Adrenal proteomics

<p dir="ltr"><b>Proteomic data comparing men1 mice +/- adrenals with men1+/+ adrenals.</b></p><p dir="ltr">The MEN1 mouse is a conventional heterozygous knockout mouse (<i>Men1</i>^{<em> </em>}^+/-), which was a kind gift from Professor Hayward of the Queensland Institute of Medical Research, Herston, Australia. Deletion of the second exon of the <i>Men1</i> gene produces heterozygous non-sense mutation.</p><p dir="ltr">Adrenals of ten MEN1 mice with ages from 10 to 13 months were used, and adrenals of ten wild type mice (<i>Men1</i>^<em>+/+</em>) of the same age range served as controls. From each animal, one gland was subjected to protein profiling and the contralateral gland was either formalin-fixed/paraffin-embedded for immunohistochemical analysis or lysate for western blot analysis.</p><p><br></p><p dir="ltr">Dissected adrenal glands (n=10/genotype) were minced and homogenized, protein was extracted, denatured, reduced and digested as previously described (14). Briefly, the samples were homogenized in the presence of octyl-ß-D-glucopyranoside, Tris-HCl, NaCl and EDTA in PBS solution. Protease cocktail inhibitor (Sigma-Aldrich, Steinheim, Germany) was added during lysis. After incubation and centrifugation of the lysate, the clear supernatant was collected for mass spectrometry and western blotting. The total protein content was determined using DC Protein Assay Kit (BioRad Laboratories, Hercules, Ca, USA) with bovine serum as standard.</p><p dir="ltr">For mass spectrometry analysis, 35 µg of protein was dissolved in 8M urea and acetonitrile (ACN) (1:1) containing NH₄HCO₃ (50 mM). A volume of 10 µL of 45 mM DTT was added to the samples for incubation at 50°C for 15 mins. After cooling, 10 µL of 100 mM IAA was added to the samples for incubation at room temp in a dark room. The samples were then transferred to 3kDa spin filters (Pall Life Sciences, Ann Arbor, MI, USA) for three steps of purification. A volume of 100 µL of 50 mM NH₄HCO₃ was added to the filter, followed by centrifugation at 14000 xg for 10 mins. Progressively, 250 µL of 1:1 2% ACN/50 mM NH₄HCO₃ and 150 µL of 50 mM NH₄HCO₃ were added separately, then filter centrifugation as above followed. Spin filters were transferred to new vials and 50 µL of 5% trypsin was applied for digestion at 37°C overnight in dark room. Samples were centrifuged and tryptic peptides collected. Residual peptides on the filters were collected by applying 100 µL of 50% ACN and 1% HAc, and the eluate was pooled with the first filtrate. Eluted peptides were dried using Speedvac system ISS110 (Thermo Scientific, Waltham, USA), and peptides re-dissolved in 30µL 0,1% formic acid (FA) before mass spectrometry analysis.</p>