Table 1.

Method parameters for IgG subclass measurements by LC-MS/MS utilizing stable labeled peptide internal standards.

Sample parametersSpecification
Specimen volume, type, and batch size20 μL of serum in a 96-well plate
Reducing and denaturing reagents added for 30 min at 55°C100 μL of 50 mmol/L (NH4)2HCO3
100 μL of 2,2,2-trifluoroethanol
25 μL of 100 mmol/L DL-dithiothreitol
Acetylating reagent added for 60 min at room temperature30 μL of 200 mmol/L iodoacetamide
Treated sample volume for digestion50 μL
Digestion reagents added with sonication and subsequent heating at 37°C for 1 h (stopped by 20 μL of formic acid)200 μL of deionized water
50 μL of 50 mmol/L (NH4)2HCO3
30 μL of 1 g/L trypsin
Digested sample volume for injection after addition of IgG subclass peptide internal standard mixture10 μL
Liquid chromatography parametersSpecification
Mobile phase A0.1% formic acid in water
Mobile phase B0.1% formic acid in 75:25 ACN/IPA
ColumnPhenomenex Aeris PEPTIDE 3.6 μm XDB-C18 50 × 2.1 mm, with an in-line filter
Column flow rate0.4 mL/min
Gradient (total run time 11 min)0% B for 1 min
0% to 50% B over 6.5 min
50% to 98% B in 0.1 min
98% B for 1.4 min
Return to 0% B in 0.1 min, hold for 2 min
MS/MS parameters (SCIEX 5500 QTrap)Specification
Ionization typeESI+
Curtain gas (CUR)40.0
Collision gas (CAD)High
Ion spray voltage (IS)5500.0
Temperature (TEM)600.0
Ion source gas 1 (GS1)35.0
Ion source gas 2 (GS2)30.0
Entrance potential10
MRM quantifier transitions and compound-specific parameters
IDMRM transition, m/zDeclustering potential, VCollision energy, VCell exit potential, V
Total IgG418.2/619.467838
Total IgG_SIS428.1/513.4802211