Clinical Chemistry
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Clinical Chemistry 51: 1300, 2005; 10.1373/clinchem.2005.050971
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(Clinical Chemistry. 2005;51:1300.)
© 2005 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry - Reply

Eric T. Fung

Ciphergen Biosystems, Inc., 6611 Dumbarton Circle, Fremont, CA 94555, Fax 510-505-2101

aE-mail efung{at}ciphergen.com


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)(2)(3)(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 ProteinChip 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.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  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;50:1544-1552.[Abstract/Free Full Text]
  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.[CrossRef][Medline] [Order article via Infotrieve]
  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;50:1939-1942.[Free Full Text]




This Article
Right arrow Extract Freely available
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PubMed
Right arrow Articles by Fung, E. T.
Related Collections
Right arrow Proteomics and Protein Markers
Right arrow Endocrinology and Metabolism

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