Analysis was performed by capillary LC-nESI-MS and HCD-MS/MS using an
Agilent 1200 Infinity Binary LC System (Santa Clara, CA) coupled to a Thermo
Scientific Q Exactive Plus Orbitrap mass spectrometer (Breman, Germany). MC
peptides were prepared at various concentrations in 2 % MeCN / 0.1 % FA, then 8
µL loaded onto a Magic AQ C18, 3 µm, 200 Å, 25 ×
0.15 mm IntegraFrit Sample Trap Column (Bruker, Billerica, MA) for 5 min at a
flow rate at 2 μL/min. Elution and separation was performed using a Magic
AQ C18, 3 μm, 200 Å, 105 × 0.15 mm PicoChip Column (Bruker,
Billerica, MA) using a two-step linear acetonitrile gradient from 2–25 %
B in 5 min and 25–60 % B in 35 min (Buffer A: 0.1 % FA; Buffer B: 95 %
MeCN / 0.1 % FA) at a flow rate of 0.8 µL/min. Full MS scans were
acquired at m/z 300–2000 with a spray voltage maintained at 2.7 kV, an
S-lens setting of 60 % and the ion transfer tube set at 300 °C. Data was
recorded at a mass resolving power of 70,000. The maximum injection time was set
at 100 ms with an AGC target value of 1.0 × 106. HCD-MS/MS
scans were obtained at a resolution of 17,500 with an AGC target value of 1.0
× 106 and an isolation window of 0.8. Evaluation of Normalized
Collision Energies (NCS’s) for the optimized identification of both
‘global’ and ‘variant-specific’ MC product ions was
performed by energy resolved HCD-MS/MS of each peptide, using repeated
injections.
Agilent 1200 Infinity Binary LC System (Santa Clara, CA) coupled to a Thermo
Scientific Q Exactive Plus Orbitrap mass spectrometer (Breman, Germany). MC
peptides were prepared at various concentrations in 2 % MeCN / 0.1 % FA, then 8
µL loaded onto a Magic AQ C18, 3 µm, 200 Å, 25 ×
0.15 mm IntegraFrit Sample Trap Column (Bruker, Billerica, MA) for 5 min at a
flow rate at 2 μL/min. Elution and separation was performed using a Magic
AQ C18, 3 μm, 200 Å, 105 × 0.15 mm PicoChip Column (Bruker,
Billerica, MA) using a two-step linear acetonitrile gradient from 2–25 %
B in 5 min and 25–60 % B in 35 min (Buffer A: 0.1 % FA; Buffer B: 95 %
MeCN / 0.1 % FA) at a flow rate of 0.8 µL/min. Full MS scans were
acquired at m/z 300–2000 with a spray voltage maintained at 2.7 kV, an
S-lens setting of 60 % and the ion transfer tube set at 300 °C. Data was
recorded at a mass resolving power of 70,000. The maximum injection time was set
at 100 ms with an AGC target value of 1.0 × 106. HCD-MS/MS
scans were obtained at a resolution of 17,500 with an AGC target value of 1.0
× 106 and an isolation window of 0.8. Evaluation of Normalized
Collision Energies (NCS’s) for the optimized identification of both
‘global’ and ‘variant-specific’ MC product ions was
performed by energy resolved HCD-MS/MS of each peptide, using repeated
injections.