Investigating the Quantitative Nature of MALDI-TOF MS

Abstract

MALDI-TOF MS has been applied by several groups to relative quantitative measurements. At the same time, the non-quantitative character of this method has been widely reported. We conducted experiments to test the reliability of this technique for quantitation using the statistical method of inverse confidence limit calculation for the first time in this context. The relationship between relative intensities of known amounts of standard peptides and their concentration ratios was investigated. We found that the concentration ratios determined by relative intensity measurements were highly inaccurate and strongly influenced by the molecular milieu of the sample analyzed. Thus, we emphasize the necessity of using the sample itself for calibration. We also performed experiments using an isotope-labeled derivative of the analyte as an internal standard for calibration line generation. As expected, the use of such standard led to a dramatic increase in precision and a less pronounced improvement in accuracy. We recommend performing a similar statistical analysis as a demonstration of reliability for every system where MALDI-TOF MS is used for quantitative measurements. Molecular & Cellular Proteomics 7: 2410 -2418, 2008.MALDI-TOF MS is a widely used analytical technique because of its excellent sensitivity, relatively high speed, and simplicity. As a consequence of producing mostly singly charged ions (1, 2) MALDI is especially suitable for mixture analysis. It is commonly used in proteomic studies (3) frequently without any prefractionation (4) as well as in the analysis of other biomolecules. Lately MALDI-TOF MS has become a popular method for quantitative analysis of biomolecules (oligonucleotides, proteins, glycoproteins, etc.) originating from various sample types (5-22) or even for imaging tissue sections (13)(14)(15)(16)(17)(18)(23)(24)(25)(26). At the same time, several studies discuss the non-quantitative nature of MALDI-TOF MS (7, 8, 13, 19, 22, 26 -30). The main problem of MALDI is its poor reproducibility (sample to sample and shot to shot), which makes most quantitative measurements quite difficult at best (19,27,31). This is especially true for complex samples. The presence of so-called hot spots or sweet spots is the main cause of the poor reproducibility and is therefore a factor strongly increasing measurement times and complicating automated measurements. Furthermore this phenomenon is one of the major factors hampering the use of MALDI MS for spatially resolved imaging (27). The reasons for sweet spot formation are not completely understood. It could be caused by variation in the amount and the crystallization of the matrix, the latter being a function of local analyte concentration. Dependence of signal intensities on the orientation of the matrix-analyte crystals relative to the spectrometer axis is also possible (27). The extent of hot spot formation depends strongly on the matrix used. For example, preparations with ␣-cyano-4-hydroxycinnamic acid (CHCA) 1 deliver relatively homogenous samples (...

Detection of Enrofloxacin and Its Metabolite Ciprofloxacin Using Gold Nanoparticles and Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…It is widely accepted that MALDI-TOF MS is not inherently quantitative, and the direct use of mass intensities for quantification is not practical. 32 However, the calibration curve is linear for target concentrations ranging from 0.05 to 50 mg kg -1 as shown in Fig. 4.…”

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

“…It also was found that although the signal intensity of tests performed using CHCA could be increased by adding a precoat of the matrix before sample loading (data for samples where precoating was not performed is not shown), H11 and H21 could be identified and differentiated only by MALDI-TOF using DHB; therefore, it was decided that DHB would be the matrix of choice for MALDI-TOF MS-H. This decision was not impeded by the fact that DHB matrix crystals are clumpy and known to produce hot spots during analysis (23,24), as these manifestations should not affect total signal intensity. Repeated tests summarized in Table 1 indicated that the DHB matrix worked well to identify examined reference strains.…”

Investigations of Some Liquid Matrixes for Analyte Quantification by MALDI

“…Figure 4a and b show the MALDI spectra of 10 pmol each of Y 5 R and Y 6 , respectively, in 25 nmol of solid CHCA. Because CHCA is a 'hot' matrix [1], most of the [Y 6 + H] + dissociated to immonium Y. Including the abundance of this ion, the total abundance of the analyte-derived ions after normalization to the matrix ion abundance is substantial for both peptides.…”

“…Tolerance to the presence of contaminants in a sample [4,5] is often cited as an important advantage of MALDI over electrospray ionization, which is another popular ionization technique for biomolecules. In spite of its popularity, MALDI is not regarded as a useful quantitative tool, mainly because the ion signals produced by MALDI display shot-to-shot variation at spots, spot-tospot variation on samples, and sample-to-sample variation [1,6]. In the MALDI of a solid sample composed of an analyte-matrix mixture, sample inhomogeneity is one of the factors responsible for such variations [7][8][9].…”