Sep 30, 2023

Public workspaceProtein Digestion and Mass Spectrometry Analysis Protocol

DOI
dx.doi.org/10.17504/protocols.io.4r3l2261ql1y/v1
  • 1University of California, Berkeley;
  • 2ASAP CRN
Annan SI Cook
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DOI: dx.doi.org/10.17504/protocols.io.4r3l2261ql1y/v1
Protocol Citation: Anthony Iavarone, Annan SI Cook 2023. Protein Digestion and Mass Spectrometry Analysis Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2261ql1y/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 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88519
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol details a protocol for the trypsin digestion of PI3KC3-C1 and subsequent analysis of digested peptides using LC-MS/MS.
Attachments
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852-2203.pdf
39KB
Materials
Materials

  • PI3KC3-C1 (mCherry-ATG14|VPS15-TSF) protein sample
  • Urea
  • tris(hydroxymethyl)aminomethane (TRIS) pH 8.0
  • tris(2-carboxyethyl)phosphine (TCEP)
  • Iodoacetamide (IAA)
  • Trypsin (Gold Trypsin, 0.5 mg/mL Promega V5280)
  • CaCl2
  • NH4CH3CO2
  • Acetonitrile
  • Formic acid (Optima LC-MS grade, 99.9% minimum)
  • Water (purified using the Milli-Q Gradient system)
  • Zorbax 300SB-C8 Micro Bore Rapid Resolution column (150 mm length, 1.0 mm inner diameter, 3.5 µm particle size, Agilent)
  • LC-MS system (e.g., Agilent 1200 series LC and Thermo Fisher Scientific LTQ-Orbitrap-XL mass spectrometer)
  • Proteome Discoverer software (version 1.3, Thermo Fisher Scientific)
Protein Denaturation and Reduction
Protein Denaturation and Reduction
20m
20m
Mix PI3KC3-C1 (mCherry-ATG14|VPS15-TSF) protein with Concentration8 Molarity (M) urea, Concentration50 millimolar (mM) TRIS Ph8.0 , and Concentration10 millimolar (mM) TCEP.
Mix
Add Iodoacetamide (IAA) to achieve a final concentration of Concentration15 millimolar (mM) .
Pipetting
Incubate the mixture at TemperatureRoom temperature for Duration00:20:00 to alkylate cysteine residues.
20m
Incubation
Trypsin Digestion Setup
Trypsin Digestion Setup
Prepare a trypsin digestion solution containing Gold Trypsin (0.5 mg/mL), Concentration50 millimolar (mM) TRIS Ph8 , and Concentration100 millimolar (mM) CaCl2.
Trypsin digestion solution
AB
Gold Trypsin (0.5 mg/mL)8 uL
50 mM TRIS pH 8160 uL
100 mM CaCl22 uL

Mix the trypsin digestion solution with the alkylated protein sample.
Mix
Digestion Incubation
Digestion Incubation
20m
20m
Incubate the digestion mixture at Temperature37 °C DurationOvernight with shaking at Shaker200 rpm .

20m
Incubation
Overnight
Confirmation by SDS-PAGE
Confirmation by SDS-PAGE
Analyze the digested proteins by SDS-PAGE to confirm successful digestion. The absence of bands corresponding to the PI3KC3-C1 components indicates successful digestion.
Analyze
Liquid Chromatography (LC) Setup
Liquid Chromatography (LC) Setup
Prepare LC mobile phase solvents: Solvent A and Solvent B.

Solvent A
A
99.9% water
0.1% formic acid

Solvent B
A
99.9% acetonitrile
0.1% formic acid

Set up the LC system with a Zorbax 300SB-C8 Micro Bore Rapid Resolution column (150 mm length, 1.0 mm inner diameter, 3.5 µm particle size).
Maintain the column compartment at Temperature50 °C .

LC-MS Analysis
LC-MS Analysis
2h
2h
Load a Amount10 µL sample onto the column.
Implement the following gradient elution program:

  • Isocratic flow at 1% (volume/volume) B for Duration00:02:00 .
  • Linear gradient to 35% B over Duration01:30:00 .
  • Linear gradient to 95% B over Duration00:01:00 .
  • Isocratic flow at 95% B for Duration00:06:00 .
  • Linear gradient to 1% B over Duration00:01:00 .
  • Isocratic flow at 1% B for Duration00:20:00 .
2h
Set the flow rate to Amount120 µL /min.
Mass Spectrometry Analysis
Mass Spectrometry Analysis
Acquire full-scan, high-resolution mass spectra in positive ion mode over the m/z range of 340 to 1800 using the Orbitrap mass analyzer with a mass resolution of 60,000 (at m/z = 400, FWHM).
Data-Dependent MS/MS Analysis
Data-Dependent MS/MS Analysis
Select the ten most intense ions exceeding an intensity threshold of 10,000 raw ion counts from each full-scan mass spectrum for tandem mass spectrometry (MS/MS) analysis using collision-induced dissociation (CID).
Acquire MS/MS spectra using the linear ion trap.
Data Analysis
Data Analysis
Use Proteome Discoverer software (version 1.3, SEQUEST algorithm) to search raw data files against the amino acid sequences of mCherry-labeled Class III phosphatidylinositol 3-kinase complex I (mCherry-PI3KC3-C1) proteins.
Consider tryptic peptides with up to three missed cleavages and specify post-translational modifications.
Validate peptide assignments by manually inspecting MS/MS spectra.