Measurement Journal Issue 6

As a peptide hormone produced in the kidneys, EPO stimulates the production of red blood cells and is released in response to decreased levels of oxygen in body tissue. It is a highly glycosylated protein with a 30 to 34 kDa molecular weight, 40 percent of which amounts to carbohydrate chains (Figure 1). The rhEPO is one of the top selling biopharmaceutical products for treating anemia caused by kidney failure. In fact, its amino acid sequence is identical to human endogenous EPO (Figure 2). One of the current EPO tests approved by the International Olympics Committee (IOC) relies on the differences in the glycosylation patterns between the recombinant EPO and the endogenous EPO.3 This test calls for the separation of the different forms of glycoproteins using isoelectric-focusing gel electrophoresis. An anti-EPO antibody helps visualize the separated gel band. Happy collaboration Huwei Liu My relationship with chromatography and Agilent’s (formerly Hewlett-Packard’s ( HP’s)) chromatographic instrumentation began in the early ’80s when I was a college student. It was further cemented in 1990, when I joined Peking University (PKU) after having received my doctorate from the Beijing Institute of Technology. There I met Hongfeng Yin, a Ph.D. student who later graduated and joined Agilent China where he was responsible for setting up a chemical analysis training center for Agilent customers. We formed a strong relationship and, in 1997, Agilent donated four sets of chromatographic instruments that were used to establish the PKU-Agilent (formerly PKU-HP) Chemical Analysis Laboratory. Today, we train Agilent customers in the area of capillary electrophoresis and test their GC and LC columns. Since 2005, PKU has received two Agilent Technologies Foundation grants and I am lucky to serve as the resident mentor for each. Thanks to the flexibility of Agilent management, the happy collaboration between PKU and Agilent continues to ﬂourish. Figure 2. rhEPO, as pictured here in its 3D structure, has led to the disqualiﬁcation of athletes from endurance sports competitions such as cycling. 2 The ﬁrst challenge to overcome is the interaction of protein molecules with the capillary wall. Such interaction can dramatically affect the separation performance. Our ongoing research has found that dynamically-coated, fused-silica capillaries with carboxymethyl chitosan can minimize the protein-to-capillary wall interaction. The second issue that requires attention is the inherent lack of detection sensitivity, when compared with antibody blotting, and the selectivity of the UV detector. A better alternative for high-sensitivity analysis is laser-induced ﬂuorescence detection. Since the EPO molecules are not inherently ﬂuorescent, they must also be derivatized from the sample. Although ﬂuorescein isothiocyanate (FITC) is a common ﬂuorescence reagent and has been used for pre-column labeling of peptides and proteins in the capillary electrophoresis with laser-induced ﬂuorescence detection (CE-LIF) method, we have found that its reaction with rhEPO is slow. In addition, FITC exhibits a high-ﬂuorescence property and pre-capillary derivatization with a higher concentration of FITC results in high background from the excess Closing in on an answer At Peking University’s College of Chemistry and Molecular Engineering, an alternative method of separating the various EPO glycoforms — capillary electrophoresis technology — is now under investigation.4 Capillary electrophoresis is widely used for the separation of protein mixtures based on their ionic charge differences. Due to the high voltage used, the separation process is generally much faster than traditional slab-gel electrophoresis. However, when it comes to using this technology for EPO testing, there are still several issues that must be addressed. 63 Agilent Measurement Journal

Table of Contents Feed for the Digital Edition of Measurement Journal Issue 6

Measurement Journal Issue 6 Using a Few Words to Convey a Deeply-Held Spirit Charting a Unique Path to Technology Innovation Contents Announcing First Commercial GC/MS Metabolites Library Assessing Nonlinear Behavior Testing Combines Form and Function Testing Multiplay Networks Higher Sensitivity LC/MS Easing Military Radio Test Stepping Up Calibration Accuracy Increasing Spectrum Analyzer Bandwidth 3GPP Tool Supports TS 36 Standards The Olympic Story Spotlights Agilent's Continuing Contribution to Technology Innovation Ensuring a Level Playing Field in Competitive Sports GPS: Coming of Age Resolving Design Issues in HSPA Mobile Devices Achieving Accurate Results with Oscilloscope-Based Jitter-Analysis Software RF Handheld Testers Guarantee Traffic Stability Under Olympic-Sized Stress Conditions Utilizing LAN-Based Instrumentation to Measure Total Harmonic Distortion in Remote Facilities IMT-Advanced: 4G Wireless Takes Shape in an Olympic Year EPO Doping: A Speed Engine, Turbo-Charged by Chemistry

For optimal viewing of this digital publication, please enable JavaScript and then refresh the page. If you would like to try to load the digital publication without using Flash Player detection, please click here.