Peptide Map of reduced and alkylated Ig-G1 on Kinetex 2.6µm C18 150 x 4.6mm ID

Application Detail (App ID: 18766)

Part No:00F-4462-E0
Dimensions:150 x 4.6 mm ID
Elution Type:Gradient
Elution A:0.1% TFA/2% ACN/Water
Elution B:0.085% TFA in ACN
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 99 1
2 40 44 56
3 41 44 56

Flow Rate:1 mL/min
Col. Temp: 40 °C
Detection: UV-Vis Abs.-Variable Wave.(UV) @ 214 nm (22°C)
Note:Application Focus: R+A Peptide mapping of Ig-G using Kinetex

Performing peptide mapping of Ig-G is required to identify all of the post translational modification that can occur with Ig-G based therapeutics. However, native Ig-G is very protease resistant so alternate sample preparation methods are needed to get good peptide fragmentation. A common method used when disulfide information is not needed is to first reduce a protein with dithiothreitol (DTT) under denaturing conditions (8M urea, 0.1M NH4HCO3, pH8.0, 10mm DTT at 45C for 15 minutes) then alkylate the protein with iodoacetic acid (100 mm IAA at RT for 15 minutes). The reduced and alkylated protein is then diluted with water to 2M Urea and digested with trypsin (1:25 E/S w:w) for 18 hours at 37C. Digest is quenched with TFA and injected on the Kinetex column (150x4.6mm). Results show good fragmentation of the Ig-G protein with numerous peptides of different hydrophobicites across the peptide map. Good peak shape is observed across the map with a large number of well resolved peaks in less than 30 minutes despite un-optimized conditions. With increase flow rates even better resolution could be realized, or run time could be significantly reduced.

Analyte Details

Analyte Detail not available

Hu-IgG1 Reduced and Alkylated