Sep 30, 2023

Protein Digestion and Mass Spectrometry Analysis Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2261ql1y/v1
  • 1University of California, Berkeley;
  • 2ASAP CRN
Annan SI Cook
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2261ql1y/v1
Protocol Citation: Anthony Iavarone, Annan 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, protocol for the trypsin digestion, digested peptide, trypsin digestion, mass spectrometry analysis protocol this protocol detail, mass spectrometry analysis protocol, protein digestion, mass spectrometry, protein, pi3kc3, protocol detail
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
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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
20m
Mix PI3KC3-C1 (mCherry-ATG14|VPS15-TSF) protein with 8 Mass Percent urea, 50 millimolar (mM) TRIS 8.0 , and 10 millimolar (mM) TCEP.
Add Iodoacetamide (IAA) to achieve a final concentration of 15 millimolar (mM) .
Incubate the mixture at Room temperature for 00:20:00 to alkylate cysteine residues.
20m
Trypsin Digestion Setup
Prepare a trypsin digestion solution containing Gold Trypsin (0.5 mg/mL), 50 millimolar (mM) TRIS 8 , and 100 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.
Digestion Incubation
20m
Incubate the digestion mixture at 37 °C Overnight with shaking at 200 rpm .

20m
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.
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 50 °C .

LC-MS Analysis
2h
Load a 10 µL sample onto the column.
Implement the following gradient elution program:

  • Isocratic flow at 1% (volume/volume) B for 00:02:00 .
  • Linear gradient to 35% B over 01:30:00 .
  • Linear gradient to 95% B over 00:01:00 .
  • Isocratic flow at 95% B for 00:06:00 .
  • Linear gradient to 1% B over 00:01:00 .
  • Isocratic flow at 1% B for 00:20:00 .
2h
Set the flow rate to 120 µL /min.
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
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
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.