Orbitrap Fusion-Lumos, DDA (EThcD pd HCD), nano LC-MS/MS

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Analytical Sample Preparation

Aliquots of dissolved proteins (plasma, CSF, cell lysate) were diluted with sodium bicarbonate in concentration 1ug/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. 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.


Digested proteins were 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 400–1800 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. 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%.


SampleSpectrum FileLC Cal. SlopeLC Cal. Int.Trans. Groups
HEK293 CellsMS_47_UO1_HEK_1_1n2.506-32.7084
HEK293 CellsMS_47_UO1_HEK_1_2n2.375-30.1519
HEK293 CellsMS_47_UO1_HEK_2_1n2.399-30.31417
HEK293 CellsMS_47_UO1_HEK_2_2n2.346-29.6491
HEK293 CellsMS_51_CM22.643-34.1154
HEK293 CellsMS_51_CM32.629-35.03911
HEK293 CellsMS_51_CM_202102101730562.313-22.8538
HEK293 CellsMS_51_HEK02_202102141730112.586-38.784
HEK293 CellsMS_51_HEK_01_EThcDpdHCD_202102040236082.488-34.97216
HEK293 CellsMS_51_HEK_CM022.573-35.0359
HEK293 CellsMS_51_HEK_acetone2.398-39.73930
HEK293 CellsMS_51_HEK_acetone22.656-49.04127
HEK293 CellsMS_51_HEK_acetone_z2incl2.594-43.91241
HEK293 CellsMS_52_HEK_secretome012.648-24.18415
HEK293 CellsMS_52_HEK_secretome022.778-30.0136
HEK293 CellsMS_52_HEK_secretome032.772-29.91414
Human SerumMS_31_UO1_MARS_MS2_1ug_longn_CE20_std2.691-30.281134
Human SerumMS_31_UO1_MS2_new_measurement_012.576-23.25911
Human SerumMS_31_UO1_MS2_new_measurement_022.602-24.56520
Human SerumMS_35_Frac_plasma_run01_fr10_012.553-31.911109
Human SerumMS_35_Frac_plasma_run01_fr11_012.53-30.4461
Human SerumMS_35_Frac_plasma_run01_fr12_012.534-30.75829
Human SerumMS_35_Frac_plasma_run01_fr13_012.782-61.0878
Human SerumMS_35_Frac_plasma_run01_fr14_012.489-30.3644
Human SerumMS_35_Frac_plasma_run01_fr1_012.483-29.9781
Human SerumMS_35_Frac_plasma_run01_fr2_012.47-29.9691
Human SerumMS_35_Frac_plasma_run01_fr3_012.478-30.0082
Human SerumMS_35_Frac_plasma_run01_fr4_012.468-29.93
Human SerumMS_35_Frac_plasma_run01_fr5_012.489-29.87627
Human SerumMS_35_Frac_plasma_run01_fr6_012.516-30.73583
Human SerumMS_35_Frac_plasma_run01_fr7_012.472-30.30673
Human SerumMS_35_Frac_plasma_run01_fr8_012.608-36.1291
Human SerumMS_35_Frac_plasma_run01_fr9_012.535-30.528118
Human SerumMS_35_Frac_plasma_run02_fr12.473-29.8961
Human SerumMS_35_Frac_plasma_run02_fr102.533-30.934106
Human SerumMS_35_Frac_plasma_run02_fr112.539-31.212124
Human SerumMS_35_Frac_plasma_run02_fr122.527-31.22108
Human SerumMS_35_Frac_plasma_run02_fr132.515-30.45883
Human SerumMS_35_Frac_plasma_run02_fr142.543-32.36925
Human SerumMS_35_Frac_plasma_run02_fr152.491-29.85913
Human SerumMS_35_Frac_plasma_run02_fr162.499-29.917
Human SerumMS_35_Frac_plasma_run02_fr172.598-34.135244
Human SerumMS_35_Frac_plasma_run02_fr22.485-30.4172
Human SerumMS_35_Frac_plasma_run02_fr42.52-31.1671
Human SerumMS_35_Frac_plasma_run02_fr52.578-32.14739
Human SerumMS_35_Frac_plasma_run02_fr62.523-30.32942
Human SerumMS_35_Frac_plasma_run02_fr72.473-29.51990
Human SerumMS_35_Frac_plasma_run02_fr82.51-30.60384
Human SerumMS_35_Frac_plasma_run02_fr92.554-32.16896
Human SerumMS_48_UO1_Erlic_E102.587-39.73136
... further results