Animals and sample collection
Blood samples were collected from horses residing at the Hoof Diagnostic and Rehabilitation Clinic (Bryan, TX) in accordance with the Texas A&M University College of Veterinary Medicine guidelines regarding research performed on non university-owned animals. All animal protocols were approved by the Texas A&M University College of Veterinary Medicine Clinical Research Review Committee (CRRC# 10–52) and were performed with the consent of the owner or the owner’s agent. Horses were diagnosed as having chronic laminitis (LMN, n = 4) by a licensed veterinarian based on clinical presentation, case history, and radiographic evidence of dorsopalmar rotation of the distal phalanx. Horses in the control group (CON, n = 4) were housed either at the clinic or at a nearby farm and had no history of laminitis. Both groups of horses, CON and LMN, were 15 to 20 years of age. Please see Additional file
1 for detailed descriptions of each animal used in the study.
Blood samples were collected from the jugular vein into evacuated blood tubes containing either heparin or sodium citrate. Tubes were inverted a minimum of eight times to ensure adequate mixing of the anticoagulant. Heparinized samples were centrifuged at 1200 x g and plasma was harvested and stored at −80°C. No protease inhibitors were added to the samples. Citrated samples were immediately transported to the Texas A&M College of Veterinary Medicine Clinical Pathology Laboratory for evaluation of coagulation parameters. All samples were collected between 9:00 and 11:00 am.
Sample preparation for DIGE
All sample preparation and DIGE experiments were conducted at the Texas A&M University Protein Chemistry Laboratory. Details of the DIGE methods used have been described previously
. Frozen plasma (CON, n = 4; LMN, n = 4) was depleted of albumin and IgG using human-specific affinity chromatography columns (Seppro, Sigma-Aldrich, St. Louis, MO) to enrich samples for low-abundance proteins. Human affinity chromatography reagents have been used successfully in horses for depletion of serum proteins
. A reference sample (containing equal amounts of protein from each of the 4 CON and 4 LMN samples) was used as a inter-gel standard. Forty-five micrograms of each CON and LMN sample was labeled with 200 pM Cy3 or Cy5 dye in a balanced design to reduce potential effects of dye bias; the reference sample was labeled with 200 pM Cy2. Equal amounts of paired LMN and CON samples, plus the reference sample, were mixed and loaded onto an isoelectric focusing strip (DryStrip, 24 cm, pH 4–7, GE Healthcare). Proteins were focused at 50 μA per strip for a total of 50,200 Vh and then separated on 12% SDS-PAGE gels. A total of four gels were used, with each gel containing equal amounts of protein from one CON sample, one LMN sample, and the reference sample.
Gel imaging and DeCyder analysis
Gels were scanned on a Typhoon Trio fluorescent imager (GE Healthcare) with an image resolution of 100 μm. The Biological Variation Analysis (BVA) and the Batch Processor modules of the DeCyder v6.5 software package were used to identify protein spots in each gel, to quantify fluorescent intensity of the spots, and to perform statistics. Criteria for signficantly differentially expressed spots included identification on at least three of the four gels and a P value < 0.05 by Student’s independent t test. Spots selected for identification by LC-MS/MS were robotically picked (Ettan Spot Picker, GE Healthcare) out of one gel, digested with trypsin, and shipped to the University of Texas – San Antonio Proteomics Core service lab for LC-MS/MS analysis.
MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version Mascot) and X! Tandem (The GPM, thegpm.org; version 2007.01.01.1). X! Tandem and Mascot were set up to search the NCBI database of nonredundant proteins (NCBInr_20091018.fasta, 9910686 entries) assuming the digestion enzyme trypsin. Scaffold (version Scaffold_3_00_04, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm
. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm
. Mass spectra have been deposited in the PRIDE database
 under accession number 22842.
The horse genome, although sequenced, is partially annotated; human orthologs of the equine proteins were therefore used to search for significant gene ontologies. Biological processes, molecular functions, cellular components, and relevant pathways were identified using the DAVID algorithm (v6.7,
[45–47]). Significance was judged by an adjusted P value less than 0.01 and a false discovery rate of less than 5%.
Capture ELISAs were performed to validate DIGE results for selected proteins. Plasma concentrations of IgA and IgM were determined using validated antibody pairs (Bethyl Labs, Montgomery, TX) and standard protocols. Briefly, high protein-binding 96 well plates (Nunc MaxiSorp, Thermo Scientific, Rockford, IL) were coated with 100 μg of unlabelled capture antibody overnight at 4°C. Plates were blocked with SuperBlock buffer (Thermo Scientific, Waltham, MA) for 30 min at room temperature. Whole plasma samples were diluted 1:4,000 in assay buffer (10% SuperBlock in PBS) and 100 μl of each diluted sample was pipetted in duplicate onto the plates, which were then incubated at 37°C for 90 min. HRP-conjugated detection antibodies were diluted 1:50,000 (IgM) or 1:150,000 (IgA) in assay buffer and incubated on the plates for 2 h at room temperature. SigmaFast OPD (Sigma, St. Louis MO) was used as a substrate for HRP. Sample concentrations were calculated using dilutions of a reference serum of known concentration. Comparisons between groups were made by Student’s t test using a variation of the S programming language called R, which is commonly used for statistical analysis (
http://www.r-project.org). A P value < 0.05 was considered sufficient to reject the null hypothesis.
In order to explore potential physiological consequences of differentially expressed proteins identified using DIGE, a standard panel of coagulation tests was performed by the Texas A&M College of Veterinary Medicine Clinical Pathology Laboratory on the 4 LMN horses used for the DIGE experiment plus an additional 3 LMN cases that were available at the time of sample collection. None of the CON horses were available for additional sample collection at this time. Reported equine-specific reference ranges are those used for clinical cases at the Texas A&M Veterinary Medical Teaching Hospital. Comparisons between groups were performed by χ2 analysis using the R programming language. A P value < 0.05 was considered sufficient to reject the null hypothesis.
Immunoblotting for APOA-IV was performed according to standard protocols. NuPage equipment, gels, and buffers (Invitrogen, Carlsbad, CA) were used for protein electrophoresis and transfer according to the manufacturer’s instructions. Briefly, 1 μl of plasma from each horse was mixed with 10x NuPage Sample Buffer, NuPage Reducing Agent, and 5.5 μl of water, heated to 70°C for 10 min, and separated by electrophoresis on 4-12% Bis-Tris gradient gels. Gels were transferred to PVDF membranes, which were then rinsed with distilled water and blocked using 5% non-fat dried milk (NFDM) in a Tris-buffered saline solution containing 0.05% Tween-20 (TBST). Primary antibody (mouse anti-human APOA-IV, Abcam, Cambridge, UK) was diluted 1:1,000 in NFDM-TBST. Secondary antibody (goat anti-mouse IgG – HRP conjugated, Abcam) was diluted 1:5,000 in NFDM-TBST. Bound antibody was visualized by addition of a chemiluminescent substrate (SuperSignal West Dura ECL, Thermo Scientific) and blots were imaged using a CCD camera system (Chemi-Doc, Bio-Rad, Hercules, CA). Densitometry was performed using Image J software
. Equal loading of all samples was confirmed by staining of the membrane for total protein using GelCode SafeBlue stain (Thermo Scientific). Comparisons between groups were made by Student’s t test. Please see Additional file
2 for a detailed description of the verification of antibody specificity and loading controls.