TABLE OF CONTENTS - BGB Analytik AG

LC and LC/MS
BioPharmaceutical Applications
Human Serum: Low Abundance Protein Isolation and
Identification by LC/MS
Column: ZORBAX 300SB-C18
Trap: 0.3 x 5 mm, 5 µm, 5065-9913
Analytical: 0.3 x 150 mm, 5 µm, 5064-8263
Mobile Phase: A: Water + 0.1% Formic acid
B: Acetonitrile + 0.1% Formic acid
Flow Rate: 6 µL/min
Gradient: 0 min 3% B
5 min 3% B (loading)
50 min 45% B
52 min 80% B
57 min 80% B
60 min 3% B
Sample: Band from 1-D in gel digest
Sample Preparation of Human Serum:
Major serum proteins removed using
Multiple Affinity Removal Column:
4.6 x 100 mm, P/N 5185-5985
Followed by 1-D gel digest
Courtesy of:
Novartis Pharma,
Biotechnology, Basel
Dr. Kurt Forrer
Patrik Roethlisberger
x107 Intens.
Monoclonal IgG1 Chains: Separation on Poroshell 300SB-C8
Column: ZORBAX Poroshell 300SB-C8
660750-906
2.1 x 75 mm, 5 µm
Mobile Phase: a: 90% water: 10% ACN + 3 mL/L
of MW 300 PEG
b: 10% water: 90% ACN + 3 mL/L
of MW 300 PEG
Flow Rate: 1.0 mL/min
Gradient: 0 min 25% B
10 min 40% B
10.1 min 25% B
12 min 25% B
Temperature: 70°C
Sample: Monoclonal IgG1
728 www.agilent.com/chem/supplies
3
2
1
0
Proteins Identified
1. Alpha-1-Antichymotrypsin
2. Antithrombin-III Precursor
3. Complement Factor B Precursor
Base Peak Chromatogram
20 25 30 35 40 45 50 55
Time (min)
Light Chains
Glycosylated Heavy Chains
a: Treated with DTT
0 2
4
6 8 10
b: Treated with DTT Peptide-N-Glycosidase F
0 2
4
6 8 10
c: Treated with DTT Peptide-N-Glycosidase F
Carboxypeptidase -B
0 2
4
6 8 10
Time (min)
LCBP014
LCBP015