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Inadequate attempts to measure the microheterogeneity of transthyretin by low-resolution mass spectrometry.

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/[DELTA][m.sub.FWHM] = 13828/52 = 266 in Fig. 1A of their report (1)], whereas Schweigert et al. (2) achieved a resolution of 90 [estimated as follows: m/[DELTA][m.sub.FWHM] = 13851 / 158 = 88 in panel A2 of Fig. 2 of their report (2)]. We therefore question whether these studies can justify their reported claims.

[FIGURE 1 OMITTED]

To provide arguments, we performed MALDI-TOF MS analysis on a gold chip ProteinChip[R] 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 - [[H.sub.2]O) + H].sup.+] and is common for SPA (5).

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].sup.+] ion of glutathionyl-TTR (peak g) from that of the [[M + (SPA - [H.sub.2]O) + H].sup.+] 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].sup.+] ions for sulfonated TTR (peak d), cysteinyl-TTR (peak e), and glycinecysteinyl-TTR (peak fl 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

(1.) Wang Z, Yip C, Ying Y, Wang J, Meng XY, Lomas L, et al. Mass spectrometric analysis of protein markers for ovarian cancer. Clin Chem 2004;50: 1939-42.

(2.) Schweigert FJ, Wirth K, Raila J. Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay. Proteome Sci 2004;2:5.

(3.) Bergquist J, Andersen 0, Westman A. Rapid method to characterize mutations in transthyretin in cerebrospinal fluid from familial amyloidotic polyneuropathy patients by use of matrixassisted laser desorption/ionization time-of-flight mass spectrometry. Clin Chem 2000;46: 1293-300.

(4.) Sass J0, Nakanishi T, Sato T, Sperl W, Shimizu A. S-Homocysteinylation of transthyretin is detected in plasma and serum of humans with different types of hyperhomocysteinemia. Biochem Biophys Res Commun 2003;310:242-6.

(5.) Bahr U, Stahl-Zeng E, Gleitsmann E, Karas M. Delayed extraction time-of-flight MALDI mass spectrometry of proteins above 25 000 Da. J Mass Spectrom 1997;32:1111-6.

Douwe de Boer *

Matthias M. Erps

Will K.W.H. Wodzig

Marja P. van Dieijen-Visser

Department of Clinical Chemistry

University Hospital Maastricht

Maastricht, The Netherlands

* Address 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@klinchem.azm.nl.

DOI: 10.1373/clinchem.2005.048108

A representative of Ciphergen Biosystems responds:

To the Editor:

The authors in their letter raise the concern that the PBSIIc ProteinChip reader (Ciphergen) lacks adequate resolution to reproducibly quantify each of the known biological forms of transthyretin, as well as artifactually generated forms of transthyretin (e.g., matrix adducts). The authors claim that a mass resolution (mass-to-width ratio at half peak-height) of at least 1000 is required to separate these forms, whereas the PBSIIc analyzer has a mass resolution between 200 and 300. Both theoretical and experimental data support some of the authors' claims, but not all (1-4).

A mass resolution of 300 enables the separation of peaks with [DELTA]m/z 47 Da or above in the mass range <14 000 Da. The mass differences between the 4 transthyretin isoforms--truncated, unmodified, cysteinylated, and glutathionylated--are well above 45 Da. These forms are well resolved in the PBSIIc system with the exception of the glutathionylated form, which experiences interference from the signal produced by the sinapinic acid matrix adduct of the cysteinylated form, which differs by 20 Da. The PBSIIc was also able to resolve the sulfonated form, which is 39 Da different from the cysteinylated form. The identification, resolution, and quantification of these forms were confirmed experimentally by high-resolution mass spectrometry using the ProteinChip Interface coupled to the QStar instrument (PE Sciex), which has a resolution of >1000.

These comments notwithstanding, we agree with de Boer's argument that better mass resolution will enable more precise quantification of the many forms of transthyretin. As we move toward clinical application of mass spectrometric analysis of proteins, we will need to address these issues rigorously. The Protein-Chip system 4000, which has a mass resolution of 700-1000 in the relevant mass range, is a step in that direction. Assay improvements that more specifically capture forms of clinical relevance will also help address these issues.

