The membrane O-glycoproteome in Streptomyces coelicolor J1929 was isolated and characterised by LC-ESI-MS/MS, using CID, HCD and ETD fragmentation. Streptomyces coelicolor J1929 was grown in phosphate limiting (F134) medium, and the membrane proteins were isolated after 20, 35, 43 and 60 h of growth. Glycoproteins were enriched using lectin affinity chromatography (Con A) and the enriched fractions were analysed by LC-ESI-CID-MS/MS on a Bruker maXis mass spectromter. The enriched glycoproteins isolated after 43 h of growth were further analysed by LC-MS/MS on an Orbitrap Fusion Tribrid mass spectrometer. The S. coelicolor glycopeptides in this experiment were analysed using both HCD and ETD fragmentation, and using both the ion trap (low resolution, high speed - mass accuracy < 0.5 Da) and Orbitrap (high resolution, lower speed - mass accuracy < 3 ppm) mass analysers. Four different data acquisitions were performed to include these options: MS in Orbitrap, HCD measured in ion trap (HCD_IT), MS in Orbitrap, ETD measured ion trap (ETD_IT), MS in Orbitrap, ETD measured in Orbitrap (ETD_OT), MS in Orbitrap, HCD measured in ion trap and ETD measured in Orbitrap (HCD_IT; ETD_OT).