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

应用详细信息 (应用ID: 18766)

尺寸:150 x 4.6 mm ID
洗脱A:0.1% TFA/2% ACN/Water
洗脱B:0.085% TFA in ACN
步骤序号 时间 (分) 图片A 图片B
1 0 99 1
2 40 44 56
3 41 44 56

流速 (毫升/分钟):1 mL/min
冷却温度: 40 °C
检测: UV-Vis Abs.-Variable Wave.(UV) @ 214 nm (22°C)
备注: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.



Hu-IgG1 Reduced and Alkylated