Tryptic
peptides were analyzed with a nanoAcquity UPLC system coupled to a
Synapt G2-Si HDMS mass spectrometer with a nanoelectrospray ionization
source (Waters Corporation, Manchester, UK). The nanoAcquity UPLC
system consisted of a C18, 5 μm, 180 μm × 20 mm trap
column and an HSS-T3 C18 1.8 μm, 75 μm × 250 mm analytical
column (Waters Corporation, Manchester, UK) set to trapping mode.
Samples containing 300 ng of protein were injected in each run. Mobile
phases A and B consisted of 0.1% formic acid and 3% dimethyl sulfoxide
in water (v/v) and 0.1% formic acid and 3% dimethyl sulfoxide in acetonitrile
(v/v), respectively. Peptide separation was done using a gradient
from 3% to 40% (v/v) of mobile phase B at a constant flow rate of
0.3 μL/min over 120 min. Lock-mass correction was performed
by spraying a lock-mass solution containing [Glu1]-fibrinopeptide
B (0.1 μM) and leu-enkephalin (1 μM) through the reference
channel every 60 s. Data were acquired using a data-independent acquisition
workflow in positive ion mode with a UDMSE method.19 (link)−21 (link) The system’s performance and stability were monitored by
injecting a commercially available HeLa digest (Thermo Scientific,
Waltham, MA) after every seventh sample injection.
peptides were analyzed with a nanoAcquity UPLC system coupled to a
Synapt G2-Si HDMS mass spectrometer with a nanoelectrospray ionization
source (Waters Corporation, Manchester, UK). The nanoAcquity UPLC
system consisted of a C18, 5 μm, 180 μm × 20 mm trap
column and an HSS-T3 C18 1.8 μm, 75 μm × 250 mm analytical
column (Waters Corporation, Manchester, UK) set to trapping mode.
Samples containing 300 ng of protein were injected in each run. Mobile
phases A and B consisted of 0.1% formic acid and 3% dimethyl sulfoxide
in water (v/v) and 0.1% formic acid and 3% dimethyl sulfoxide in acetonitrile
(v/v), respectively. Peptide separation was done using a gradient
from 3% to 40% (v/v) of mobile phase B at a constant flow rate of
0.3 μL/min over 120 min. Lock-mass correction was performed
by spraying a lock-mass solution containing [Glu1]-fibrinopeptide
B (0.1 μM) and leu-enkephalin (1 μM) through the reference
channel every 60 s. Data were acquired using a data-independent acquisition
workflow in positive ion mode with a UDMSE method.19 (link)−21 (link) The system’s performance and stability were monitored by
injecting a commercially available HeLa digest (Thermo Scientific,
Waltham, MA) after every seventh sample injection.
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