RP-HPLC tryptic mapping of IgG1 proteins with post-column fluorescence derivatization

Peptide mapping is an important analytical technique widely used to study t he primary structure of proteins. In quality control settings, it is employ ed as an identity test to probe for small changes in protein primary struct ure. A great challenge in peptide mapping is to minimize the detection limi t for peptides due to the low detectability of smaller peptides based on th eir ultraviolet absorbance. The detection of peptide fragments can be enhan ced by pre-or post-column derivatization with fluorescent tags. The use of post-column o-pthalaldehyde (OPA) and fluorescamine chemistries for on-line derivatization of peptide fragments from the RP-HPLC tryptic maps of sever al IgG1 monoclonal antibodies was explored. This paper describes the simple and sensitive peptide mapping technique for structural confirmation of pro teins using picomoles of samples by post-column fluorescence derivatization . A comparison of UV and fluorescence detection of a peptide map is also pr esented. The method includes post column OPA derivatization of tryptic pept ides from RP-HPLC tryptic maps with fluorescence detection. The conclusion reached that fluorescence detection gave relative detectability for tryptic peptides that range from 10- to 100-fold better than those observed with U V detection. The sensitivity of the peptide map increased by about 200-500 fold, i.e. peptide maps could be obtained using 2-5 pmol of digest instead of 1 nmol of digest. A roughly equal fluorescence response for all peptides (equal peak areas) was generally observed. (C) 1999 Elsevier Science B.V. All rights reserved.