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Efficient chromatographic separation of intact proteins derivatized with a fluorogenic reagent for proteomics analysis
Author(s) -
Ichibangase Tomoko,
Yazawa Itaru,
Imai Kazuhiro
Publication year - 2013
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.2952
Subject(s) - chemistry , chromatography , chromatographic separation , reagent , column (typography) , proteomics , protein purification , glutathione , high performance liquid chromatography , biochemistry , enzyme , organic chemistry , structural engineering , connection (principal bundle) , engineering , gene
To achieve more efficient separation of intact proteins for proteomics applications, three columns of differing diameters (4.0, 4.6 and 6.0 mm internal diameter) were chosen for comparison and investigated to identify optimal conditions. The column with the largest diameter gave the largest peak capacity, showing the efficient separation of intact proteins, such as two protein standards, glutathione S ‐transferase and β ‐lactoglobulin. On the other hand, a low‐molecular‐weight compound was separated effectively on the smaller diameter column, demonstrating that the separation mechanism seems to differ between high‐ and low‐molecular‐weight compounds. Finally, using the 6.0 mm i.d. column, 680 protein peaks were observed in mouse liver extracts, demonstrating that a wider diameter separation column is effective for intact protein separations. Copyright © 2013 John Wiley & Sons, Ltd.