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Letters to the Editor |
Department of Clinical Chemistry, University Hospital Maastricht, Maastricht, The Netherlands
aAddress correspondence to this author at: Department of Clinical Chemistry, University Hospital Maastricht, PO Box 5800, Maastricht, 6202 AZ The Netherlands. Fax 31-43-387-4692; e-mail ddb{at}klinchem.azm.nl.
To the Editor:
Recently, the authors of 2 studies have claimed to measure accurately the microheterogeneity of transthyretin (TTR) variants by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) immunoassays (1)(2). Here we report that the mass spectrometric performance in those studies was not adequate to measure the respective TTR variants.
A mass resolution of at least 1000 is required to separate the majority of the currently known posttranslational and mutation-based modifications of TTR (3). Wang et al.(1) performed their analysis at a resolution of 270 [estimated as follows: m/
mFWHM = 13828/52 = 266 in Fig. 1A of their report (1)], whereas Schweigert et al. (2) achieved a resolution of 90 [estimated as follows: m/mFWHM = 13851/158 = 88 in panel A2 of Fig. 2 of their report (2)]. We therefore question whether these studies can justify their reported claims.
To provide arguments, we performed MALDI-TOF MS analysis on a gold chip ProteinChip® array of a commercially available TTR preparation with an instrument typically used in SELDI-TOF MS (PBS IIc analyzer; Ciphergen). We applied internal calibration, using bovine heart cytochrome C and equine apomyoglobin as calibrants. Because pure TTR variants were not available, assignment of the TTR variants to the peaks in the mass spectra was based on the measured m/z values vs reported m/z values of the respective [M + H]+ ions in the literature (3).
The results obtained for the untreated TTR preparation are shown in Fig. 1
, as are the results for the TTR preparation treated with dithiothreitol to reduce all TTR variants containing a disulfide linkage to the native variant (4). Our achieved mass resolutions were in the range of 250–300. The data for the reduced TTR were essential because they confirm the correctness of the applied m/z value assignments by the disappearance and appearance of TTR variants. Moreover, it becomes evident that in the m/z range for native TTR (Fig. 1
, peak c), at least 2 other variants (peaks a and b) overlap, which both may be artifacts of the procedure used to isolate the commercial TTR preparation. Of particular interest is the presence of the sinapinic acid (SPA) adduct of the native TTR (peak c") and cysteinyl-TTR (peak e"). This type of adduct corresponds to [M + (SPA – H2O) + H]+ and is common for SPA (5).
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Although our own achieved resolution was also insufficient to distinguish all TTR variants, it can be stated that with respect to the study of Wang et al. (1), it is doubtful that they could have distinguished the [M + H]+ ion of glutathionyl-TTR (peak g) from that of the [M + (SPA – H2O) + H]+ ion of cysteinyl-TTR (peak e"). The same point of criticism can be made for the report by Schweigert et al. (2), who also could not have distinguished the [M + H]+ ions for sulfonated TTR (peak d), cysteinyl-TTR (peak e), and glycinecysteinyl-TTR (peak f) from each other.
The mass accuracy for the assigned cysteinyl-TTR in our study was <0.03%. The claimed mass accuracies in the studies of Wang et al. (1) and Schweigert et al.(2) for the cysteinyl-TTR variant were 0.38% and 0.22%, respectively, and thus were significantly inferior to our data. One explanation could be the method of calibration, which in our case was internal calibration over a small range and in both studies of interest (1)(2) was external calibration over a wide range. Because identification of the TTR variants was based on m/z value assignment, the method of calibration is crucial. We therefore conclude that the presented low-resolution SELDI-TOF MS immunoassays are not appropriate to distinguish accurately all of the TTR variants.
Our source of TTR was a certified purified preparation, whereas Wang et al. (1) and Schweigert et al.(2) measured their TTR in immunologically enriched serum and plasma, respectively. Moreover, we analyzed the TTR variants in higher concentrations or larger volumes than are routinely found in physiologic or pathologic situations. Consequently, we were able to apply lower laser intensities than, for example, Wang et al. (1) and Schweigert et al. (2) might have done. Use of higher intensities and lower amounts of TTR does not justify inadequate attempts to measure the microheterogeneity of TTR.
References
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-cyano-4-hydrocinnamic acid as energy-absorbing matrix [measured m/z values at 13 723.3 (peak a), 13 759.5 (peak c), 13 840.8 (peak d), 13 880.2 (peak e), 13 933.5 (peak f), and 14 064.7 (peak g)]. Spectrum III, measured spectrum of the TTR preparation after reduction with dithiothreitol and with SPA as energy-absorbing matrix [measured m/z values at 13 726.1 (peak a), 13 761.6 (peak c), 13 840.9 (peak d), and 14 067.8 (peak c")].