Mass spectrometry data from Caulobacter crescentus. Strains include: wild type, ccna_01217 deletion, ccna_01218 deletion, ccna_01219 deletion, and the respective complementation strains. Lipids were extracted by the method of Bligh and Dyer and analyzed by normal phase LC/MS. MS/MS analysis was used to determine the structure of the ceramide phosphoglycerate lipid.
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Bacterial strains were grown overnight in peptone-yeast extract media. 1 ml of culture was extracted by the method of Bligh and Dyer. Lipids were analyzed by LC/MS using the following protocol. Normal phase LC was performed on an Agilent 1200 Quaternary LC system equipped with an Ascentis Silica HPLC column, 5 μm, 25 cm × 2.1 mm (Sigma-Aldrich, St. Louis, MO). Mobile phase A consisted of chloroform/methanol/aqueous ammonium hydroxide (800:195:5, v/v); mobile phase B consisted of chloroform/methanol/water/aqueous ammonium hydroxide (600:340:50:5, v/v); mobile phase C consisted of chloroform/methanol/water/aqueous ammonium hydroxide (450:450:95:5, v/v). The elution program consisted of the following: 100% mobile phase A was held isocratically for 2 min and then linearly increased to 100% mobile phase B over 14 min and held at 100% B for 11 min. The LC gradient was then changed to 100% mobile phase C over 3 min and held at 100% C for 3 min, then returned to 100% A over 0.5 min and held at 100% A for 5 min. The LC eluent (with a total flow rate of 300 μl/min) was introduced into the ESI source of a high resolution TripleTOF5600 mass spectrometer (Applied Biosystem, Foster City, CA). Instrumental settings for negative ion ESI and MS/MS analysis of lipid species were as follows: IS= −4500 V; CUR= 20 psi; GSI= 20 psi; DP= −55 V; and FP= −150 V. The MS/MS analysis used nitrogen as the collision gas. Data analysis was performed using Analyst TF1.5 software (Applied Biosystems, Foster City, CA).