Project : PXD000133



Equine Cerebrospinal Fluid


Cerebrospinal fluid is a diagnostic biofluid that is reflective of the overall health of the patient. Proteomic profiling of human CSF has been performed on a variety of disease states, but a comprehensive proteomic profile of equine CSF has not been completed until now. A total of 320 proteins were confidently identified in a pooled sample representative of 6 healthy horses. Gene Ontology terms were mapped in Uniprot, and normalized spectral abundance factors were calculated as a measure of relative abundance. Briefly, CSF was collected from healthy horses via subarachnoid catheter or manual draw, protease inhibitors were added (Pierce, Rockford, IL), and samples were frozen at -80oC (Figure 1). Protein concentrations were determined via Bradford Assay (Thermo Scientific, Rockford, IL) and 30 ug of each sample underwent in-solution digestion using ProteaseMAX (Promega, Madison, WI) and urea. Samples were solubilized in 8 M urea, 0.2% protease max, then reduced, alkylated, and digested with 1% protease max and trypsin at 37oC for 3 hours. Samples were dried in a Speed VacĀ® vacuum centrifuge, desalted using Pierce PepClean C18 spin columns (Pierce, Rockford, IL), dried and resuspended in 30 ul 3% ACN, 0.1% formic acid. All solvents, water, and acid were LC-MS/MS grade from Sigma (St. Louis, MO). Online 2-dimensional LC-MS/MS with SCX (strong cation exchange) and subsequent reverse phase chromatography was performed as follows. 10 ug of digested peptides from a sample were loaded onto a Zorbax BIO-SCX II 3.5 umm, 50 x 0.8 mm column (Agilent Technologies, Santa Clara, CA). Peptides were eluted off of the SCX column step-wise using increasing concentrations of NaCl in 0.3% ACN, 0.1% FA (20 ul NaCl salt injections: 15, 30, 45, 60, 75, 90, 120, 150, 300, 500 mM). Peptides from each individual salt injection were then purified and concentrated using an on-line enrichment column (Agilent Zorbax C18, 5 um, 5 x 0.3mm). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Agilent 1100 nanoHPLC, Zorbax C18, 5um, 75 um ID x 150mm column) using a 60 minute linear gradient from 25%-55% buffer B (90% ACN, 0.1% formic acid) at a flow rate of 300 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific LTQ linear ion trap) and spectra were collected over a range of 200-2000 m/z using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Bioworks 3.0 software (Thermo Scientific) with an intensity threshold of 5,000 and 1 scan/group. This workflow generates 10 raw data files per sample. MS/MS spectra were searched against the NCBInr equine database concatenated to a reverse database (14,473 entries) using the Mascot database search engine (Matrix Science, version 2.3.02) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research). The following search parameters were used: average mass, peptide mass tolerance of 2.5 Da, fragment ion mass tolerance of 1.0 Da, complete tryptic digestion allowing one missed cleavage, variable modification of methionine oxidation, and a fixed modification of cysteine carbamidomethylation. Peptide identifications from both of the search engines were combined using protein identification algorithms in Scaffold 3 (Version 3.6.4, Proteome Software, Portland, OR). All data files were then combined using the MudPIT option in Scaffold 3 generating a composite listing for all proteins identified across all runs. Thresholds were set to 99% and 95% protein and peptide probability respectively, and a 2 unique peptide minimum was required. The peptide false discovery rate (FDR) was less than 0.2% after manual validation of all proteins identified by 2 unique peptides. Criteria for manual validation included the following: 1) a minimum of at least 3 theoretical y or b ions in consecutive order that are peaks greater than 5% of the maximum intensity; 2) an absence of prominent unassigned peaks greater than 5% of the maximum intensity; and 3) indicative residue specific fragmentation, such as intense ions N-terminal to proline and immediately C-terminal to aspartate and glutamate.

Sample Processing Protocol

Not available

Data Processing Protocol

Not available


Carolyn Broccardo, Proteomics and Metabolomics Facility

Submission Date



Not available




Not available


Not available

Experiment Type

Bottom-up proteomics


Publication pending

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