All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver.
Pentobartial sodium was purchased from Abbott Labs (North Chicago, IL). Polyethylene tubing was purchased from Intrametic, Fisher Scientific. Heparin was purchased from American Pharmaceutical Partner, In (Schaumburg, IL). DIGE experiment reagents were purchased from GE healthcare. All other reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise specified.
Controlled hemorrhage was induced to male Sprague-Dawley rats weighing 218 mg to 351 mg (Colorado State University) that had been housed in climate controlled barrier facility with 12 hr light/dark cycles with free access to food and water. The animals were anesthetized with 50 mg/kg pentobarbital sodium via intraperitoneal injection. The femoral artery and vein were then cannulated with polyethylene (PE) 50 tubing and the blood pressure and mean arterial pressure were monitored using a ProPaq invasive monitoring device (Welch Allyn Inc., Skaneateles Falls, NY). A separate skin puncture was created to tunnel the catheters prior to closure of the groin incision. A 3 cm midline laparotomy was performed. The bowel was eviscerated and rotated to the left, and the mesenteric duct and accessory duct (located adjacent to the superior mesenteric artery) were isolated by blunt dissection. The main lymphatic duct was cannulated with PE 100 tubing and secured with 7-0 prolene suture. The accessory duct was then ligated with suture, and the catheter was tunneled posteriorly through the skin. The laparotomy incision was closed in a two layer fashion and lymph collection took place in half-hour intervals into 1.6 mL tubes containing 1.0 mg ethylenediaminetetraacetic acid (EDTA) followed by rapid freezing in liquid nitrogen. After 1 hour of lymph collection, hemorrhagic shock was induced by controlled blood loss to maintain a mean arterial pressure (MAP) of 30 mmHg and sustained for 40 min. Euthermic body temperature was maintain with a heat lamp and monitored rectally in regular intervals. Resuscitation was performed by infusing 2x shed blood volume in normal saline over 30 min, followed by 1/2 shed blood volume returned over 30 min, then completed with 2x shed blood volume in normal saline over 60 min. Lymph collection continued for one hour post completion of resuscitation and all lymph samples were then centrifuged at 5000 × g for 10 min to remove cellular components. The lymph supernatant was collected and frozen in liquid nitrogen, and all lymph samples were stored at -80°C until processing. The fractions collected between 2-3 hours following resuscitation were consistently bioactive by a number of priming, signaling and physiological tests . Protein quantification was performed using the BCA protein assay kit (with BSA as standard) to create a regression analysis to estimate overall protein concentration for each hourly sample . In general post-shock mesenteric lymph was approximately 1/5th as concentrated as pre-shock lymph.
Lymph Sample Preparation and Protein Isolation
Lymph samples collected with EDTA were methanol-chloroform precipitated  and the resulting protein pellet was re-suspended in rehydration buffer at room-temperature overnight . For preparative gel analysis, equal protein weights of lymph from three animals were pooled prior to precipitation. A small aliquot of lymph at each time point was kept unprecipitated. Protein concentration was quantified using the Bradford assay as previously described .
Cy Dye Labeling and 2D Electrophoresis
A pooled internal standard approach was used, and two sets of analytical technical replicates were run, each representing an individual rat . An equal fraction from each animal of 500 μg total protein was combined, aliquoted, frozen with LN2, and kept at -80°C until used, providing an internal standard for all subsequent 2D gel experiments. Each analytical gel represents one animal differentially comparing the pre (initial collection) and post shock (3 hours from the start of resuscitation) states. The pooled internal standard was consistently Cy2 labeled; individual samples were alternatively labeled with Cy3 and Cy5 dyes between technical runs to control for any dye-specific labeling artifacts. Along with the second set of analytical gels, a preparative gel was run, consisting of 500 μg of a pre-shock protein pool and 500 μg of a post-shock protein pool made with equal protein amounts from lymph collected with EDTA from all three animals, along with the 50 μg Cy2 labeled internal standard.
All Cy labeling was done according to the manufacturer's protocol, where 200 pmol of dye was used to label 50 μg of protein (Cy dyes DIGE Fluors, GE Healthcare, Piscataway, NJ), under standard minimal dye labeling conditions .
