Comparative mapping of recombinant proteins and glycoproteins by plasma desorption and matrix-assisted laser desorption/ionization mass spectrometry.
The mass spectrometric (MS) techniques of 252Cf-plasma desorption (PD) and matrix-assisted laser desorption/ionization (MALDI) are compared in the molecular weight determination and the mapping analysis of several recombinant proteins and glycoproteins. MALDI MS analysis exhibited better sensitivity and mass measurement accuracy and a remarkably short analysis time compared with PD MS analysis. The latter was not successful in the analysis of rhIFN-gamma and the higher mass mammalian cell-derived IL-5 glycoproteins. Mapping of the Escherichia coli-derived rhIFN alpha-2b and rhIL-4 proteins, by direct PD or MALDI MS analysis of the trypsin-generated peptide mixtures provided signals for ca. 95% and 88% of the expected tryptic peptides, respectively. Peptide signals below m/z 1500 were generally more intense in the PD mass spectra, while higher mass signals were more intense in the MALDI mass spectra. Both PD and MALDI MS analyses provided a rapid confirmation of the existing two and three disulfide bonds in the rhIFN alpha-2b and rhIL-4 proteins, respectively. In the mapping of the CHO IL-4 glycoprotein, detection of the trypsin-generated glycopeptides was only possible by MALDI, where their detection was greatly improved by using the super-DHB (sDHB) matrix, a 9:1 mixture of 2,5-dihydroxybenzoic acid (DHB) with 2-hydroxy-5-methoxybenzoic acid. This sDHB matrix also generated significantly enhanced and better resolved MALDI peptide signals, which in turn resulted in a much improved mass measurement accuracy.
Tsarbopoulos
A
A
Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
Karas
M
M
Strupat
K
K
Pramanik
B N
BN
Nagabhushan
T L
TL
Hillenkamp
F
F
D000818:Animals; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007834:Lasers; D013058:Mass Spectrometry; D051379:Mice; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis*
D006023:Glycoproteins; D011994:Recombinant Proteins