HPLC Analysis of Carboxyamidomethylated Lysozyme Tryptic Digest on BIOshell™ A160 Peptide C18

HPLC Analysis of Carboxyamidomethylated Lysozyme Tryptic Digest on BIOshell™ A160 Peptide C18

Conditions

column BIOshell A160 Peptide C18; 15 cm x 2.1 mm I.D., 2.0 µm particles (67243-U)
column temp. 35 °C
mobile phase [A] 98:2 (0.1% TFA in water: 0.1% TFA in acetonitrile); [B] 50:50 (0.1% TFA in water: 0.1% TFA in acetonitrile)
gradient 0 to 63% B in 60 min
flow rate 0.3 mL/min
pressure 7440 psi (513 bar)
sample Lysozyme tryptic digest, 1.0 mg/mL, 0.1% TFA in water
inj. temp. 20 µL
detector UV, 215 nm

Description

Analysis Note One way to study the primary structure of a protein is to proteolytically digest the protein into peptides and then resolve the peptides by U/HPLC or LC/MS analysis. The BIOshell A160 Peptide C18 column, packed with 2.0 μm, superficially porous particles, can provide baseline resolution of peptides generated from a protein digest. This application utilizes this column in resolving peptides generated from a tryptic digest of hen egg white lysozyme.
Categories Analytical Chromatography, Peptides
Featured Industry Life Science and Biopharma
General description The following protocol was used to digest lysozyme: Lysozyme (10 mg) was mixed with a 1:1 solution (20 μL) of trifluoroethanol (TFE) and TCEP (20 mM in 50 mM ammonium bicarbonate). The sample was incubated at 60 °C for 1 hour. After incubation, the sample was centrifuged (14,000 x g) for 30 s. To the sample, iodoacetamide (IAM, 200 mM, 5 μL) was added, and the sample was incubated at room temperature, in the dark, for 1 hour. After incubation, ammonium bicarbonate (25 mM, 220 μL) was added to the sample. Trypsin (1 mg/mL, 500 μL) was added to the sample in order to make the ratio of trypsin to lysozyme 1:20. The digestion reaction was performed overnight at 37 °C in an automated shaker set at 300 rpm. The reaction was quenched by adding 2.0 μL of concentrated formic acid. The LC sample was prepared by mixing 100 μL digested lysozyme with 900 μL 0.1% TFA in water.
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
suitability application for HPLC

Materials

     
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