Improved understanding of dental enamel development will benefit not only dentistry but also biomedicine more generally. Rat and mouse models of enamel development are relatively well characterized ...and experimentally powerful. However, the diminutive size of murine teeth makes them difficult to study using standard proteomics approaches. Here, we describe gel-based proteomic methods that enable parallel quantification, identification, and functional characterization of proteins from developing rat and mouse teeth. These refined methods are applicable to other scarce samples including human enamel defects.
While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of ...this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•We developed a mass spectrometry method that allows robust quantification of orexin A in mouse CSF samples as small as 1 µL.•Analysis of individual mouse CSF samples confirmed a diurnal rhythm in ...orexin A levels.•Orexin A levels in mouse CSF were increased during acute sleep deprivation and reduced in the following dark phase.•Application of this LC-ESI-MRM method presents an opportunity to increase understanding of the role of orexin A in neurological and neuropsychiatric disease using mouse models.
Sleep-wakefulness is disrupted in most neurological and psychiatric disorders. Although clinical data implicate orexin (hypocretin), a crucial sleep/wake regulatory neuropeptide, in such disorders, limited sample volumes effectively prevent quantification of cerebrospinal fluid (CSF) levels of orexin A in mouse models of brain disorders. Current enzyme- and radio-immunoassays for orexin A generally require 50–100 µL CSF, whereas typical CSF sample volumes from mice are ~5–10 µL/mouse. We therefore aimed to develop and validate a liquid chromatography (LC) targeted mass spectrometry (MS) method for the absolute quantification of orexin A in the CSF of individual mice. LC coupled to tandem MS (LC-MS/MS) and a triple quadrupole (QQQ) mass spectrometer were used to develop a LC electrospray ionization multiple-reaction monitoring (LC-ESI-MRM) method. CSF orexin A levels of C57BL/6JARC mice were quantified using this method at the predicted peak and trough of diurnal orexin A release and following sleep deprivation. The LC-ESI-MRM assay was robust and sensitive, with an intra-assay variation <9% CV, inter-assay variation of 10% CV and limit of quantitation of 1.65 fmoles. CSF orexin A concentrations in C57/Bl6JARC mice were higher in the late active period (2.5 ± 0.5 fmoles/µL) versus the late inactive period (1.2 ± 0.5 fmoles/µL, p < 0.001). Sleep deprivation significantly dysregulated diurnal rhythm, up-regulating orexin A acutely, followed by down-regulation 16 hours after sleep deprivation. We anticipate this validated LC-ESI-MRM assay for the absolute quantification of orexin A in the CSF of individual mice will enhance research using relevant rodent models of sleep or arousal-related brain disorders.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The biomedical need for streamlined approaches to monitor proteome dynamics is growing rapidly. This study examined the ability of a knowledge-based triplex-profiling strategy (i.e., three ...functionally distinct chaperones, ERp29/PDI/BiP) to clarify uncertainties about how cancer affects the endoplasmic reticulum (ER) proteome. Investigating a wide range of samples at the tissue and cellular levels (>114 samples from 9 tissues of origin), we obtained consistent evidence that the ER proteome undergoes a major but variable expansion in cancer. Three factors having a strong influence on the ER proteome were identified (cancer-cell type, growth rate, culture mode), and the functionally enigmatic chaperone ERp29 was linked distinctively to histogenetic aspects of tumorigenesis. These findings justify pursuit of the ER-proteome as a medical target in cancer, validate ERp29/PDI/BiP profiling as a streamlined yet powerful measure of ER-proteome dynamics, and suggest that biomarker sets based on distinct functionalities could have broader biomedical utility.
