Impact hd 2 mass spectrometer

Amino acids released from AN by acid hydrolysis was analyzed by mass spectrometry essentially as previously described by Gamon et al. [29 ]. Briefly, protein samples (25 μg) were precipitated with trichloroacetic acid (w/v 8 %) and spiked with stable isotope-labelled amino acid standards before drying down using a centrifugal vacuum concentrator for 30 min at 30 °C. The resulting pellet was hydrolyzed overnight in 4 M methanesulfonic acid with 0.2 % w/v tryptamine (50 μL) under vacuum at 110 °C. Amino acids were partially purified by solid-phase extraction using 30 mg/1 mL mixed-mode strong cation exchange Strata X–C cartridges (Phenomenex). Eluted fractions were dried at 30 °C under vacuum overnight, and then dissolved in 50 μL of 0.1 % formic acid. Analytes were quantified by ESI LC-MS in positive ion mode using a Bruker Impact HD II mass spectrometer. Samples were separated by gradient elution using an Imtakt Intrada Amino Acid 100 × 3.0 mm column with acetonitrile/formic acid (Solvent A; 100/0.3) and acetonitrile/100 mM ammonium formate (Solvent B; 20/80). MS analyses and quantification was performed at the MS¹ level.

Samples of rhV3 (2 μg, 1.42 μM) were treated with increasing molar ratios of ONOO⁻/ONOOH or SIN-1 to protein followed by precipitation with trichloroacetic acid (8% w/v). Samples were spiked with stable isotope-labelled amino acid standards and dried down using a centrifugal vacuum concentrator for 30 min at 30 °C. The pellet was hydrolysed overnight in 4 M methanesulfonic acid with 0.2% (w/v) tryptamine (50 μL) under vacuum at 110 °C. The hydrolysed amino acids were partially purified by solid-phase extraction using 30 mg/1 mL mixed-mode strong cation exchange Strata X–C cartridges (Phenomenex), and the eluted fractions were dried overnight at 30 °C under vacuum. The amino acids were reconstituted in 25 μL of 0.1% (v/v) formic acid and quantified by electrospray ionization LC-MS in the positive ion mode using a Bruker Impact HD II mass spectrometer. Samples were separated by gradient elution using an Imtakt Intrada Amino Acid 100 × 3.0 mm column with acetonitrile/formic acid (Solvent A; 100/0.3) and acetonitrile/100 mM ammonium formate (Solvent B; 20/80). MS analyses and quantification was performed at the MS¹ level [42 ].