Mass spectrometry data acquisition
Digested samples were analyzed on an Orbitrap Exploris 480 mass spectrometry system (Thermo Fisher Scientific) equipped with an Easy nLC 1200 ultra-high pressure liquid chromatography system (Thermo Fisher Scientific) interfaced via a Nanospray Flex nanoelectrospray source. For all analyses, samples were injected on a C18 reverse phase column (25 cm x 75 μm packed with ReprosilPur 1.9 μm particles). Mobile phase A consisted of 0.1% FA, and mobile phase B consisted of 0.1% FA/80% ACN. Peptides were separated by an organic gradient from 5% to 30% mobile phase B over 112 minutes followed by an increase to 58% B over 12 minutes, then held at 90% B for 16 minutes at a flow rate of 350 nL/minute. Analytical columns were equilibrated with 6 μL of mobile phase A. To build a spectral library, one sample from each set of biological replicates was acquired in a data dependent manner. Data dependent analysis (DDA) was performed by acquiring a full scan over a m/z range of 400-1000 in the Orbitrap at 60,000 resolving power (@200 m/z) with a normalized AGC target of 300%, an RF lens setting of 40%, and a maximum ion injection time of 60 ms. Dynamic exclusion was set to 60 seconds, with a 10 ppm exclusion width setting. Peptides with charge states 2-6 were selected for MS/MS interrogation using higher energy collisional dissociation (HCD), with 20 MS/MS scans per cycle. For phosphopeptide enriched samples, MS/MS scans were analyzed in the Orbitrap using isolation width of 1.3 m/z, normalized HCD collision energy of 30%, normalized AGC of 200% at a resolving power of 30,000 with a 54 ms maximum ion injection time. Similar settings were used for data dependent analysis of samples used to determine protein abundance, with an MS/MS resolving power of 15,000 and a 22 ms maximum ion injection time. Data-independent analysis (DIA) was performed on all samples. An MS scan at 60,000 resolving power over a scan range of 390-1010 m/z, a normalized AGC target of 300%, an RF lens setting of 40%, and a maximum injection time of 60 ms was acquired, followed by DIA scans using 8 m/z isolation windows over 400-1000 m/z at a normalized HCD collision energy of 27%. Loop control was set to All. For phosphopeptide enriched samples, data were collected using a resolving power of 30,000 and a maximum ion injection time of 54 ms. Protein abundance samples were collected using a resolving power of 15,000 and a maximum ion injection time of 22 ms.