DOI: 10.5061/dryad.nk98sf7pz
title: Differential Proteomic Expression of Equine Cardiac and Lamellar Tissue During Insulin-Induced Laminitis

description: Endocrinopathic laminitis is pathologically similar to the multi-organ
 dysfunction and peripheral neuropathy found in human patients with
 metabolic syndrome.  Similarly, endocrinopathic laminitis has
 been shown to partially result from vascular dysfunction. However, despite
 extensive research, the pathogenesis of this disease is not well
 elucidated and laminitis remains without an effective treatment. Here, we
 sought to identify novel proteins and pathways underlying the development
 of equine endocrinopathic laminitis. Healthy Standardbred horses
 (n=4/group) were either given an electrolyte infusion, or a 48-hour
 euglycemic-hyperinsulinemic clamp. Cardiac and lamellar tissues were
 analyzed by mass spectrometry (FDR=0.05). All hyperinsulinemic horses
 developed laminitis despite being previously healthy. We identified 538
 and 737 unique proteins in the cardiac and lamellar proteomes,
 respectively. In the lamellar tissue, we identified 14 proteins which were
 significantly upregulated and 13 proteins which were significantly
 downregulated in the hyperinsulinemic group as compared to controls. These
 results were confirmed via real-time reverse-transcriptase PCR. A STRING
 analysis of protein-protein interactions revealed that these upregulated
 proteins were primarily involved in coagulation and complement cascades,
 platelet activity, and ribosomal function, while downregulated proteins
 were involved in focal adhesions, spliceosomes, and cell-cell matrices.
 Novel significant differentially expressed proteins associated with
 hyperinsulinemia-induced laminitis include talin -1, vinculin,
 cadherin-13, fibrinogen, alpha-2-macroglobulin, and heat shock protein 90.
 In contrast, no proteins were found to be significantly differentially
 expressed in the heart of hyperinsulinemic horses compared to controls.
 Together, these data indicate that while hyperinsulinemia induced, in
 part, microvascular damage, complement activation, and ribosomal
 dysfunction in the lamellae, but a similar effect was not seen in the
 heart. In brief, this proteomic investigation of a unique equine model of
 hyperinsulinemia identified novel proteins and signaling pathways, which
 may lead to the discovery of molecular biomarkers and/or therapeutic
 targets for endocrinopathic laminitis.
description: <strong>Proteomic
 Analysis</strong> Tissues were homogenized in RIPA
 buffer containing protease inhibitor cocktail, and protein contents were
 quantified by bicinchoninic assay, as previously described
 (Maria,
 Campolo et al. 2015).  Aliquots (25 micrograms) of
 each lysate were precipitated with TCA/acetone, redissolved and reduced
 for 1 hr in 8 M urea, 5 mM TECP, 100 mM Tris pH 8.5, and then alkylated
 for 20 min by adding 10 mM IAA.  Reactions were then diluted to 2 M urea,
 trypsin was added to 4 micrograms / ml, and digestions were incubated
 overnight at 37 degrees C.  After digestion, peptides were desalted by
 solid phase extraction on monolithic C18 pipet tips, and lyophilized to
 dryness. For LC-MS/MS, one
 microgram of peptides was injected onto a 0.15 x 50 mm vented trap column,
 followed by separation on a 0.075 x 400 mm Picofrit column/emitter (New
 Objective).  Both columns were packed with 3-micron Magic AQ C18 particles
 (Bruker).  Peptides were separated at room temperature using a 5-40%
 ACN/0.1% formic acid gradient performed over 100 min at a flow rate of 250
 nL/min, eluting directly into a New Objective PV-550 nanoelectrospray ion
 source.  Peptides were analyzed uing an LTQ OrbitrapXL mass spectrometer
 (Thermo) via a Top Six data-dependent acquisition method as previously
 described (Voruganti, Lacroix et al. 2013) Proteins were
 identified and quantified by using MaxQuant (Cox and Mann 2008) to search
 the raw instrument files against a database of  22,718 reference equine
 protein sequences downloaded from UniProt on 12/27/15.  Searches used the
 fixed modification carbamidomethyl (C) and the following variable
 modifications:  oxidized (M), acetyl (protein N-terminus) and glutamine
 cyclization to glutamate).  Searches were performed using default MaxQuant
 settings, with the addition of label-free quantitation via the LFQ
 algorithm and match between runs to transfer MS/MS identifications between
 LC-MS/MS data files.  Statistical
 analyses were performed using the Perseus software package (Max Planck
 Institute of Biochemistry) (Tyanova, Temu et al. 2016) to analyze
 log(2)-transformed MaxQuant LFQ protein intensities. Samples were
 identified as being significantly differentially expressed via Student’s
 t-test using the Benjamini-Hochberg multiple tests correction, with a
 false discovery rate of 0.05 (Benjamini and Hochberg 1995).
  Protein-protein interactions and KEGG (Kyoto Encyclopedia of Genes and
 Genomes) pathways were identified via STRING 10.5 (Search Tool for the
 Retrieval of Interacting Genes/Proteins).