TuO-13


GRIDLESS DELAYED EXTRACTION MALDI-TOF SPECTRA OF LARGER PROTEINS

Detlev Suckau, Anja Resemann, Klaus Köster, Uwe Rapp and Armin Holle

Bruker-Franzen Analytik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
email: bfasoft!ds@germany.eu.net


The analysis of large molecules beyond ca. 20 kDa by mass spectrometric techniques is complicated by the formation of adducts. Although salt adducts are not as problematic for Matrix-Assisted Laser Desorption/Ionization (MALDI) spectra as they are for ESI, salts and matrix adducts can cause major mass uncertainties in the analysis of larger proteins. E.g., the well-known MALDI matrix sinapic acid causes increasing abundances of MH+ + n*206 Da photo-adducts at increasing masses. This prevents the observation of clearly resolved molecular ions and limits the mass accuracy for proteins like BSA to about 0.1 %. ln addition, the capability to resolve individual components in heterogeneous samples like glycoproteins or chemically modified proteins is rather limited in the "classical" continuous extraction (CE) MALDI-TOF experiment.

The "rediscovery" of "delayed extraction" (DE) by Brown et al. dramatically improved the resolving powers and mass accuracies obtained for small molecules (< 8000 Da), which can be isotopically resolved in MALDI-TOF instruments equipped with reflector. We demonstrate in this work the effects of gridless delayed extraction (GDE) in the analysis of larger molecules especially in the mass range of 20-50 kDa. Both mass accuracy and resolving power benefit largely as compared to CE.

The linear GDE-MALDI-TOF spectrum of Protein A (MW 44.6 kDa) provided a resolving power of 600 of the MH+ which allowed its clear separation from adducts (see Fig.). However, the choice of calibrant was found to be fundamentally critical for proper calibration. The main reason for calibration inaccuracies is the different extent of adduct formation of calibrant vs. analyte and, more importantly, the different ability to resolve the adducts on these signals.

Mass Spectrum BSA was clearly inappropriate to calibrate proteins in the mass range 20-50 kDa because sinapic acid adducts could not be resolved. However, using monomer and dimer signals of trypsinogen the molecular weight of Protein A was determined to be MW=44608±9 Da (n=5) with a mass error of 4 Da (0.01 %). This protein promises to be a well suited calibrant for medium sized proteins under delayed extraction conditions.

Furthermore the capability to resolve individual components of protein mixtures with narrow molecular weight distributions was evaluated in GDE-MALDI experiments with proteins, which were chemically modified.