Protein spots were excised from both preparative gels to maximize the opportunity
for protein identification. The picker head was calibrated using the picking references
located on the margins of the preparative gel plates. Excised gel plugs were automatically
placed in barcoded 96-well plates for in-gel trypsin digestion. Plugs were washed
twice with 200mM ammonium bicarbonate/40% acetonitrile, dehydrated with 75% acetonitrile,
and dried to completion by speedvac. Proteins were digested overnight incubation
at 37°C in 20?g/?L trypsin in 100?M HCl/36mM ammonium bicarbonate, 8% acetonitrile.
Following digestion, peptides were extracted with 50% acetonitrile/0.1% trifluoroacetic
acid at 37°C and then dried to completion by speedvac. Digests were then resuspended
in 0.5% trifluoroacetic acid and desalted and concentrated using C18 ZipTips (Millipore,
Billeria, MA). Peptides were spotted onto OptiTof 384-position MALDI plate inserts
(Applied Biosystems, Foster City, CA) in 1.2?L volumes and overlayed with 0.7?L 20?g/?L
?-Cyano-4-hydroxycinnamic acid (ACH-cinnamic acid) matrix (GE Healthcare) in 60%
acetonitrile/6mM ammonium phosphate. Peptides were analyzed in the Penn State Mass
Spectrometry Facility by MALDI-TOF/TOF using a 4800 Proteomics Analyzer (Applied
Biosystems) calibrated with 4700 Proteomics Mass Standards (Applied Biosystems).
For each sample, an initial mass spectrum was collected. MS scans were collected
in the reflector positive mode between 800 and 4000 m/z with a signal-to-noise filter
of 10, mass exclusion tolerance of 0.2Da, and a peak density filter of 50 peaks per
200Da. Based on the initial mass spectrum, up to 10 precursors were selected for
tandem mass spectrometry (MS/MS) analysis, excluding trypsin autolysis peptides,
matrix, and tryptic peptides of human keratin, as well as precursor ions indentified
in a “blank” gel plug. MS/MS was performed both with and without collision-induced
decay in a mass range from 60Da to 20Da below the precursor mass with a fragment
tolerance of 0.2Da for +1 charged ions. Using GPS Explorer 3.6 software (Applied
Biosystems), the MS and MS/MS data were submitted to a MASCOT (v2.0.00) search engine
for identification. The NCBI nonredundant database with the Rattus taxonomy (downloaded
February 16, 2008; 107,758 entries searched) and a concatenated, reversed “decoy”
version were used for the searches with a mass accuracy of 50ppm, one missed trypsin
cleavage, fixed carbamidomethylation of Cysteine residues and variable oxidation
of methionine residues. A protein was considered identified if the MASCOT confidence
interval was >95th percentile, and those proteins with a MASCOT confidence interval
<95% were excluded from subsequent analyses.

Ten gel plugs, from one of the two preparative gels, matching to the ten largest
magnitude, statistically significant changes were submitted to a commercial protein
identification service provided by Proteomic Research Services (NextGen Sciences,
Ann Arbor, MI). This additional analysis was performed to maximize protein identification
of these protein spots of interest. Protein identification
was performed using an on-line nano LC/MS/MS and ThermoFisher Orbitrap XL. Tryptic
peptides were loaded on a 75mm C12 vented column at a flow-rate of 10?L/min and eluted
at 300nL/min with the following gradient (time in minutes:% elution buffer): 0.1
min:3%, 30 min:23%, 38 min:32%, 42 min:50%, 44 min:95%, 47 min:95%, 47.5 min:1.0%,
55 min: 1.0%.
MS/MS data for confidently identified proteins were complied as follows: information
regarding molecular weight and theoretical isoelectric point was obtained from MASCOT
reports. Percent sequence coverage and MOWSE scores of these proteins were determined
with the MS-Fit database search component of ProteinProspector v5.3.3 (http://prospector.ucsf.edu/);
University of California, San Francisco). The NCBI gi number sequence identifiers
provided for identified proteins by the MASCOT database search were converted to
UniProt/Swiss-Prot accession numbers using the ID Mapping function of the Universal
Protein Resource Knowledgebase (UniProtKB; http://www.uniprot.org).