GC-MS Measurements of ACC and KBA

The standards for 1-aminocyclopropane-carboxylic acid (ACC; EMD 149101-1G) and 2-ketobutyric acid (KBA; K401, Sigma-Aldrich, Vienna, Austria) were purchased from Sigma-Aldrich. The solvents methanol LC-MS grade (Honeywell 34966) and pyridine p.A. (Merck 1.09728.0500) were obtained from Merck. The derivatisation chemicals methoxyamine hydrochloride (MOX; 226904, Sigma-Aldrich, Vienna, Austria) and N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA; 701270.201, Macherey-Nagel) were purchased from Sigma Aldrich and Macherey-Nagel, respectively. The stock solutions of ACC and KBA were prepared in 50% aqueous methanol.

The unpurified protein extracts were centrifuged for 10 min at 14,000 rpm at 4°C, 10 μL of the supernatant were transferred into micro-inserts in GC/HPLC vials and dried overnight using a centrifugal evaporator (Labconco, Kansas City, MO) at 15°C. Subsequently an automated two step derivatisation was carried out using the GC auto sampler (PAL LHX-xt, CTC Analytics, Carrboro, NC). To this end the residue was resuspended in 50 µL MOX (20 mg/ml pyridine) and agitated for 90 min at 90°C. After addition of 50 µL MSTFA the mixture was again agitated at 90°C for 60 min.

For GC-MS analysis, 1 µL of the derivate was injected into the split-/splitless injector of an Agilent 7890A gas chromatograph coupled to a 5975C inert XL MSD detector (Agilent, Waldbronn, Germany), equipped with ChemStation software (version E.02.01.1177) and operated in pulsed splitless mode at 250°C. Chromatographic separation was carried out on an HP5-ms column (30 m x 0.25 mm x 0.25 µm; Agilent Technologies, Waldbronn, Germany) at a constant flow of 1 ml/min helium. Temperature program: 50°C, 2 min hold, to 120°C (10°C/min, 5 min hold), to 150°C (5°C/min), to 325°C (70°C/min, 10 min hold). The MSD was operated with interface set to 335°C, the EI source to 230°C and the MSD quadrupole to 150°C, solvent delay 6 min, scan range of m/z 50–300, dwell time 100 ms. SIM mode was used for quantification of the ACC 1TMS derivate m/z 83 (quant ion), 130, 173; ACC 2TMS derivate m/z 147 (quant ion), 202, 230 and KBA 1TMS derivate m/z 89, 172, 188 (quant ion).