Sep 30, 2023
Electrospray ionization analysis of eluted nucleotides
- Annan Cook1,2,
- Xuefeng Ren3,2,
- Anthony T. Iavarone1
- 1University of California, Berkeley;
- 2Aligning Science Across Parkinson's;
- 3of California, Berkeley
Protocol Citation: Annan Cook, Xuefeng Ren, Anthony T. Iavarone 2023. Electrospray ionization analysis of eluted nucleotides. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qz6jvo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 22, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88517
Keywords: ASAPCRN, eluted nucleotide, electrospray ionization, denaturation of pi3kc3, nucleotides this protocol, ionization analysis, ionization mass spec, pi3kc3
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol describes the denaturation of PI3KC3-C1, and subsequent analysis of eluted nucleotides by electrospray ionization mass spec.
Attachments
852-2200.pdf
38KB
Materials
Materials
- PI3KC3-C1(VPS15-TSF) and PI3KC3-C1(mCherry-ATG14|VPS15-TSF) protein samples
- 5 mL PD-10 column (Cytiva)
- 0.5 M NH4CH3CO2 buffer
- Nanodrop spectrophotometer
- Liquid chromatography (LC) system (Agilent 1200 series)
- LTQ-Orbitrap-XL mass spectrometer with ESI source (Thermo Fisher Scientific)
- Ammonium acetate (≥98% purity)
- Methanol (Optima LC-MS grade, ≥99.9% purity)
- Purified water (resistivity of 18.2 MΩ·cm)
- Ultra C18 column (length: 150 mm, inner diameter: 2.1 mm, particle size: 3 µm)
- Pierce LTQ ESI positive ion calibration solution (Thermo Fisher Scientific)
- Xcalibur software (version 2.0.7, Thermo Fisher Scientific)
Buffer Exchange
Wash a 5 mL desalting (PD-10) column with 0.5 Mass Percent NH4CH3CO2.
Load PI3KC3-C1(VPS15-TSF) and PI3KC3-C1(mCherry-ATG14|VPS15-TSF) protein samples onto a 5-mL PD-10 column.
Exchange the protein sample buffer by passing the protein through the column.
Protein Denaturation
10m
Heat the buffer-exchanged protein samples to 90 °C for 00:10:00 to denature the proteins.
10m
Centrifugation
15m
After denaturation, centrifuge the samples at 21000 x g, 00:15:00 to separate out any precipitated protein.
15m
Nucleotide Concentration Assessment
Measure the A260 absorbance of the denatured samples using a Nanodrop spectrophotometer.
Setup of LC-MS System
Connect the LC system (Agilent 1200 series) to the LTQ-Orbitrap-XL mass spectrometer equipped with an ESI source (Thermo Fisher Scientific).
LC Column Equilibration
Equilibrate the Ultra C18 column with the LC mobile phase solvents according to the manufacturer's instructions.
Mobile Phase Preparation
Prepare mobile phase solvents A and B:
- Solvent A: Water with 10 millimolar (mM) ammonium acetate.
- Solvent B: Methanol with 10 millimolar (mM) ammonium acetate.
LC Elution Program
25m
Set up the LC system with the following gradient program:
Isocratic flow at 0.5% (volume/volume) B for 00:02:00 .
2m
Linear gradient to 99.5% B over 00:01:00 .
1m
Isocratic flow at 99.5% B for 00:04:00 .
4m
Linear gradient to 0.5% B over 00:00:30 .
30s
Isocratic flow at 0.5% B for 00:17:30 .
17m 30s
Maintain a flow rate of 150 µL /min and a column temperature of 40 °C .
Inject 20 µL of the sample into the LC system.
Mass Spectrometer Calibration
Perform external mass calibration in the positive ion mode using the Pierce LTQ ESI positive ion calibration solution.
Data Acquisition
Acquire full-scan, high-resolution mass spectra in the positive ion mode over the range of mass-to-charge ratio (m/z) = 300 to 1000 using the Orbitrap mass analyzer.
Set the mass resolution to 60,000 (at m/z = 400, FWHM).
Data Analysis
Analyze the acquired data using Xcalibur software (version 2.0.7, Thermo Fisher Scientific) for peak identification and interpretation.