2
Equipment
• UltiMate
™
3000 ×2 Dual Biocompatible Analytical LC
system, including:
- DGP-3600BM Biocompatible Dual-Gradient
Micro Pump
- WPS-3000TBFC Thermostatted Biocompatible
Pulled-Loop Well Plate Autosampler with Integrated
Fraction Collection
- TCC-3000SD Thermostatted Column Compartment
- DAD-3000 Diode Array Detector with 13 µL flow cell
• Thermo Scientific MSQ Plus Mass Spectrometer
with electrospray ionization (ESI) source
• Thermo Scientific Dionex Chromeleon
Chromatography Data System software version 6.80,
SR9 or higher
Reagents and Standards
• Deionized (DI) water, 18.2 MΩ-cm resistivity
• Acetonitrile (CH
3
CN), HPLC grade (Fisher Scientific
P/N AC610010040)
• Formic acid, ~98% (Fluka P/N 94318, Sigma-Aldrich
®
)
• Albumin from chicken egg white (Ovalbumin), ≥98%
(Sigma-Aldrich P/N A5503)
• Peroxidase from horseradish (HRP)(Sigma-Aldrich
P/N P6782)
• Ribonuclease B (Worthington Biochemical
P/N LS005710)
• Trypsin from bovine pancreas (Sigma-Aldrich
P/N T1426)
• Endo H, 500,000 units/mL (New England BioLabs
P/N P0702S)
• DL-Dithiothreitol, ≥99.0% (RT) (Fluka P/N 43819,
Sigma-Aldrich)
• Iodoacetamide, ≥99% (HPLC) (Sigma-Aldrich
P/N I6125
Conditions
Columns:
ProSwift ConA-1S Affinity (5 × 50 mm,
P/N 074148)
Thermo Scientific Acclaim Polar Advantage II
(PA2), 3 µm Analytical (3.0 × 150 mm,
P/N 063705)
Mobile Phase:
ProSwift Column:
A: 50 mM sodium acetate, 200 mM sodium
chloride, 1 mM calcium chloride, pH 5.3
B: 100 mM
α
-methyl mannoside in mobile
phase A
Acclaim
™
PA2 Column:
A: Water with 0.05% formic acid
B: Acetonitrile with 0.04% formic acid
Gradient: ProSwift Column: 0–5.0 min, 0% B;
5.0–5.5 min, 0–100% B; 5.5–15 min, 100% B
Acclaim PA2 Column: 0–5.0 min, 0% B;
5–35.0 min, 0–50% B; 35.5–45.0 min, 90% B
Flow Rate:
ProSwift Column: 0.5 mL/min
Acclaim PA2 Column: 0.425 mL/min
Inj. Volume:
20 µL
Temperature:
30 °C
Detection:
UV absorbance at 214 nm
MS SIM mode at
m/z
163, 204, and 366
Sample Preparation: Protein Samples: Dilute protein stock solution
(2 mg/mL in DI water) in mobile phase A to
1 mg/mL before injection
Peptide Samples: After tryptic digestion, dilute
the peptide sample in mobile phase A to
1 mg/mL before injection
MSQ Plus
™
Mass Spectrometer Conditions
Ionization Mode:
ESI
Operating Mode:
Positive Scan
Probe Temperature: 400 °C
Needle Voltage:
3.5 kV
Detection Mode: SIM at
m/z
163, 204, and 366
Dwell Time:
0.5 sec
Cone Voltage:
140 V
Nebulizer Gas:
Nitrogen at 75 psi
