Glu fibrinopeptide

On-tissue digestion of samples was performed by spraying 4 layers of trypsin (V5111, Promega, Madison, WI, USA) 0.1 µg/mL in 25 mM NH₄HCO₃, 10% TFE, at a flow of 10 mL/min with a SunCollect (Sunchrom, Germany) sprayer. Once the on-tissue digestion was finished, slides were dried at vacuum for 30 min before matrix deposition. MALDI matrices were deposited onto tissue using a solution based on α-cyano-4-hydroxycinnamic acid and CHCA (70990, Sigma Aldrich, Steiheim, Germany) (7 mg/mL 60% ACN 0.2% TFA). The internal calibrant Glu-Fibrinopeptide (Sigma Aldrich, Steiheim, Germany) was added to the matrix solution at a final concentration of 625 fmol/mL. The final matrix solution was sprayed in 8 layers: first layer at 10 mL/min, second layer at 20 mL/min, third layer at 30 mL/min, and the fourth to eighth layers at 45 mL/min; all layers were at a z-axis equal to 27.05. Once completed, slides were vacuum-dried for 30 min before analysis was performed on the mass spectrometer.

dithiothreitol (DTT), pronase, ethylenediamine tetra-acetic acid (EDTA), collagen IV, urea, thio-urea, orthovanadate, glycerol, TRIS, iodoacetamide, NaCl, detergents (CHAPS, sodium deoxychotate, Triton X100), NH4CO3 and the Glu-fibrinopeptide were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Anti-proteases were from Roche (Neuilly sur Seine, France). Bromophenol blue, SDS, acrylamide, silver staining kit were from Biorad (Marnes la Coquette, France). Trypsin for mass spectrometry was of highest grade purchased from Promega (Charbonnieres-les-bains, France). Acetonitril (ACN) and Tri-fluoro-acetic acid (TFA) were from Carlo Erba (Val de Reuil, France), Alpha-Cyano-4-Hydroxycinnamic Acid (HCCA) from Laser Biolabs (Sophia-Antipolis, France). iTRAQ reagents were from Applied Biosystems (Courtaboeuf, France)