View source for ME000013

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== Analytical Sample Preparation ==

Aliquot of dissolved proteins was diluted with sodium bicarbonate in concentration 0.5ug/ul. Protein solution was reduced with 5 mM DTT for 60 min at 60°C and alkylated with 15 mM iodoacetamide for 30 min in the dark. Residual iodacetamide was quenched by 5mM DTT. Trypsin Gold (Promega, Madison, WI) digestion (2.5 ng/μl) was carried out at 37°C in Barocycler NEP2320 (Pressure BioSciences, South Easton, MA) for 1 hour (pressure 35kPa for 50s and 0kPa for 10s). 

== Chromatography ==

Digested protein was separated using a 90 minute ACN gradient on a 250 mm x 75 μm C18 pepmap column at a flow rate of 0.3 μL/min. In brief, peptide and glycopeptide separation was achieved by a 5 min trapping/washing step using 99% solvent A (2% acetonitrile, 0.1% formic acid) at 10 μL/min followed by a 90 min acetonitrile gradient at a flow rate of 0.3 μL/min: 0-3 min 2% B (0.1% formic acid in ACN), 3-5 min 2-10% B; 5-60 min 10-45% B; 60-65 min 45-98% B; 65-70 min 98% B, 70-90 min equilibration by 2% B.

== Mass Spectrometry ==

Glycopeptides were analyzed using Orbitrap Lumos Fusion mass spectrometer. The electrospray ionization voltage was 3 kV, the capillary temperature 275°C. MS1 scans were performed over m/z 300–1800 with the wide quadrupole isolation on a resolution of 120,000 (m/z 200), RF Lens at 40%, intensity threshold for MS2 was set to 2.0e4, selected precursors for MS2 were with charge state 3-8, Dynamic exclusion 30s and selected mass range for MS2: 700 – 1800. Data-dependent EThcD tandem mass spectra were collected with a resolution of 30,000 in Orbitrap with fixed first mass 120 and normalized collision energy 25%. Data-dependent HCD scan was triggered by presence of 204.0867, 138.0545 or 366.1396 m/z in EThcD spectra. HCD tandem mass spectra were collected with a resolution of 30,000 in Orbitrap with fixed first mass 120 and normalized collision energy 20%.
{{Method|Orbitrap Fusion-Lumos, DDA (EThcD pd HCD), nano LC-MS/MS}}