Traditionally, MALDI-TOF mass spectrometry has been used very little for quantitative studies. Analysts have usually assumed that potential differences in ionization efficiency between analyte and internal standards and an inherent heterogeneity of the sample spot would limit MALDI-TOF mass spectrometry as a quantitative technique. In addition, conventional MALDI-TOF spectral acquisition has not allowed rejection of saturated peaks which, when averaged with unsaturated peaks, do not provide peak areas or heights suitable for quantitation. However, with the introduction of sophisticated software and high precision spotting robots, the use of MALDI-TOF mass spectrometry for small organic molecule and peptide quantitation has become a viable analytical procedure. Robotic or manual sample preparation combined with automated MALDI-TOF mass spectrometry, using single-shot averaging, is now a viable method for quantitation of small organic molecules and peptides.
Automatic and intelligent spectra acquisition and evaluation
Acceptable calibration curve linearity from robotic or manual sample preparation
Visual Basic for Applications macro for quantitative analysis
Small Organic Molecule Quantitation
Stock standard solutions were prepared in ethanol. Working analyte (4-acetoamidophenol) standard solutions were prepared by serial dilution of the stock analyte solutions with acetonitrile/water 50:50 (v/v). Working internal standard (theophylline) solutions were prepared directly with matrix solution (2 g/L DHB prepared in 50% acetonitrile/50% 0.1% TFA). Equal volumes of the working and internal standard solutions were mixed. The SymBiot I robot was used to spot 0.5 μL of each solution in triplicate onto the MAL DI-TOF target plate. After the sample spots had dried completely, each one was overlayed with 0.2 μL of acetonitrile. This produced a thin film of homogeneous crystals.
Stock solutions containing 100 μM internal standard (FLEEV) and analyte (FLEEI) concentration ranging from 5 to 900 μM were prepared in 50% acetonitrile. Working solutions were diluted by a factor of 10 with matrix. The matrix was prepared by mixing 3 parts of 13.3 mM ammonium citrate (50% acetonitrile) with one part of 10 g/L THAP (100% acetonitrile). Spotting procedure: One microliter of 10 g/L THAP (100% acetonitrile) was spotted by hand and allowed to crystallize. Next, 1.5 μL of each working solution was manually deposited onto the dried matrix and allowed to air dry.
Automatic Single-Shot Averaging Spectrum Acquisition
For each sample, a Voyager-DE PRO Workstation was used to automatically acquire 100 single-shot MALDI-TOF mass spectra in linear, positive ion mode.
The spectra were evaluated and averaged in the following manner:
The laser power was automatically adjusted to ensure no saturation of the analyte or internal standard peaks (Figure 1). Evaluation of single-shot mass spectra for averaging was done automatically by the acquisition software. The criteria for acceptance were based on minimum and maximum peak height limits that satisfied a signal-to-noise (S/N) standard relative to the base peak intensity. The S/N criterion was included to reject spurious peaks resulting from sodium and potassium adducts and noise spikes. A single-shot spectrum was accepted and averaged only if all of the acceptance criteria illustrated in Figure 1 were satisfied. Analyte/internal standard peak area ratios were calculated as response factors. Response factors were automatically plotted as a function of analyte concentration us ing a macro coded in Visual Basic for Applications.
Results and Discussion
Figures 2 and 3 illustrate the calibration curves obtained for 4-acetoamidophenol (r2 = 0.9995) and the tetrapeptide FLEEV (r2 = 0.9976) demonstrating excellent linearity over the concentration range analyzed. A reproducibility study was performed using 4-acetoamidophenol at 1.5 and 0.38 μg/mL. Figure 4 illustrates the high precision of single-shot averaging at both concentrations for 25 consecutive measures of the response factor. Each measure was calculated from 100 single-shot spectra averaged together.
There are a number of points to consider in performing small molecule quantitation with MALDI-TOF. First, choose an internal standard close to the mass of the molecule being quantitated. Note that structural resemblance between analyte and internal standard is not necessary to achieve quantitative results. We recommend that you acquire and average a large number of single-shot spectra. Also, you should limit the acceptable analyte and internal standard signal intensity to below saturation. Sample spotting that favors formation of homogeneous crystals (e.g. overlay sample spots with acetonitrile) should also be employed. Lastly, it isnt necessary to use robotic spotting to achieve excellent standard curve linearity.