Extracted proteolytic peptides were analyzed by LC-MS. Peptide separation was carried out with gradient elution (water-acetonitrile-0.1% formic acid) from a 75m ID capillary reversed phase C18 column (New Objective) using an Agilent 1100 nano HPLC system (Agilent Technologies); the flow rate was 280 nl/min. In order to optimize peptide separation, a three hour long gradient was used. Peptide molecular weight data as well as peptide fragment ion mass spectra were acquired with an LTQ ion trap mass spectrometer (ThermoFischer Scientific).
Agilent 1100 nano hplc system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1100 nano HPLC system is a liquid chromatography instrument designed for high-performance liquid chromatography (HPLC) analysis. It is capable of performing nano-scale separations with high sensitivity and resolution.
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3 protocols using agilent 1100 nano hplc system
1
Proteolytic Peptide Analysis by LC-MS
2
Proteomic Peptide Analysis by LC-MS
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Extracted proteolytic peptides were analyzed by LC‐MS. Peptide separation was carried out with gradient elution (water‐acetonitrile‐0.1% formic acid) from a 75m ID capillary reversed phase C18 column (New Objective) using an Agilent 1100 nano HPLC system (Agilent Technologies); the flow rate was 280 nL/min. In order to optimize peptide separation, a three hour long gradient was used. Peptide molecular weight data as well as peptide fragment ion mass spectra were acquired with an LTQ ion trap mass spectrometer (ThermoFischer Scientific).
3
Protein Reduction, Alkylation and Tryptic Digestion
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The half of the protein was reduced with 10 mM DTT for 30 min at 60 °C and alkylated with 55 mM IAA for 30 min in the dark, following by digested with trypsin. The digested peptides were resuspended in 0.1% TFA and loaded onto Zorbax 300SB-C18 75 μm i.d. × 15 cm column via a trap column (Zorbax 300SB-C18 300 μm i.d. × 5 mm column). Peptides were then separated in an acetonitrile gradient (buffer A – 0.1% formic acid; buffer B – 100% acetonitrile and 0.1% formic acid) at a flow rate of 200 nl/min with an Agilent 1100 nanoHPLC system (Agilent, USA) and applied on-line to an Q Star XL mass spectrometer (AB Sciex, USA). The gradient was increased from 5% to 40% solution B over 110 min, followed by an increase to 95% B over 1 min, and then 95% B isocratic for 15 min. MS spectra were collected in full scan mode (350–1400 Da) followed by three MS/MS scans of the most intense ions.
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