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Peptide Map of unreduced Human Ig-G2 on Kinetex 2.6µm C18 150 x 2.1mm ID

Application Detail (App ID: 18764)

Part No:00F-4462-AN
Dimensions:150 x 2.1 mm ID
Elution Type:Gradient
Elution A:0.1% TFA/2% ACN/Water
Elution B:0.09% TFA/98% ACN/Water
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 98 2
2 2 98 2
3 37 52 48

Flow Rate:1 mL/min
Col. Temp: 40 °C
Detection: UV-Vis Abs.-Variable Wave.(UV) @ 214 nm (22°C)
Note:Application Focus: To demonstrate dual digestion of unreduced Ig-G to overcome protease resistance and characterize disulfide bonds

As Ig-G is a large protein (150KDa), peptide maps can be rather complex due to the large number of peptide generated; thus a high resolution media is needed to get the maximum peptide resolution. Ig-G is also protease resistant and a method is presented for unreduced peptide mapping. Human Ig-G was digested with Lys-C for 18 hours at 37C (E/S 1:30) in 4M guanidine/ 0.1M ammonium bicarbonate. Sample was diluted to 1M guanidine then digested with Asp-N for 12 hours (E/S 1:50). The reaction was quenched with TFA and 20 µg aliquots were injected on HPLC.

App ID # 18764 shows the Lys-C/ Asp-N peptide map run on a compared to the Kinetex™ 2.6 µm C18 column to show the high resolution that Kinetex delivers in separating peptide. The combination of enzymes is widely used in the industry to digest human Ig-G therapeutics as tryptic digests fail to fully digest the non-reduced protein. When compared to a fully porous 3u media (data not shown) Kinetex resolves a larger number of peptide from very complex peptide maps.

Analyte Details

Analyte Detail not available

1
Hu-IgG2 Lys-C / Asn-N