Each set of analytical samples were passively rehydrated into Immobiline DryStrips 24 cm pH3-10 (GE Healthcare) overnight or for at least 18 hours, followed by isoelectric focusing using an IPGphor IEF unit (Amersham Biosciences/GE Healthcare). Focusing was performed at 20°C, at 50 μA per strip, according to the following step and hold sequence: 1) 500 V for 500 Vhr, 2) 1000 V for 1000 Vhr, 3) 8000 V for 24 000 Vhr, 4) 8000 V for 64 000 Vhr and 5) 8000 V for 64 000 Vhr.
For the preparative gel, labeling and rehydration was performed as it was with the analytical gels, with the exception that after the labeling step, 450 μg of each sample was added. The focusing parameters were the same, and included the following step and hold voltages: 1) 250 V for 1000 Vhr, 2) 500 V for 1000 Vhr, 3) 1000 V for 1000 Vhr, 4) 8000 V for 66 000 Vhr, 5) 8000 V for 66 000 Vhr and 6) 8000 V for 66 000 Vhr.
After focusing and prior to eletrophoresis, each strip was incubated at room temperature for 15 hours in reducing and alkylating solutions as previously described . Strips were then loaded onto second dimension 9-16% tris-glycine gels (Jules Gels, Milford, CT), sealed with agarose (SDS equilibrium buffer, 0.5% (w/v) agarose, and 0.25% (v/v) of saturated aqueous bromophenol blue) and run at 20 W per gel on the Ettan Dalt System (Amersham/GE Healthcare) for approximately 4 to 6 hours.
Imaging was done on a Typhoon 9400 Variable Mode Laser Imager (Amersham/GE Healthcare) . The gels that were used for protein identification were then fixed for 1 hour in 7.5% acetic acid/10% methanol, and stained overnight with Sypro Ruby protein gel stain (Invitrogen/Molecular Probes, Eugene, OR). Following destaining (7.5% acetic acid/30% methanol), gels were re-imaged at 100 μm resolution (laser excitation 532 nm, emission 560 nm, LP Gen. Purple).
Gel image analysis
Images were analyzed using Progenesis SameSpots v 3.1 (Nonlinear Dynamics, Durham, NC) software. One Cy2 image was selected as the reference image, and all gels were mapped to this reference image. Approximately 20 vectors were hand-placed on each additional gel image to facilitate the gel-to-gel matching; afterwards, automatic software matching was performed. Alignment was verified manually, matching artifacts deleted, and misalignments corrected. Following alignment, statistical analysis was performed, using normalized volume as a representation of protein abundance. Resulting ANOVA p and q values were used to assign statistical significance to detected changes in the pre and post states; both were limited to values < 0.05. The corresponding spots were then matched to a Sypro stained image of the preparative gel, which was first mapped to the reference image (Figure 1, Additional file 1: Table S1).
In addition, one set of analytical gels were analyzed independently, and a preliminary set of spots were selected to be picked on one individual replicate gel (animal R32) based on visual inspection and basing picks on viewed changes and spot abundance (Figure 3, Additional file 1: Table S1).
Spot Picking and Tryptic Digestion
Proteins of interest were excised from the two gels using an robotic spot picker (Ettan SpotPicker software v 1.10, GE Healthcare/Amersham Bioscience) fitted with a 1.0 mm deep, 1.4 mm in diameter picker head, and placed in 96 well plates, which were then transferred to an Ettan Digester (software v 1.10, GE Healthcare/Amersham Bioscience). Excised spots were washed twice with 100 μL of 50 mM ammnonium bicarbonate, once with 100 μL of 75% acetonitrile and once with 100 μL of 100% acetonitrile and left to dry at room temperature. Sequencing grade modified trypsin in 25 mM ammonium bicarbonate (1:4 v/v; Promega, Madison, WI) was added to each gel plug, plates were sealed and after a 30 minute incubation at 4°C were left at room temperature overnight for digestion. Following digestion, the peptides were extracted with 1.0% FA solution and then again with 50% ACN and 1.0% FA.
Matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry was carried out on an Applied Biosystems 4700 mass spectrometer, or an Applied Biosystems 4800 mass spectrometer. A saturated solution of alpha-cyano-4-hydroxycinnamic acid was prepared in acetonitrile/water (0.1% TFA). The equal parts of sample and α-cyano-4-hydroxycinnamic acid (7%) were manually spotted onto 100 well and/or 384 well stainless steel target plates (Applied Biosystems, Foster City, CA) and allowed to air dry prior to insertion into the mass spectrometer. Mass spectra were obtained for mass range from 800 to 4000 Daltons in reflector mode. All spectra were processed in Data Explorer v 5.0 (Applied Biosystems), and internally calibrated to a minimum of three monoisotopic trypsin autolysis peptides. Spectra were then used to interrogate sequences in the Swiss-Prot database using Mascot Daemon software v 2.2.2 (Matrix Science, Boston, MA) running the Mascot server (V 2.2). The search parameters were as follows: mass tolerance 100 ppm, Rattus taxon, enzyme specificity to trypsin and one missed cleavage. Trypsin specificity was used allowing for 1 missed cleavage. The modifications of Met oxidation, protein N-terminal acetylation, peptide N-terminal pyroglutamic acid formation were allowed for (used for all searches below).
