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Biology

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A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

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20:23 min

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October 1st, 2009

DOI :

October 1st, 2009

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Eric Johansen¹, Birgit Schilling², Michael Lerch¹, Richard K. Niles¹, Haichuan Liu¹, Bensheng Li², Simon Allen¹, Steven C. Hall¹, H. Ewa Witkowska¹, Fred E. Regnier³, Bradford W. Gibson², Susan J. Fisher¹, Penelope M. Drake¹

¹Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, ²Buck Institute for Age Research, ³Department of Chemistry, Purdue University

Summary

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Chapters in this video

0:00

Title

0:55

Introduction

1:42

Preparing Lectin-Conjugated POROS Columns

5:15

Packing the Lectin-POROS Beads into a PEEK Column

8:11

HPLC of Samples

11:49

Desalting the Collected Fractions and PNGase F-digestion

14:50

Ziptip Desalting of PNGaseF Samples

16:34

Glycopeptide Analysis Representative Results

19:45

Conclusion

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