TITLE: The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis
AUTHORS: Quentin M. Nunes, Dunhao Su, Phuong Thao Bui, Deborah M. Simpson, Philip J. Brownridge, Changye Sun, Yong Li, Zhang X, Wei Huang, Daniel J. Rigden, Robert J. Beynon, Robert Sutton, David G. Fernig

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, driven by changes in communication between cells. Heparin-binding proteins (HBPs) play a central role in cell communication. Therefore, we used heparin affinity proteomics to identify extracellular HBPs in pancreas and plasma of normal mice and in a caerulein mouse model of AP. Many new extracellular HBPs (360) were discovered in the pancreas, taking the total number of HBPs known to 786. Extracellular pancreas HBPs form highly interconnected protein-protein interaction networks in both normal pancreas (NP) and AP. Thus, HBPs represent an important set of extracellular proteins with significant regulatory potential in the pancreas. HBPs in NP are associated with biological functions such as molecular transport and cellular movement that underlie pancreatic homeostasis. However, in AP HBPs are associated with additional processes such as acute phase response signalling, complement activation and mitochondrial dysfunction. Plasma HBPs in AP included known AP biomarkers such as serum amyloid A, as well as emerging targets such as histone H2A. Pancreas HBPs are extracellular and so easily accessible and are potential drug targets in AP, whereas plasma HBPs represent potential biomarkers for AP. These need further investigation for potential applications in the management of AP.</div><style>
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[STEPS]
?. [Heparin affinity chromatography]
The frozen murine plasma was defrosted on ice and centrifuged at 16,100 g for 10 min, at 4 ˚C.The resulting supernatant was diluted (1:8) in 75 mM NaCl, 6.85 mM Na2HPO4, 3.15 mM NaH2PO4, pH 7.2.The diluted supernatant was centrifuged at 5,000 g for 5 min.and the resulting supernatant was then applied on a 1 mL Hi-Trap heparin column equilibrated in 75 mM NaCl, 6.85 mM Na2HPO4, 3.15 mM NaH2PO4, pH 7.2.The heparin column was subsequently washed extensively with 50 mL of modified PBS (150 mM NaCl, 13.7 mM Na2HPO4, 6.3 mM NaH2PO4, pH7.2). The heparin-bound fraction was then eluted with 2 M NaCl in modified PBS.
[16,100 g]
This method was effective in reducing the high abundance protein, albumin, down to levels that would not affect the MS analysis of plasma HBPs.
[5000 g]
The receipt for PBS has been modified here. Please check carefullly.
[NaCl]
?. [Protein adsorption using StrataClean for mass spectrometry]
In order to eliminate the high concentration of electrolytes (2.15 M NaCl) in the eluate, so as not to influence MS analysis, StrataCleanTM beads (Agilent Technologies, UK) were used to adsorb proteins.Briefly, 100 µg of the heparin-bound fraction was mixed with 30 µL StrataClean slurry, vortexed for 2 min. and centrifuged at 2,000 g for 2 min at 4 ˚C.The supernatant was removed carefully and discarded.The pellet was first washed with PBS and then further washed twice with H2O.
?. [Sample preparation for mass spectrometry ]
In the case of plasma samples, the StrataCleanTM beads were resuspended in 80 μL of 25 mM ammonium bicarbonate and 5 μL of 1 % (w/v) Rapigest (Waters, Manchester, UK) added and the samples shaken at 450 rpm for 10 min at 80 °C.Samples were reduced by the addition of 5 μL of 60 mM  (DTT) and incubated at 60°C for 10 min and alkylated (addition of 5 μL of 180 mM iodoacetamide and incubation at room temperature for 30 min in the dark). Trypsin (Sigma, Poole, UK, proteomics grade) was reconstituted in 50 mM acetic acid to a concentration of 0.2 μg/μL and 5 μL (1 μg)  added to the sample followed by overnight incubation at 37 °C. The following day the digestion was terminated and Rapigest removed by acidification with TFA (1 μL) and incubation at 37 °C for 45 min. Samples were centrifuged at 17,200 g for 30 min and the clarified supernatants transferred to tubes.
[for the denature by RapiGest]
60 Molarity (m)
[enzyme]
[overnight]
Samples were mixed on a rotating mixer
[17,200 g centrifuge]