Interferon Alpha digests reduced & non-red on Jupiter 3u C18

LC Conditions

Column

Jupiter 3 µm C18 300 Å, LC Column 150 x 2 mm, Ea

Brand

Jupiter

Part No

00F-4263-B0

Phase Name

C18

Elution Type

Gradient

Mobile Phase

A: 0.1% TFA and 2% Acetonitrile in Water

B: 0.085% TFA, 90% Acetonitrile in Water

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

99

1

2

35

50

50

3

40

20

80

Flow Rate

0.30 mL/min

Temperature

25°C

Detection

UV-Vis Abs.-Diode Array (PDA) @ (25°C)

Detection Info

Sample Notes

Application Focus: Using peptide mapping with Jupiter 300 3u C18 to identify disulfide bonds Peptide mapping using different proteases is a good analytical method to screen PTMs of therapeutic proteins and is shown on Jupiter 300 3u C18 for a biogeneric alpha interferon. Interferon-alpha is a 19.2 kDa protein containing two disulfide bonds (cys1-cys98; cys29-cys138) and was tryptic digested (1:20 E/S ratio, 18 hrs at 37C) and then half of the digest was reduced with DTT (3 mM DTT at 45°C for 30 min). Both native and reduced tryptic digests injected on the Jupiter 300 C18 columns and overlaid. App ID 18072 shows that peptides retention time shifting due to disulphide bonds breakage after reduction reaction. Such shifts are clearly observed in the peak distribution at different retention times of the trypsin digested Interferon- alpha(lower chromatogram) and Interferon-alpha digested and reduced (upper chromatogram). Such shifts are indicative of a disulfide containing peptide and are easily verified by mass spectrometry. The high resolution that the Jupiter 300 3u C18 column delivers makes it an excellent choice for peptide mapping.