New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database

F. Basilico, D. Di Silvestre, S. Sedini, A. Petretto, I. Levreri, G. Melioli, C. Farina, F. Mori, P. L. Mauri

Research output: Contribution to journalArticle

Abstract

The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a 'step-by-step' procedure, we have developed a 'single step' approach for the identification of Hb variants present in biological samples. This is based on the μHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin.

Original languageEnglish
Pages (from-to)288-292
Number of pages5
JournalJournal of Mass Spectrometry
Volume42
Issue number3
DOIs
Publication statusPublished - Mar 2007

Keywords

  • Hemoglobin variants
  • LC-MS/MS
  • SEQUEST
  • Tryptic peptide database

ASJC Scopus subject areas

  • Biophysics
  • Organic Chemistry
  • Spectroscopy

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  • Cite this

    Basilico, F., Di Silvestre, D., Sedini, S., Petretto, A., Levreri, I., Melioli, G., Farina, C., Mori, F., & Mauri, P. L. (2007). New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database. Journal of Mass Spectrometry, 42(3), 288-292. https://doi.org/10.1002/jms.1160