Relative abundance of phosphorylated α-crystallin in different regions of bovine and rabbit lenses

<p><b>Copyright information:</b></p><p>Taken from "Distribution of bovine and rabbit lens α-crystallin products by MALDI imaging mass spectrometry"</p><p></p><p>Molecular Vision 2008;14():171-179.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254960.</p><p></p> Extracted representative mass spectra of phosphorylation of intact αA- and αB-crystallin from outer cortex (, ), middle cortex (, ), and outer nucleus (, ) of bovine (-) and rabbit (-) lenses are shown. Phosphorylation of both subunits of α-crystallin is most abundant in the middle cortex of bovine and rabbit lenses

MALDI mass spectra of αA-crystallin truncation in bovine and rabbit lens

<p><b>Copyright information:</b></p><p>Taken from "Distribution of bovine and rabbit lens α-crystallin products by MALDI imaging mass spectrometry"</p><p></p><p>Molecular Vision 2008;14():171-179.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254960.</p><p></p> Extracted representative mass spectra from the outer cortex (, ), middle cortex (, ), and outer nucleus (, ) of bovine (-) and rabbit (-) lenses are shown. Degradation products increased toward the nucleus of both species, although higher mass degradation products are more prominent in the rabbit lenses than in the bovine lenses

Spatially-Directed Protein Identification from Tissue Sections by Top-Down LC-MS/MS with Electron Transfer Dissociation

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small molecules and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the analysis and limits the interpretation of underlying biology of tissue specimens. In this work, spatially directed tissue microextraction of intact proteins followed by LC-MS/MS with electron transfer dissociation (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 μL extraction volume was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50–100 proteins in a single experiment. Additionally, multiple modified proteins were identified; including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein separation and identification are expected to improve with advances in intact protein fractionation/chromatography and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations

MALDI Imaging and in Situ Identification of Integral Membrane Proteins from Rat Brain Tissue Sections

Transmembrane proteins are greatly underrepresented in data generated by imaging mass spectrometry (IMS) because of analytical challenges related to their size and solubility. Here, we present the first example of MALDI IMS of two highly modified multitransmembrane domain proteins, myelin proteolipid protein (PLP, 30 kDa) and DM-20 (26 kDa), from various regions of rat brain, namely, the cerebrum, cerebellum, and medulla. We utilize a novel tissue pretreatment aimed at transmembrane protein enrichment to show the in situ distribution of fatty acylation of these proteins, particularly of post-translational palmitoylation. Additionally, we demonstrate the utility of protease-encapsulated hydrogels for spatially localized on-tissue protein digestion and peptide extraction for subsequent direct coupling to LC-MS/MS for protein identification

L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins

<div><p>Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.</p></div

Imbalanced Expression of <i>Vcan</i> mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

<div><p>The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the <i>Vcan</i> null mouse called heart defect (<i>hdf</i>). Total absence of the <i>Vcan</i> gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, <i>Vcan</i>^(tm1Zim), of heart defects that results from deletion of exon 7 in the <i>Vcan</i> gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the <i>Vcan</i> gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of <i>Vcan</i> exon 7. The <i>Vcan</i>^(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.</p></div

Western blot comparison of four proteins with altered abundance by itraq in <i>Vcan</i>^(tm1Zim) mutant and wild-type hearts.

<p>Proteins that showed different levels of altered abundance in the <i>Vcan</i>^(tm1Zim) hearts are shown in the blot. Annexin A6 and Stathmin are proteins expressed in the heart that showed a relative decrease (0.85x and 0.73x respectively) in abundance in the <i>Vcan</i>^(tm1Zim) mutant by iTRAQ and serphin1 (Hsp 47) that by iTRAQ showed increased abundance (1.55x). Additionally, Desmin that did not change with any significance between mutant (Mut) and wild-type (Wt) by iTRAQ, showed no significant change by western blot. Results of average relative density measurements of separately analyzed hearts are shown in the graph for each protein (for Stmn, Hsp47, Desmin n = 3; p<0.01; for Annexin A6 n = 2; p<0.0045).</p

Splice forms of versican.

<p>The <i>Vcan</i> gene consists of 15 exons. Four mRNA and corresponding protein variants of the core protein (V0, V1, V2, V3) are derived from alternative splicing of exons 7 (blue boxes) and/or 8 (green boxes) into mRNA of <i>Vcan</i> gene. The glycosaminoglycan attachment domains GAG α & β are encoded by exon 7 (blue) and 8 (green) respectively. Deletion of exon 7 (blue) results in the loss of both the V2 and V0 variants. Exons 2–6 comprise the G1 domain that binds hyaluronan and 9–15 the G3 domain that also can interact with other ECM molecules.</p

Three-dimensional reconstructions and quantitative measurements of wild-type and <i>Vcan</i> exon 7 AV associated cushions in E13.5 pc hearts.

<p>The differences found in the mesenchymalized AV cushions, visually apparent in the 3-dimensional comparisons (Panel A, ventral view; B, dorsal view), were also quantified using the AMIRA imaging software to measure the cushion volumes. A significant reduction in volume was measured in the central AV cushion comprising the aortic leaflets (AL in panels A, B; reduced 35%; *p<0.034 n = 3 for each genotype, panel C) and septal (SL in panels A,B; reduced 30% p<0.046 n = 3 for each genotype in panel D). A significant (*p value 0.05 panel G) decrease in volume (0.58x) of the dorsal mesenchymal protrusion (DM) was measured. The other cushions showed no significant difference and the overall size of the E13.5 <i>Vcan</i>^(tm1Zim) and wild-type hearts (measured by tissue weight) was not significantly different (74 mg and 75.6 mg respectively; n = 3 for each genotype). AL-aortic leaflet (red); PL-parietal leaflet (blue); SL-septal leaflet (pink); ML-mural leaflet (green); DM-dorsal mesenchyme (white).</p

Histological comparison of cushions in the <i>Vcan</i>^(tm1Zim).

<p>Hematoxylin/eosin stained sections of postnatal day 1 (B, D) and <i>Vcan</i>^(tm1Zim) (A, C) hearts were compared. Boxed region in A and B of the cushion is shown higher magnification in C and D. Note the smaller cushions in the <i>Vcan</i>^(tm1Zim) heart. Panel A and B are the same magnification as are C and D; magnification bars = 200 µm.</p