Materials and Reagents

1. Gloves
   
2. Lab coat
   
3. Culture tubes (16 x 100 mm) (VWR, catalog number: 47729-576 )
   
4. 4 ml glass vials and caps (13-425 thread finish, borosilicate glass) (VWR, catalog number: 46610-706 )
   
5. HPLC autosampler vials and caps (9-425 thread finish) (VWR, catalog number: 89523-482 )
   
6. Pipette tips (standard 1,000 µl and 200 µl)
   
7. Gel loading pipette tips (VWR, catalog number: 37001-150 )
   
8. Joint clips to attach the round bottom flask to Allihn condenser (VWR, catalog number: 89426-304)
   Manufacturer: GLASSCOLABS, catalog number: 007.470.05A .
   
9. Eppendorf tubes (standard 1.5 ml)
   
10. Ammonium acetate (for LC-MS LiChropur^®) (EMD Millipore, catalog number: 533004 )
    
11. Water (HPLC-grade) (VWR, BDH^®, catalog number: BDH23595.400 )
    
12. Ammonium hydroxide (28% NH₃ in H₂O, ≥ 99.99%) (Sigma-Aldrich, catalog number: 338818 )
    
13. 3,3,3’,3’-d₄-L-cystine (98%) (Cambridge Isotope Laboratories, catalog number: DLM-1000-1 )
    
14. L-cystine (≥ 98%) (Sigma-Aldrich, catalog number: C8755 )
    
15. DL-cystine dimethyl ester dihydrochloride (≥ 95%) (Sigma-Aldrich, catalog number: 857327 )
    
16. Acetonitrile (HPLC-grade) (VWR, BDH^®, catalog number: BDH83639.400 )
    
17. Ammonium acetate solution (see Recipes)
    

    Note: Specific brand and catalog number (as used in our work) have been provided for the materials and reagents listed. However, we do not explicitly endorse any of these brands or products and most items can be obtained from other reputed vendors. Further, most of these product/catalog numbers are based on the current availability in the United States, so researchers in other countries may find it necessary to obtain materials from another vendor.

Equipment

1. pH meter (standard)
   
2. Safety goggles
   
3. Chemical safety hood
   
4. Glass bottles (standard 500 ml and 1 L)
   
5. Glass measuring cylinders (standard 1 L, 250 ml, and 100 ml)
   
6. Glass round bottom flask (standard 24/40 ground joint, 100 ml)
   
7. Magnetic stir-bar (standard)
   
8. Reflux apparatus (standard, consisting of stirring-heating mantle, Allihn condenser with a water jacket and 24/40 junction, running water source, rubber tubing, and clips for attachment). Follow vendor instructions for assembly
   
9. Pipettes (standard 1,000 µl and 200 µl)
   
10. Vortex (standard)
    
11. -20 °C freezer (standard)
    
12. 4 °C incubator or fridge (standard)
    
13. HPLC-MS system:
1. HPLC: Shimadzu UFLC prominence system (HPLC) (Shimadzu, model: Prominence UFLC ) fitted with following modules: CBM-20A (Communication bus module), DGU-A₃ (degasser), two LC-20AD (liquid chromatograph, binary pump), SIL-20AC HT (auto sampler)
   
2. HPLC column: Luna^® NH₂ column (2 x 50 mm, 3 µm, 100 Å) (Phenomenex, catalog number: 00B-4377-B0 )
   
3. Mass Spectrometer: AB Sciex 4000 LC-MS/MS mass spectrometer (AB Sciex, model: QTRAP^® 4000 ) fitted with a Turbo V^TM ion source
   
14. Centrifuge (standard, with attachment for 4 ml vials)
    
15. Weighing scale (standard)
    

    Note: Standard lab equipment should be used for this assay as indicated. Specific brand and catalog numbers (as used in our work) have been provided for components of the HPLC-MS setup–these can be replaced by equipment from other vendors; arguably any HPLC-MS setup will work with this assay, but may need additional optimization based on vendor-provided instructions.

Software

1. AB Sciex’s Analyst^® v1.6.1 for data acquisition, development of LC method, and optimization of analyte-specific MRM (multiple reaction monitoring) transitions
   
2. AB Sciex’s Peakview^® v2.1 and Skyline^® v3.6²³ for LC-MS/MS data analysis
   

Procedure