For mass spectrometry analysis, a Waters (Baden, Switzerland) nanoAcquity pump equipped with Waters LCT Premier XE and a Acquity UPLC Protein C4 column, 300 Å, 1.7 μm, 1 x 50 mm was used in ESI positive mode at a flow rate of 70 μL/min. Mobile phases used were (A) water/acetonitrile 9:1 with 0.1% trifluoroacetic acid and (B) acetonitrile/water 9:1 with 0.1% trifluoroacetic acid, from 10% (B) to 65% (B) within 10 minutes. Proteins have been analysed (if not otherwise mentioned) reduced and deglycosylated by the use of Rapid PNGase F (New England Biolabs, Ipswich, MA, USA) according to the following representative procedure: 5.7 μL miliQ water was added to 10.3 μL (12 μg) protein solution. 4 μL Rapid PNGase F buffer, containing dithiothreitol (DTT) for mild reduction of disulfide bonds (5x) and 1 μL Rapid PNGase F [18 (link)] for deglycosylation was added. The mixture was incubated at 50°C for 15 minutes.
Nanoacquity pump
Manufactured by Waters Corporation
Sourced in Switzerland
The NanoAcquity pump is a high-performance liquid chromatography (HPLC) pump designed for nanoscale applications. It is capable of delivering precise and consistent flow rates for nano-scale separations and analysis. The pump's core function is to provide accurate and reliable solvent delivery for HPLC systems used in various analytical applications.
Automatically generated - may contain errors
2 protocols using nanoacquity pump
1
Mass Spectrometry Analysis of Deglycosylated Proteins
2
Proteomics Workflow for ERLIC Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
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ERLIC samples were digested
prior to ERLIC and did not require any additional sample PREPL prior
to LC–MS. Gel slices from PAGE separation were extracted and
then digested with trypsin overnight. The resulting peptide mixture
was separated from any residual gel slices and analyzed on an Orbitrap
Velos hybrid ion trap mass spectrometer (Thermo Fisher Scientific).
Regions between 395 and 1600 m/z ions were collected at 60K resolving power for the MS1, and these
data were used to trigger MS/MS in the ion trap for the top 20 ions
in the MS1 (i.e., top 20 experiment). Active dynamic exclusion of
500 ions for 90 s was used throughout the LC–MS/MS method.
Samples were trapped for 15 min with flow rate of 2 μL/min on
a trapping column 100 μm ID packed for 5 cm in-house with 5
μm Magic C18 AQ beads (Waters) and eluted onto 20 cm ×
75 μm ID analytical column (New Objective) packed in-house with
3 μm Magic C18 AQ beads (Waters). Peptides were eluted with
300 nL flow rate using a NanoAcquity pump (Waters) using a binary
gradient of 2–32% B over 90 min (A: 0.1% formic acid in water;
B: 0.1% formic acid in acetonitrile).
prior to ERLIC and did not require any additional sample PREPL prior
to LC–MS. Gel slices from PAGE separation were extracted and
then digested with trypsin overnight. The resulting peptide mixture
was separated from any residual gel slices and analyzed on an Orbitrap
Velos hybrid ion trap mass spectrometer (Thermo Fisher Scientific).
Regions between 395 and 1600 m/z ions were collected at 60K resolving power for the MS1, and these
data were used to trigger MS/MS in the ion trap for the top 20 ions
in the MS1 (i.e., top 20 experiment). Active dynamic exclusion of
500 ions for 90 s was used throughout the LC–MS/MS method.
Samples were trapped for 15 min with flow rate of 2 μL/min on
a trapping column 100 μm ID packed for 5 cm in-house with 5
μm Magic C18 AQ beads (Waters) and eluted onto 20 cm ×
75 μm ID analytical column (New Objective) packed in-house with
3 μm Magic C18 AQ beads (Waters). Peptides were eluted with
300 nL flow rate using a NanoAcquity pump (Waters) using a binary
gradient of 2–32% B over 90 min (A: 0.1% formic acid in water;
B: 0.1% formic acid in acetonitrile).
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