Analysis of fatty acid content by negative ion ES-MS survey scans

Lipid extractions from purified recombinant proteins were achieved by two different methods. Method A: 3 successive vigorous extractions with 10 volumes of diethyl ether after treatment with 2 volumes of 6M HCl overnight. The ether extracts were evaporated under nitrogen and analysed by electrospray mass spectrometric and tatty acid methyl ester analysis as described below.

Method B: 3 successive vigorous extractions with ethanol to fully denature proteins (final 90% v/v) [54]. The pooled extracts were dried by nitrogen gas in a glass vial and analysed by electrospray mass spectrometry.

For electrospray mass spectrometry analysis, extracts were analyzed on a Absceix 4000 QTrap, a triple quadrupole mass spectrometer equipped with a nanoelectrospray source as described previously [55].

Quantification of the fatty acids from method A were done by conversion to the corresponding fatty acid methyl esters (FAME) followed by GC-MS analysis as described previously [56] using the following GC temperature program: 70°C for 12 min followed by a gradient to 220°C at 4°C/min and held at 220°C for a further 10 min. Mass spectra were acquired from 50–500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern with mixtures of FAME standards.