A normative study of the synovial fluid proteome from healthy porcine knee joints

  • Tue Bjerg Bennike (Creator)
  • Ugur Ayturk (Contributor)
  • Carla M Haslauer (Contributor)
  • John William Froehlich (Contributor)
  • Benedikt L Proffen (Contributor)
  • Omar Barnaby (Contributor)
  • Svend Birkelund (Contributor)
  • Martha M Murray (Contributor)
  • Matthew L Warman (Contributor)
  • Allan Stensballe (Contributor)
  • Hanno Steen (Contributor)



Synovial fluid in an articulating joint contains proteins derived from blood transudates, and proteins that are produced by cells within joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid is not fully understood. Also not fully delineated are the cellular origins of synovial fluid components. We here present a normative proteomics study for synovial fluid optimized in pig.

Sample Processing Protocol
Six adolescent Yucatan minipigs (Coyote CCI, Douglas, MA, USA), aged 12-15 months, were obtained for use in this study. All minipigs were housed and monitored by the Animal Resources at Children’s Hospital (ARCH, Boston, MA, USA) and handled according to approved Institutional Animal Care and Use Committee (IACUC) protocols. Minipigs were acclimated to the ARCH environment for a minimum of 3 days prior to experimental handling. At time of euthanasia, synovium tissue from both knee joints of each minipig was harvested. Each tissue specimen was submerged in a cryovial, then flash frozen in liquid nitrogen and stored at -80°C until gene expression analysis. Synovial fluid was also obtained from the joints, and centrifuged at 3,000 g for 10 min to remove any cells. In some cases, 3 mL sterile saline was injected to facilitate fluid extraction, after which the knee was bent 10 times to mix and the saline/synovial fluid mix was extracted and then centrifuged. The supernatant was removed, and approximately 240-500 μL sample was stored in 120 μL aliquots in cryovials at -80°C. Three protocols, employing different strategies were evaluated for the tryptic digestion of synovial fluid samples: urea filter assisted sample preparation (FASP), urea in-solution digestion, and in-gel digestion. 1) Urea FASP Digestion: Performed using the FASP Protein Digestion Kit (Protein Discovery, San Diego, CA, USA) according to manufacturer’s instructions using 30 kDa cutoff spin filters. 90 µg total synovial fluid protein was digested using two µg sequencing grade modified trypsin (Promega, Fitchburg, USA), and the samples were digested overnight at 37°C. To assess the need of glycan removal when working with synovial fluid, 500 U peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase (PNGase F) (New England BioLabs Inc, Ipswich, USA) was added to these samples prior to the trypsin digestion step, and incubated overnight at 37°C, after which the normal FASP protocol was continued. After trypsin digestion, the samples were desalted with TAGRA C18 columns (Nest Group Inc, Southborough, MA, USA) and resuspended in 5% ACN 5% formic acid (FA) prior to analysis. 2) Urea in-solution Digestion: Performed according to Gallien et al. 90 µg total synovial fluid protein was diluted with 8 M urea in 100 mM ammonium bicarbonate to a final volume of 25 µL. The sample was reduced with DTT at a final concentration of 12 mM for 30 min at 37°C, and alkylated with iodoacetamide at a final concentration of 40 mM for 1 h at 25°C in the dark. The samples were diluted with 100 mM ammonium bicarbonate to a total volume of 100 µL, 2 µg trypsin was added and the sample was digested overnight at 37°C. The samples were desalted with Nest group TAGRA C18 columns and resuspended in 5% ACN 5% FA prior to analysis. 3) In-gel Digestion: Three gel-lanes, each loaded with 150 µg total synovial fluid protein, were divided into 10 sections each and subjected to in-gel tryptic digestion, followed by analysis. Two state of the art mass spectrometers were utilized: 1) A 5600 TripleTOF (AB Sciex, Framingham, USA) connected online by a nanoflow HPLC NanoFlex system (Exigent, Redwood, USA) by a nanospray ion source. The samples were loaded onto a 15 cm reversed phase C18 200 µm chip with 2 µL/min in 100% solvent A (0.1% FA). The samples were then separated using a 15 cm reversed phase C18 75 µm chip, and eluted with a linear gradient of 2% solvent B (0.1% FA in ACN) which was raised to 35% solvent B over 120 min (60 min for in-gel digested samples) at a constant flow rate of 500 nL/min. 2) The urea FASP digested synovial fluid samples were also analyzed on a Q Exactive (Thermo Scientific, Waltham, USA) connected online to an EASY-nLC 1000 (Thermo Scientific, Waltham, USA) by a nanospray ion source. The samples were loaded onto a 10.5 cm reversed phase C18 PicoChip with a flow rate of approximately 1 µL/min in 98% solvent A and 2% solvent B, and eluted with eluent B using a linear gradient which was raised to 35% over 120 min at a constant flow rate of 300 nL/min.

Data Processing Protocol
The AB Sciex .wiff data-files were analyzed using ProteinPilot 4.5 (Rev. 1656, Paragon Algorithm To identify the most commonly single observed PTMs, data-files were searched in thorough-mode with a focus on biological modifications in ProteinPilot to include more than 300 different PTMs [ProteinPilot Online Help]. The Thermo Scientific .raw data-files were analyzed using MaxQuant All standard settings were employed with carbamidomethyl(C) as a static modification and deamidation (NQ), and protein N-terminal acetylation was included as variable modifications. Label-free quantitation of all proteins was performed in MaxQuant based on integrated precursor intensities. Protein abundances are represented as protein intensity-based absolute quantitation values (iBAQ), and are reported for all proteins having at least two quantifiable unique peptides in at least three LC-MS runs.

Tue Bjerg Bennike, Aalborg University
Hanno Steen, Department of Pathology and Proteomics Center, Boston Children's Hospital, Harvard Medical School, MA, USA (lab head)

Submission Date

Publication Date

Project PXD000935
Date made available9 Sept 2014
PublisherPRoteomics IDEntifications Database (PRIDE)

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