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IJS, KILJ, NUK, PNG, UL, UM
Impaired therapeutic responses to anti-inflammatory glucocorticoids (GC) in chronic respiratory diseases are partly attributable to interleukins and transforming growth factor β1 (TGF-β1). However, ...previous efforts to prevent induction of GC insensitivity by targeting established canonical and non-canonical TGF-β1 pathways have been unsuccessful. Here we elucidate a TGF-β1 signaling pathway modulating GC activity that involves LIM domain kinase 2-mediated phosphorylation of cofilin1. Severe, steroid-resistant asthmatic airway epithelium showed increased levels of immunoreactive phospho-cofilin1. Phospho-cofilin1 was implicated in the activation of phospholipase D (PLD) to generate the effector(s) (lyso)phosphatidic acid, which mimics the TGF-β1-induced GC insensitivity. TGF-β1 induction of the nuclear hormone receptor corepressor, SMRT (NCOR2), was dependent on cofilin1 and PLD activities. Depletion of SMRT prevented GC insensitivity. This pathway for GC insensitivity offers several promising drug targets that potentially enable a safer approach to the modulation of TGF-β1 in chronic inflammatory diseases than is afforded by global TGF-β1 inhibition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ERp29 is a recently characterized resident of the ER (endoplasmic reticulum) lumen that has broad biological significance, being expressed ubiquitously and abundantly in animal cells. As an apparent ...housekeeper, ERp29 is thought to be a general folding assistant for secretory proteins and to probably function as a PDI (protein disulphide isomerase)-like molecular chaperone. In the present paper, we report the first purification to homogeneity and direct functional analysis of native ERp29, which has led to the unexpected finding that ERp29 lacks PDI-like folding activities. ERp29 was purified 4800-fold in non-denaturing conditions exploiting an unusual affinity for heparin. Two additional biochemical hallmarks that will assist the classification of ERp29 homologues were identified, namely the idiosyncratic behaviours of ERp29 on size-exclusion chromatography (M(r)<globular homodimer) and SDS/PAGE (M(r)>monomeric mass). In contrast with PDI and parallel-purified co-residents (calreticulin, ERp60), native ERp29 lacked classical chaperone, disulphide reductase and isomerase, and calcium-binding activities. In the chaperone assays, ERp29 neither protected substrate proteins against thermal aggregation nor interacted stably with chemically denatured proteins as detected by cross-linking. ERp29 also did not exhibit helper activity toward calreticulin (chaperone) or PDI and ERp60 (disulphide reductase). By refuting long-standing predictions about chaperone activity, these results expose ERp29 as a functionally distinct member of the ER machinery and prompt a revised hypothesis that ERp29 acts as a non-classical folding assistant. The native preparation and biochemical hallmarks established here provide a useful foundation for ongoing efforts to resolve the functional orphan status of ERp29.
•First proteomic analysis of cisternal CSF and comparison of ventricular and cisternal CSF composition.•First group to successfully quantify d-dimer in CSF using mass spectroscopy.•Absence of ...significant differences between ventricular and cisternal CSF suggests a homogenous environment.•Studies analysing CSF for vasospasm biomarkers can confidently analyse ventricular sources.
Analysis of cerebrospinal fluid (CSF) using mass spectrometry is a relatively novel analytical tool, and comparisons of ventricular and cisternal proteomes are yet to be performed. This may have implications for clinical medicine, particularly in demonstrating continuity of the ventricular system with preserved flow in the presence of ventricular blood. Other uses include the identification of novel biomarkers, including for diagnosis of subarachnoid haemorrhage and of aetiology. The primary objective was therefore to characterise and compare the proteomes of ventricular and CSF after haemorrhagic stroke.
Paired CSF samples were prospectively collected from the optico-carotid cistern and the frontal horn of the lateral ventricle at the time of craniotomy and clipping in 8 patients with haemorrhagic stroke. Six patients had an aneurysmal subarachnoid haemorrhage (aSAH) from a ruptured saccular aneurysm, one patient had an aSAH after rupture of a mycotic aneurysm and one patient had a spontaneous intracerebral haemorrhage (IPH) with an adjacent unruptured saccular aneurysm. Samples were processed and proteins identified and quantified using data-dependent liquid chromatography tandem mass spectrometry (DDA LC-MSMS).
There was no systematic difference between the cisternal and ventricular proteomes. However, blinded principal component analysis (PCA) of the cisternal and ventricular samples separated patients according to pathophysiology. Additionally CSF D-Dimer levels were not detected in the IPH patient but were reliably measured in aSAH patients.
Ventricular CSF is representative of cisternal CSF after aSAH. CSF proteomic PCA analysis can distinguish between haemorrhage types. CSF D-dimer levels may represent a novel diagnostic marker for aSAH. Label free DDA LC-MSMS CSF analysis may inform possible biomarkers.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Craniofacial disorders are associated with one‐third of human birth defects but the underlying molecular and cellular causes remain poorly understood. Proteomics seems well‐placed to benefit this ...medically important area but the scarcity of embryonic tissues poses a major challenge. In this study, we applied a microsample proteomics strategy to investigate the first branchial arch, an embryonic structure crucial for facial development, and found that proteome analysis is both practicable and informative despite the scarcity of tissue. Exploiting the embryonic chick as a tractable source of accurately staged tissue, we developed a sequential extraction procedure to interface with one‐dimensional polyacrylamide gel electrophoresis (1‐D PAGE) and 2‐D PAGE. In 2‐D gels, about 8% of the visible proteome changed between embryonic days 3 and 5, and the identities determined for 21 proteins accorded with the rapid growth during this period. These results led to the first molecular identification of chicken alpha‐fetoprotein, and an unusual localisation of vimentin to endoderm. With over 470 protein spots accessible, this comparative proteomics approach has good prospects for providing new markers, functional hypotheses and genes to target in functional tests. A broader value of extending these approaches to facial development in other species and to other areas in embryology can be anticipated.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are ...thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin28kDa. Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin28kDa-null mutants. An unchanged rate of calcium transport was verified by measurements of 45Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin28kDa was not compensated by other cytosolic calcium-binding proteins as detectable by 45Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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