Increased Resolution in Peptide Separations using UPLC™

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Beth L. Gillece-Castro;Ziling Lu;Thomas E. Wheat;and Jeffrey R. Mazzeo [Waters]
Second Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries, Practical MS
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Practical MS
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Peptide maps are used for protein characterization, identity testing, purity assays, and sequence confirmation with LC/MS. More highly resolving peptide mapping without sacrificing speed or sensitivity is required for developing well-characterized biopharmaceuticals. This demanding analytical technique requires extreme resolution. HPLC peptide maps are usually developed using relatively long columns, 150 to 250mm, with small particle packing materials, 2.5 to 3.5µm. The gradients are extremely shallow, often 1-3 hours. It is still difficult to confirm that all the variants have been separated. There is, therefore, a need for greater resolution. The new category of separation science, UltraPerformance LC™ (UPLC™), may apply to this analytical problem. This technology takes advantage sub-2µm particles using an instrument optimized for such columns. UPLC improves resolution, speed, and sensitivity for many HPLC methods. The physical basis of this technique is defined in the van Deemter equation and is explicitly related to reduced diffusion distances. Peptides, however, exist over a range of sizes and chemical properties such that the principles of UPLC™ do not apply to peptides in the same way as for smaller molecules. The relatively low diffusion rate of peptides presents the major complexity. We have examined the diffusion-related variables that affect peptide chromatography. Linear velocity and gradient slope were varied independently to find optimum resolution. The samples include are tryptic digests of standard proteins. The set of peptides, therefore, ranges up to about 25 residues over a wide range of polarities. Oxidized and deamidated analogues were produced with forced degradation. Glycopeptides were also examined. Separations were monitored with oa-Tof MS to track the individual peaks. Ultra Performance LC™ improves resolution for peptide mapping. Deamidated peptides, including isoforms, are readily resolved. The glycopeptides give increased resolution symmetrical peak shapes. This experimental approach leads to increased resolution, sensitivity, and speed in peptide mapping.

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