Anal. Chem. 19B1, 63, 2909-2915
2909
Characterization of the Tryptic Map of Recombinant DNA Derived Tissue Plasminogen Activator by High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry Victor Ling, Andrew W. Guzzetta, Eleanor Canova-Davis, John T. Stults, and William S. Hancock* Genentech, Znc., 460 Point S a n Bruno Boulevard, South S a n Francisco, California 94080
Thomas R. Covey and Bori I. Shushan Sciex, 55 Glen Cameron Road, Thornhill, Ontario, Canada L3T 1P2
A detaned tryptic map Is presented for recombinant human tiswe plasminogen actlvator (rt-PA). Electrospray Ionization mass spectrometry b utlllzed as an on-line HPLC detector for tryptic mapplng of thls glycoproteln. The additional dhnenslon provkted by mass spectrometry glves conskierably more detail about the complex tryptk map and slgnlfkantly enhances the high-resolution chromatographic separatlon by dlstlngulshlng by mass any coelutlng components. Through this Improvement, the proilne Isomers of a tryptic peptide were observed W n g over a broad range of retentlon tlmes. The glycopeptides of &PA are observed as well as any corresponding nonglycosyiated peptides. I n addltlon, the carbohydrate heterogeneity Is readily observed, ailowlng analysis of the carbohydrate composttion. The characteristic diagonal patterns formed by gtycopeptldes In a contour plot of the data allow rapid recognition of the glycopeptides.
INTRODUCTION Extensive structural characterization of proteins, particularly those produced by recombinant DNA techniques, has become an important aspect of pharmaceutical research, since detailed knowledge of the primary structure is required as part of the licensing process for proteins to be used as therapeutics. Detailed structural analysis is also necessary in order that minor variant proteins may be identified. Reversed-phase high-performance liquid chromatography (RP-HPLC) has been shown to give high-resolution separation of tryptic digests of proteins ( 1 ) and has been applied to the characterization of a number of recombinant proteins (2-6). This analytical technique has played a major role in the characterization and quality control of protein pharmaceuticals manufactured by recombinant DNA technology (7). However, it was recently recognized that tryptic mapping has some limitations (8), principally with sensitivity to low levels of variant sequences, especially if coelution of a diagnostic peptide occurs. Tryptic mapping conventionally utilizes RP-HPLC separation with UV-absorbance detection, typically operated at 214 nm. Mass spectrometric detection has been shown to provide an extra dimension of information (mass-to-charge ratio) that is invaluable for identifying the eluting components. The most commonly used interfaces for on-line liquid chromatography-mass spectrometry (LC-MS) are thermospray (9, 10) and continuous-flow fast atom bombardment (flowFAB) (11-13). Thermospray ionization is useful for peptides as large as 2000 Da and in quantities as small as 100 pmol.
* Corresponding author. 0003-2700/91/0363-2909$02.50/0
Flow-FAB has been demonstrated for peptides as large as 5OOO Da and in quantities as small as 5 pmol. Detection of small molecules (
