The main problems associated with matrix-assisted laser desorption/ionization (MALDI) quantitative analysis are poor shot-to-shot reproducibility, crystal inhomogeneity, signal degradation, and data acquisition system nonlinearity. These problems are addressed in this paper. The instrumental setup was modified to obtain required linearity and measurement accuracy over a dynamic range

The protocol and various matrices were examined for quantification of biomolecules in both the low ca. 1200 amu and mid mass 6000-12000 amu ranges using an internal standard. Comparative studies of different matrices of MALDI quantitative analysis showed that the best accuracy and standard curve linearity were obtained for two matrices: (a) 2,5-dihydroxybenzoic acid (DHB) combined with a comatrix of fucose and 5-methoxysalicylic acid (MSA) and (b) ferulic acid/fucose. In the low mass range, the quantitative limit was in the 30 fmol range and in the mid mass range the quantitative limit was in the 250 fmol range. Linear response was observed over 2-3 decades of analyte concentration. The relative error of the standard curve slope was 1.3-1.8% with correlation coefficients of 0.996-0.998.The main problem for quantitative measurement was suppression of the signal of the less concentrated component (analyte or internal standard) by the more concentrated component. The effect was identified with saturation of the matrix by the analyte. The threshold of matrix saturation was found to be in the range of 1/(3000-5000) analyte/matrix molar ratio. To avoid matrix saturation the (analyte+internal standard) to matrix molar ratio should be below this threshold. Thus the internal standard concentration should be as low as possible.DHB/MSA/fucose and ferulic acid/fucose matrices demonstrated good accuracy and linearity for standard curves even when the internal standard had chemical properties different from the analyte. However, use of an internal standard with different chemical properties requires highly stable instrumental parameters as well as constant (analyte+internal standard)/matrix molar ratio for all samples.

No abstract

No abstract

Quantitative analysis of peptides using internal standards was carried out with the use of matrix-assisted laser desorption/ionization (MALDI) on a time-of-flight mass spectrometer in a ferulic acid matrix. Spectra were collected and averaged over several areas of the sample surface at constant laser energy, and a defocused beam was used to improve reproducibility. A calibration curve was plotted, showing a correlation coefficient of 0.992 and a relative standard deviation of 1.83%. The accuracy and precision of this technique were sensitive to the crystal structure of the matrix and saturation effects of the instrument's detector.