TITLE: Protein Digestion and Mass Spectrometry Analysis Protocol
AUTHORS: Anthony Iavarone, Annan SI Cook

[DESCRIPTION]
This protocol details a protocol for the trypsin digestion of PI3KC3-C1 and subsequent analysis of digested peptides using LC-MS/MS.

[STEPS]
SECTION: Protein Denaturation and Reduction
1. Mix PI3KC3-C1 (mCherry-ATG14|VPS15-TSF) protein with 8 Molarity (M) urea, 50 millimolar (mM) TRIS pH 8.0, and 10 millimolar (mM) TCEP.
SECTION: Protein Denaturation and Reduction
2. Add Iodoacetamide (IAA) to achieve a final concentration of 15 millimolar (mM).
SECTION: Protein Denaturation and Reduction
3. Incubate the mixture at Room temperature for 20 min to alkylate cysteine residues.
SECTION: Trypsin Digestion Setup
4. Prepare a trypsin digestion solution containing Gold Trypsin (0.5 mg/mL), 50 millimolar (mM) TRIS pH 8, and 100 millimolar (mM) CaCl2. 
 Trypsin digestion solution
      Gold Trypsin (0.5
  mg/mL)  8 uL    50 mM TRIS pH 8  160 uL    100 mM CaCl2  2 uL  

SECTION: Trypsin Digestion Setup
5. Mix the trypsin digestion solution with the alkylated protein sample.
SECTION: Digestion Incubation
6. Incubate the digestion mixture at 37 °C 20 min with shaking at  .

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

Solvent A
 

Solvent B
 

SECTION: Liquid Chromatography (LC) Setup
9. 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).
SECTION: Liquid Chromatography (LC) Setup
10. Maintain the column compartment at 50 °C.

SECTION: LC-MS Analysis
11.  Load a 10 µL sample onto the column.
SECTION: LC-MS Analysis
12. Implement the following gradient elution program:

Isocratic flow at 1% (volume/volume) B for 2 min.
Linear gradient to 35% B over 90 min.
Linear gradient to 95% B over 1 min.
Isocratic flow at 95% B for 6 min.
Linear gradient to 1% B over 1 min.
Isocratic flow at 1% B for 20 min.
SECTION: LC-MS Analysis
13. Set the flow rate to 120 µL/min.
SECTION: Mass Spectrometry Analysis
14. 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).
SECTION: Data-Dependent MS/MS Analysis
15. 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).
SECTION: Data-Dependent MS/MS Analysis
16. Acquire MS/MS spectra using the linear ion trap.
SECTION: Data Analysis
17. 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.
SECTION: Data Analysis
18. Consider tryptic peptides with up to three missed cleavages and specify post-translational modifications.
SECTION: Data Analysis
19. Validate peptide assignments by manually inspecting MS/MS spectra.