Journal: Bio-protocol

Article Title: Measurement of redox states of the β3 integrin disulfide bonds by Mass Spectrometry

doi: 10.21769/BioProtoc.3156

Figure Lengend Snippet: Monoisotopic m/z ratios for the β3 integrin YCNCTTR peptide alkylated with 12 C-IPA and 13 C-IPA

Article Snippet: Ziptip ® C18 pipette tip (Millipore, catalog number: ZTC18S96) 1.5 ml tubes Natural human αIIbβ3 protein purified from human platelets (Abcam, catalog number: ab95130) Anti-integrin β3 (GPIIIa, CD61) PSI domain [AP-3] antibody (Kerafast, catalog number: EBW106) 2-iodo-N-phenylacetamide ( 12 C-IPA) (Cambridge isotopes, catalog number: ULM-8131-PK) 13 C-2-iodo-N-phenylacetamide ( 13 C-IPA) (Cambridge isotopes, catalog number: CLM-8130-0.1) Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418) NuPAGE TM 4x LDS sample loading buffer (Thermo Fisher Scientific, catalog number: NP0007) NuPAGE TM 4-12% Bis-Tris gel 1.5 mm, 10-well (Thermo Fisher Scientific, catalog number: NP0335BOX) NuPAGE TM MOPS running buffer 20x (Thermo Fisher Scientific, catalog number: NP0001) Brilliant Blue G solution (Sigma, catalog number: B8522-1EA) Ammonium bicarbonate (Sigma, catalog number: A6141) Dithiothreitol (DTT) (Sigma, catalog number: D0632) Formic acid LC-MS grade (Thermo Fisher Scientific, catalog number: 28905) Trifluoroacetic acid (Sigma, catalog number: 302031) Formic acid (Sigma, catalog number: F0507) Acetonitrile HPLC grade (Fisher Chemical, catalog number: 10407440) PNGase F Proteomics grade enzyme (Sigma, catalog number: P7367) Sequencing grade bovine chymotrypsin (Roche, catalog number: 11418467001) Trypsin Gold, Mass spectrometry grade (Promega, catalog number: V5280) HCl Acetic acid Calcium chloride Methanol Coomassie blue NaCl KCl Na 2 HPO 4 KH 2 PO 4 PBS (see Recipes) Gel destaining solution (see Recipes) 50 mM 12 C-IPA/DMSO solution (see Recipes) 50 mM 13 C-IPA/DMSO solution (see Recipes) 5 mM 13 C-IPA/25 mM ammonium bicarbonate (see Recipes) 25 mM ammonium bicarbonate buffer (see Recipes) 25 mM ammonium bicarbondate/50% acetonitrile buffer (see Recipes) 25 mM ammonium bicarbonate/10 mM calcium chloride buffer (see Recipes) 1 M calcium chloride solution (see Recipes) 40 mM DTT/25 mM ammonium bicarbonate (see Recipes) 1 mM HCl solution (see Recipes) 50 mM acetic acid (see Recipes) 5% formic acid (see Recipes) 5% formic acid/50% acetonitrile buffer (see Recipes) 1% trifluoroacetic acid solution (see Recipes) Wetting solution (see Recipes) Equilibration solution (see Recipes) 0.1% formic acid (see Recipes) HPLC Buffer A (see Recipes) HPLC Buffer B (see Recipes)

Techniques: Sequencing