Tryptic mapping of recombinant proteins by matrix-assisted laser desorption/ionization mass spectrometry.
Various matrix mixtures have been used for matrix-assisted laser desorption/ionization mass spectrometry to characterize the tryptic maps of recombinant human growth hormone (rhGH) and recombinant human tissue plasminogen activator (rt-PA). Carbohydrate-containing comatrices give improved results over single-component matrices. Of those studied, fucose plus 2,5-dihydroxybenzoic acid (DHB) produced a signal for 24 out of 25, or 96%, of the tryptic peptides of rhGH in a single spectrum. These results were obtained for analyses of as little as 280 fmol of unfractionated material measured in digestion buffer. Analysis of 150 fmol showed a decrease in the relative abundance of higher molecular weight peptides. The incorporation of 5-methoxysalicylic acid (5MSA) as a comatrix in a molar ratio of analyte:fucose:DHB:5MSA = 1:5000:5000:50 gave signals for 45 out of 51 peptides for 4.5 pmol of a tryptic digest of rt-PA, corresponding to 88% of the expected fragments. Unobserved peptides were typically di- and tripeptides. Three glycopeptides were observed with peaks corresponding to the known major glycoforms. The fucose/DHB and 5MSA/DHB comatrices produced significant enhancements in spectral quality over DHB alone, including suppression of matrix peaks, increased ion signal, improved resolution, increased number of useful laser shots per crystal, and minimization of baseline slope. Spectra obtained with fucose/DHB generally surpassed DHB/5MSA in quality, though both matrix mixtures were clearly superior to neat DHB. Fucose/DHB demonstrated an increase in tolerance to ionic contaminants by producing a 10-fold reduction in the abundance of (M + Na)+ions. A trimatrix, DHB/5MSA/fucose, produced the highest quality spectra to date, although only marginally better than the fucose/DHB comatrix.
Billeci
T M
TM
Protein Chemistry Department, Genentech, Inc., South San Francisco, California 94080.
Stults
J T
JT
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D013006:Growth Hormone / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry*; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D014357:Trypsin
D011994:Recombinant Proteins; D013006:Growth Hormone; D014357:Trypsin; D010959:Tissue Plasminogen Activator