References

(1.) Terazaki H, Ando Y, Suhr O, Ohlsson PI, Obayashi K, Yamashita T, et al. Post-translational modification of transthyretin in plasma. Biochem Biophys Res Commun 1998;249:26-30.

(2.) Bergen HR 3rd, Zeldenrust SR, Butz ML, Snow DS, Dyck PJ, Klein CJ, et al. Identification of transthyretin variants by sequential proteomic and genomic analysis. Clin Chem 2004;50: 1544-52.

(3.) Schweigert FJ, Wirth K, Raila J. Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay. Proteome Sci 2004;2:5.

(4.) Wang Z, Yip C, Ying Y, Wang J, Meng XY, Lomas L, et al. Mass spectrometric analysis of protein markers for ovarian cancer. Clin Chem 2004;50: 1939-42.

Eric T. Fung

Ciphergen Biosystems, Inc.

6611 Dumbarton Circle

Fremont, CA 94555

Fax 510-505-2101

E-mail efungCciphergen.com

DOI: 10.1373/clinchem.2005.050971

Drs. Schweigert and Raila also respond to the letter by de Boer et al.:

To the Editor:

In general, we agree with the authors' conclusion that mass resolution in mass spectrometry greatly affects discriminating power. The obtained separation results on the microheterogeneity of transthyretin (TTR) were therefore not surprising and correlate with findings from other studies (1). Both of the reports criticized by the authors claimed only to separate the most prominent variants of TTR, namely the native, cysteinylated, and glutathionylated forms, which differ in mass by ~120 and ~190 Da. Other mass signals produced by modifications that lead to smaller mass changes were not claimed to be detectable.

The basic difference in the 2 reports lies in the fact that Wang et al. (2) used a modified immunoprecipitation method, whereas we applied an on-chip immunocapture format (3). The point in using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) instead of matrix-assisted laser desorption/ionization (MALDI)-TOF MS, with its higher resolution, is the capability of SELDI to enrich low-abundance proteins from complex matrices such as plasma, through, for example, the coupling of specific antibodies to the chip surfaces (4). Such an approach is an important methodologic aspect in "second phase" proteomics, which are characterized by repetitive investigation of the same protein to validate the protein phenotype in large population-based studies. This provides a basis for diagnostic progress in personalized medicine (5). For TTR, such an approach is relevant not only in the diagnosis of TTR-related amyloidosis but also in other diseases.

The true problem with on-chip immunopurification is not the resolution of the MS, which can be solved by use of specific available interfaces, but the efficient coupling of the antibody to the chip surface. When an on-chip immunoassay format is being used, it is important that the protein chip retains the antibody in an active state at high density. Results are greatly affected by functionality characteristics, such as the stability, affinity, and specificity of the antibody. On the basis of studies relating to microarrays, only 5%-20% of commercially available antibodies are suitable for one or the other microarray format (6).

References

(1.) Theberge R, Connors LH, Skinner M, Costello CE. Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: a clinical application of mass spectrometry. J Am Soc Mass Spectrom 2000;11:172-5.

(2.) Wang Z, Yip C, Ying Y, Wang J, Meng XY, Lomas L, et al. Mass spectrometric analysis of protein markers for ovarian cancer. Clin Chem 2004;50: 1939-42.

(3.) Schweigert FJ, Wirth K, Raila J. Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay. Proteome Sci 2004;2:5.

(4.) Schweigert FJ. Characterization of protein microheterogeneity using mass spectrometry based immunoassays. Brief Funct Genomic Proteomic 2005;4:7-15.

(5.) Hanash S. Disease proteomics. Nature 2003; 422:226-32.

(6.) MacBeath G. Protein microarrays and proteomics. Nat Genet 2002;32:526-32.

Florian J. Schweigert * Jens Raila

Department of Physiology and Pathophysiology

University of Potsdam

Potsdam-Rehbrucke, Germany

* Address correspondence to this author at: Institute of Nutritional Science, University of Potsdam, Scheunert-Allee 114-116, Potsdam-Rehbrucke, D-14558 Germany. E-mail fjschwei@rz.uni-potsdam. de.

DOI: 10.1373/clinchem.2005.051714
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Title Annotation:Letters
Author:de Boer, Douwe; Erps, Matthias M.; Wodzig, Will K.W.H.; van Dieijen-Visser, Marja P.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jul 1, 2005
Words:1911
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