Lyophilized, HPLC-purified daptides 1–3 were resuspended in aq. 60% acetonitrile/0.1% acetic acid. Samples were directly infused onto a ThermoFisher Scientific Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate 100. Calibration was performed with Pierce LTQ Velos ESI Positive Ion Calibration Solution (ThermoFisher). The MS was operated with the following parameters: 100,000 resolution, 2 m/z isolation width (MS/MS), 0.4 activation q value (MS/MS), and 30 ms activation time (MS/MS). Fragmentation was performed using collision-induced dissociation (CID) at normalized collision energy. Each peptide was subjected to 30, 70, and 100 normalized collision energy (MS/MS). Data analysis was conducted using the Qualbrowser application of Xcalibur software (ThermoFisher Scientific).
Triversa nanomate 100
Manufactured by Advion
The TriVersa Nanomate 100 is a self-contained nanoelectrospray ionization source. It is designed for automated sample introduction and ionization for mass spectrometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ltq velos esi positive ion calibration solution, by Thermo Fisher Scientific (4 mentions)
Orbitrap fusion esi ms, by Advion (4 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Yeast extract, by Research Products International (1 mentions)
Deoxynucleotides, by New England Biolabs (1 mentions)
Tryptone, by Research Products International (1 mentions)
Ultraflextreme maldi tof mass spectrometer, by Thermo Fisher Scientific (1 mentions)
C18 column, by Macherey-Nagel (1 mentions)
S1000 thermal cycler, by Bio-Rad (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
Agilent 1200 series hplc, by Agilent Technologies (1 mentions)
5 protocols using triversa nanomate 100
1
Characterization of Daptide Peptides
2
Mass Spectrometry Protocol for Protein Analysis
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The reactions were desalted using a ZipTip and eluted into 70% aq. MeCN. The eluents were directly infused into a ThermoFisher Scientific Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate 100. The MS was calibrated and tuned with Pierce LTQ Velos ESI Positive Ion Calibration Solution (ThermoFisher). The MS was operated using the following parameters: resolution, 100,000; isolation width (MS/MS), 1 m/z; normalized collision energy (MS/MS), 70; activation q value (MS/MS), 0.4; activation time (MS/MS), 30 ms. Fragmentation was performed using collision-induced dissociation (CID) at 35 to 70%. The resulting data were averaged and analyzed using the Qualbrowser application of Xcalibur software.
3
Mass Spectrometry Protocol for Protein Analysis
4
Purification and Characterization of StsA
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Materials and reagents were purchased from
Gold Biotechnology, Fisher Scientific, or Sigma-Aldrich unless otherwise
noted. Yeast extract and tryptone were purchased from Research Products
International. Molecular biology reagents for cloning (e.g., restriction
enzymes, Q5 polymerase, T4 DNA ligase, and deoxynucleotides) were
purchased from New England Biolabs. Oligonucleotide primers and gene
blocks were obtained from Integrated DNA Technologies. DNA spin columns
were purchased from Epoch Life Sciences. Sanger sequencing was performed
by the Roy J. Carver Biotechnology Center (University of Illinois
at Urbana-Champaign). Polymerase chain reactions were performed using
a Bio-Rad S1000 thermal cycler. Escherichia coli DH5α and BL21(DE3) strains were used for plasmid maintenance
and protein overexpression, respectively. Expressed StsA was purified
using an Agilent 1200 series HPLC fitted with a 10 × 250 mm C18
column (Macherey Nagel). Mass spectroscopy was performed using a Bruker
Daltonics UltrafleXtreme MALDI-TOF mass spectrometer and a ThermoFisher
Scientific Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate
100.
Gold Biotechnology, Fisher Scientific, or Sigma-Aldrich unless otherwise
noted. Yeast extract and tryptone were purchased from Research Products
International. Molecular biology reagents for cloning (e.g., restriction
enzymes, Q5 polymerase, T4 DNA ligase, and deoxynucleotides) were
purchased from New England Biolabs. Oligonucleotide primers and gene
blocks were obtained from Integrated DNA Technologies. DNA spin columns
were purchased from Epoch Life Sciences. Sanger sequencing was performed
by the Roy J. Carver Biotechnology Center (University of Illinois
at Urbana-Champaign). Polymerase chain reactions were performed using
a Bio-Rad S1000 thermal cycler. Escherichia coli DH5α and BL21(DE3) strains were used for plasmid maintenance
and protein overexpression, respectively. Expressed StsA was purified
using an Agilent 1200 series HPLC fitted with a 10 × 250 mm C18
column (Macherey Nagel). Mass spectroscopy was performed using a Bruker
Daltonics UltrafleXtreme MALDI-TOF mass spectrometer and a ThermoFisher
Scientific Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate
100.
5
High-Resolution Tandem Mass Spectrometry Workflow
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Samples for high-resolution electrospray
ionization tandem mass spectrometry (HR-ESI-MS/MS) were either desalted
by ZipTip or purified by HPLC as described above. Next, samples were
diluted 1:1 into an ESI mix (80% methanol, 19% H2O, 1%
acetic acid). Samples were directly infused into a ThermoFisher Scientific
Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate 100. The
MS was calibrated and tuned with Pierce LTQ Velos ESI Positive Ion
Calibration Solution (ThermoFisher). The MS was operated using the
following parameters: resolution, 100,000; isolation width (MS/MS),
1 m/z; normalized collision energy (MS/MS), 35; activation q value
(MS/MS), 0.4; activation time (MS/MS), 30 ms. Fragmentation was performed
using CID at 30%. Data analysis was conducted using the Qualbrowser
application of Xcalibur software (ThermoFisher Scientific).
ionization tandem mass spectrometry (HR-ESI-MS/MS) were either desalted
by ZipTip or purified by HPLC as described above. Next, samples were
diluted 1:1 into an ESI mix (80% methanol, 19% H2O, 1%
acetic acid). Samples were directly infused into a ThermoFisher Scientific
Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate 100. The
MS was calibrated and tuned with Pierce LTQ Velos ESI Positive Ion
Calibration Solution (ThermoFisher). The MS was operated using the
following parameters: resolution, 100,000; isolation width (MS/MS),
1 m/z; normalized collision energy (MS/MS), 35; activation q value
(MS/MS), 0.4; activation time (MS/MS), 30 ms. Fragmentation was performed
using CID at 30%. Data analysis was conducted using the Qualbrowser
application of Xcalibur software (ThermoFisher Scientific).
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