High-Resolution Cation-Exchange
Alternative to Peptide Mapping for
Protein ID and QA/QC
Dai Zhenyu,
1
Xu Qun,
1
and Jeffrey Rohrer
2
1
Thermo Fisher Scientific, Shanghai, People’s Republic of China;
2
Thermo Fisher Scientific, Sunnyvale, CA, USA
Application Update 188
Key Words
Ion Exchange, ProPac SCX-10 Column, MabPac SCX-10 Column,
Fast Separation
Goal
To develop an efficient and high-resolution peptide mapping
method in cation-exchange mode as an alternative to
reversed-phase separation
Introduction
Peptide mapping is commonly used to demonstrate
protein identity. In later phases of pharmaceutical
development and in quality assurance/control (QA/QC),
peptide mapping with UV detection of the protein drug
serves as a primary protein QC method. Although a
reversed-phase separation is the typical choice for
separating peptides, high-resolution ion-exchange
chromatography is an alternate method that provides
additional information and a different selectivity.
In proteomics applications, strong cation-exchange
chromatography is routinely the choice for the first
dimension separation of peptides.
1
The Thermo Scientific ProPac SCX-10 and MabPac SCX-10
Analytical Columns are constructed by grafting cation-
exchange groups onto a nonporous polymeric particle
that has a hydrophilic coating. This construction results
in high-efficiency peaks and high-resolution separations
of protein variants.
2
The work shown here demonstrates
that strong cation-exchange chromatography can
provide good resolution for peptide mapping, and thereby
serve as an alternate or supplementary method for
separating peptides.
Equipment
• Thermo Scientific Dionex UltiMate 3000 ×2 Dual
Biocompatible Analytical Liquid Chromatography (LC)
System, including:
– DGP-3600BM Biocompatible Dual-Gradient Micro
Pump with SRD-3600 Integrated Solvent and
Degasser Rack
– WPS-3000TBFC Thermostatted Biocompatible
Pulled-Loop Well Plate Autosampler with Integrated
Fraction Collection
– TCC-3000SD Thermostatted Column Compartment
– DAD-3000 Diode Array Detector
• Thermo Scientific Dionex Chromeleon
Chromatography Data System software version 6.80,
SR9 or higher
• Thermo Scientific Orion 2-Star Benchtop pH Meter
Reagents and Standards
• Deionized (DI) water, 18.2 MΩ-cm resistivity
• Acetonitrile (CH
3
CN), HPLC grade (Fisher Scientific
P/N AC610010040)
• Formic acid, analytical grade, SCRC, China
• Sodium perchlorate, analytical grade, SCRC, China
• Triethylamine, HPLC grade, CNW Technologies
GmbH, Germany
• Trypsin from bovine pancreas (Sigma-Aldrich
®
P/N T1426)
• Myoglobin from equine heart (Sigma-Aldrich
P/N M1882)
• DL-Dithiothreitol, ≥99% (Fluka P/N 43819,
Sigma-Aldrich)
• Iodoacetamide, ≥99% (Sigma-Aldrich P/N I6125)
