Samples were defrosted, acidified with 0.2% formic acid, and loaded onto reversed-phase solid phase extraction (SPE) columns (Bond Elute C18 LRC 10 ml, 200 mg, 40 μm; Agilent) at ~1 ml min−1. SPE columns were preactivated with one column volume of methanol, followed by one column volume of water with 0.2% formic acid. After desalting columns with 5 ml Milli-Q water with 0.2% formic acid, columns were eluted with 750 μl of 50% methanol (aq). The eluent was left in the reservoir for 1 hour to lyse and extract cells. An additional 750 μl of 50% methanol (aq) was added after a 5-min soak step to maximize yield. Samples were analyzed by isocratic elution with 25% acetonitrile (aq) supplemented with 0.1% formic acid at 0.2 ml min−1 on an Agilent 1200 LC system fitted with RP-MS column, 150 mm × 2.1 mm, 2.6 μm (Thermo Scientific) coupled to an Agilent 6410 triple quadrupole detector. The ion source was operated at 300°C and 4500 V in negative mode with a nitrogen flow of 5.8 liters min−1 at 20 psi.

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