Nano-liquid chromatography tandem mass spectrometry analysis was performed using an LTQ-XL Linear Ion Trap Mass Spectrometer or an LTQ-FT Ultra Hybrid ion cyclotron resonance mass spectrometer (ThermoFisher; San Jose, CA).
2 μL of tryptic digest sample was injected onto a reverse-phase column using a cooled (9°C) autosampler (Eksigent; Dublin, CA) connected to a HPLC system run at 120 μL/min before the T-split and ~400 nL/min post-split (Aligent; Santa Clara, CA). The column was made from an in-house pulled 100 μm i.d. × 150 mm fused silica capillary packed with Jupiter C18 resin (Phenomex; Torrance, CA) kept at a constant 40°C using an in-house built column heater . A gradient of 12% to 30% of ACN over a sixty minute run was employed for peptide separation. The column effluent was coupled directly to a LTQ-XL Linear Ion Trap mass spectrometer with an in-house built nanospray ion source. Data acquisition was performed using the instrument supplied Xcalibur (version 2.0.6) software. The sixty minute LC runs were monitored by sequentially recording the precursor scan (MS) followed by three collision-induced dissociation (CID) acquisitions (MS/MS). Normalized collision energies were employed using helium as the collision gas.
In addition, samples were analyzed on a LTQ-FT hybrid mass spectrometer. Peptide desalting and separation was achieved using a dual capillary/nano pump HPLC system (Agilent 1200, Palo Alto, CA). On this system 8 μL of sample was loaded onto a trapping column (ZORBAX 300SB-C18, dimensions 5 μm i.d. × 5 mm, Agilent Technologies, Santa Clara, CA) and washed with 5% ACN, 0.1% FA at a flow rate of 15 μL/min for 5 minutes. At this time the trapping column was put online with the nano-pump at a flow rate of 350 nL/min. An 85 minute gradient of 8 - 40% ACN was used to separate the tryptic peptides on an in house packed column. Data acquisition and analysis was performed as described above with the following modifications: for every MS scan four CID-induced MS/MS scans were acquired; MS mass tolerance was set to +/- 10 ppm for precursors; and +/- 0.6 Da for MS/MS fragment ions.
An in-house script was used to create de-isotoped centroided peak lists from the raw spectra (.mgf format). These peak lists were then interrogated against all rodent entries in the Swiss-Prot database using Mascot Daemon software v 2.2.2 (Matrix Science, London, UK) using an in-house Mascot server (v 2.2). Mass tolerances were +/- 1.2 Da for precursor ions, and +/- 0.6 Da for MS/MS fragment ions for spectra acquire from the LTQ-XL; +/- 10 ppm for MS peaks, and +/- 0.6 Da for MS/MS fragment ions for spectra acquired from the FT-ICR.
Western Blot Analysis
Proteins (approximately 20 μg per lane for lymph samples) were separated by 1D SDS-PAGE on 4-20% bisacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. The filters were stained with 1% Ponceau S in 5% acetic acid to confirm proper transfer. For destaining, the blot was washed with alkaline water. Blocking was performed for 1 hour at room temperature in 5% nonfat dried milk, in 100 mM PBS. Incubation with antibodies to MUP (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, Cat. # R-181), Apo E (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, Cat. # R-20) or β-actin (Cell Signaling Technologies Inc., Danvers, MA, Cat. #4967) were performed overnight at 4°C in 5% nonfat dried milk in 100 mM PBS containing 0.5% Tween 20. Bands were detected with goat anti-rabbit (Thermo Scientific, Rockford, IL, Cat. # 31460) or goat anti-mouse horseradish peroxidase (Thermo Scientific, Rockford, IL, Cat. #31430) using West Pico enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL), and visualized with the ChemiDoc XRS gel documentation system (Bio-Rad, Hercules, CA). Quantification of band intensities was performed with Quantity One analysis software (Bio-Rad, Hercules, CA).