BioHPLC Column Selection Guide Cover - Agilent Technologies

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
Peptide phosphorylation sites LC
and LC/MS using Capillary LC columns
Column: ZORBAX 300SB-C18
5064-8268
0.5 x 150 mm, 3.5 µm
Mobile Phase: A: Water + 0.1% Formic acid
B: Acetonitrile + 0.1% Formic acid
Flow Rate: 5.5 µL/min
Gradient: 5-55% B in 50 min, to
85% B from 55-57 min
Detector: UV 206 nm
MS Conditions: LC/MS: Pos. Ion ESI
with LC/MSD trap
Vcap: 4000 V
Drying gas flow:
Drying gas
7 L/min
temperature: 250 °C
Nebulizer: 15 psi
Capillary Exit Volt: 50 V Max
Accum Time: 300 ms
Total Averages: 3
Isolation Width: 3 m/z
Frag Ampitude: 1.0 V
Sample: Beta case in digest, 100 nL (4 pmol)
x107 Intens.
3
2
1
Base Peak Chromatogram
Capillary and Nano Columns
Proteins Identified
1. Alpha-1-Antichymotrypsin
2. Antithrombin-III Precursor
3. Complement Factor B Precursor
0
20 25 30 35 40 45 50 55
Time (min)
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
MSFull Scan MS
% Relative Abundance
% Relative Abundance
x10
0.8
0.6
0.4
0.2
7
0.0
100
80
60
40
20
0
UV at 206 nm UV
phosphopeptide
10 15 20 25 30 35 40 45 50
Time (min)
10 15 20 25 30 35 40 45 50
Time (min)
343.0 559.5
x10
2.5
2.0
1.5
688.4
1032.0
1.0
0.5
1275.4 1582.0
5
0.0
400 800 1600 1600
LCBP014
m/z
982.5
1022.9
[MH2-H20]
747.3 1106.7
1490.8
1690.0
2+
[MH2-H2PO4] 2+
MS/MS of [M+2H] 2+ at m/z 1032
x10
2.5
2.0
1.5
1.0
0.5
m/z
4
0.0
400 800 1600 1600
% Relative Abundance
LCBP037
ANALYSIS SCALE
109