High pH reversed-phase liquid chromatography separation
Samples were fractionated to increase the proteomic depth, using high pH
reverse phase separation. The peptides resuspended in buffer A (2%
acetonitrile, containing ammonium hydroxide solution, at pH 10) were
separated by ACQUITY UPLC (Waters, Milford, MA, USA) connected to a high
pH UPLC column. Gradient elution was achieved at a rate of 200 μl
min-1 for 66 min with buffer B (80% acetonitrile,
containing ammonium hydroxide solution, at pH 10). Twenty fractions were
collected from each sample and were pooled into 10 total fractions.
Liquid chromatography-mass
spectrometry (LC-MS/MS) analysis
Mass spectrometry analysis was
performed on a Q Exactive mass spectrometer coupled
with Easy-nLC 1200 (ThermoFisher
Scientific). The 2 μg peptides loaded onto the C18 column in buffer A
(2% acetonitrile, 0.1% formic acid) were separated with a linear
gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow
rate of 300 nl min-1 in Easy-nLC 1200. The eluted
peptides were injected into a Q Exactive mass spectrometer (ThermoFisher
Scientific), using 1.8 kV
electrospray voltage. The
acquisition of survey full-scan MS spectra (m/z 350-1300) was attributed
to a mass resolution of 70,000, followed by 20 sequential high energy
collisional dissociation MS/MS scans at a resolution of 35,000.
For protein identification, MS/MS
spectra were searched using Protein
DiscovererTMSoftware 2.2 and parent proteins were identified by the highest score
for a given peptide mass. The
following settings were used: a
maximum of two miscleavages;
carbamidomethylation of cysteines as fixed modification;
protein N-terminal acetylation and
oxidation of methionines as variable modifications. At least one unique
peptide containing a minimum of six amino acids sequence was required
for per protein. The false discovery
rate (FDR) was set to 1% to
validate peptide spectra.