approach, accurate mass distributions can be measured for heterogeneous and high-molecular-weight samples that are usually not amenable to analysis by conventional mass spectrometry. Recent applications include heavily glycosylated proteins, protein complexes, protein aggregates such as amyloid fibers, infectious viruses, gene therapies, vaccines, and vesicles such as exosomes.

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology.

Amyloid fibres attract considerable interest due to their biological role in neurodegenerative diseases and their potential as functional biomaterials. Here, we describe an intrinsic signal of amyloid fibres in the near-infrared range. When combined with their recently reported blue luminescence, it paves the way towards new blueprints for the label-free detection of amyloid deposits in in vitro and in vivo contexts. The blue luminescence allows for staining-free characterization of amyloid deposits in human samples. The near-infrared signal offers promising prospects for innovative diagnostic strategies for neurodegenerative diseases—to improve medical care and for the development of new therapies. As a proof of concept, we demonstrate direct detection of amyloid deposits within brains of living, aged mice with Alzheimer’s disease using non-invasive and contrast-agent-free imaging. Ultraviolet–visible–near-infrared optical properties of amyloids open new research avenues for amyloidosis as well as for next-generation biophotonic devices.

Ultraviolet–visible–near-infrared optical properties of amyloid fibrils shed light on amyloidogenesis

Mass spectrometry is a central technology in the life sciences, providing our most comprehensive account of the molecular inventory of the cell. In parallel with developments in mass spectrometry technologies targeting such assessments of cellular composition, mass spectrometry tools have emerged as versatile probes of biomolecular stability. In this review, we cover recent advancements in this branch of mass spectrometry that target proteins, a centrally important class of macromolecules that accounts for most biochemical functions and drug targets. Our efforts cover tools such as hydrogen-deuterium exchange, chemical cross-linking, ion mobility, collision induced unfolding, and other techniques capable of stability assessments on a proteomic scale. In addition, we focus on a range of application areas where mass spectrometry-driven protein stability measurements have made notable impacts, including studies of membrane proteins, heat shock proteins, amyloidogenic proteins, and biotherapeutics. We conclude by briefly discussing the future of this vibrant and fast-moving area of research.

Mass Spectrometry Methods for Measuring Protein Stability.

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.

/pdf/broadly-conserved-roles-of-tmem131-family-proteins-in-4dxmnkurxo.pdf

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy in need of more effective treatment approaches. One potential therapeutic target is Wnt/β-catenin signaling, which plays important roles in PDAC tumor initiation and progression. Among Wnt inhibitors with suitable in vivo biologic activity is vitamin D, which is known to antagonize Wnt/β-catenin signaling in colorectal cancer and have antitumor activity in PDAC. For this study, the relationship between vitamin D signaling, Wnt/β-catenin activity, and tumor cell growth in PDAC was investigated through the use of calcipotriol, a potent non-hypercalcemic vitamin D analogue. PDAC tumor cell growth inhibition by calcipotriol was positively correlated with vitamin D receptor expression and Wnt/β-catenin activity. Furthermore, vitamin D and Wnt signaling activity were found to be reciprocally linked through feedback regulation. Calcipotriol inhibited autocrine Wnt/β-catenin signaling in PDAC cell lines in parallel with decreased protein levels of the low-density lipoprotein receptor-related protein 6 (LRP6), a requisite coreceptor for ligand-dependent canonical Wnt signaling. Decrease in LRP6 protein seen with calcipotriol was mediated through a novel mechanism involving transcriptional upregulation of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1). Finally, changes in LRP6 or LDLRAP1 expression directly altered Wnt reporter activity, supporting their roles as regulators of ligand-dependent Wnt/β-catenin signaling. Implications: This study provides a novel biochemical target through which vitamin D signaling exerts inhibitory effects on Wnt/β-catenin signaling, as well as potential biomarkers for predicting and following tumor response to vitamin D–based therapy. Mol Cancer Res; 13(11); 1509–19. ©2015 AACR.

/pdf/calcipotriol-targets-lrp6-to-inhibit-wnt-signaling-in-25vw9a4vbb.pdf

Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer

Heterogeneity and polymorphism are generic features of amyloid fibers with some important effects on the related disease development. We report here the characterization, by charge detection mass spectrometry, of amyloid fibers made of three polypeptides involved in neurodegenerative diseases: Aβ1–42 peptide, tau and α-synuclein. Beside the mass of individual fibers, this technique enables to characterize the heterogeneity and the polymorphism of the population. In the case of Aβ1–42 peptide and tau protein, several coexisting species could be distinguished and characterized. In the case of α-synuclein, we show how the polymorphism affects the mass and charge distributions.

/pdf/mass-and-charge-distributions-of-amyloid-fibers-involved-in-1xrv4d2dgl.pdf

Mass and charge distributions of amyloid fibers involved in neurodegenerative diseases: mapping heterogeneity and polymorphism

In this study, we identify transmembrane protein 131–like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA–mediated silencing of TMEM131L in human CD34 + hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rγ null mice, resulted in both thymocyte hyperproliferation and multiple pre– and post–β-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and β-catenin intranuclear accumulation. Using T cell factor reporter assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/β-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome-dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent mechanisms.

/pdf/identification-of-tmem131l-as-a-novel-regulator-of-thymocyte-5f9r1r7tvv.pdf

Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans

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Mass and charge distributions of amyloid fibers involved in neurodegenerative diseases: mapping heterogeneity and polymorphism

Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans