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J Biomol Tech. 2007 February; 18(1): 74.
PMCID: PMC2291912

P213-T Comparison of Methods for Hemoglobin Variant Analysis that Provide a High Degree of Automation


Many of the available methods for the analysis of hemoglobin using mass spectrometry rely upon mathematical reconstruction of the data to generate information about the different isoforms. Although these produce the ability to speciate fine mass differences between various isoforms, they are computationally intensive and time consuming. These forms of mathematical reconstruction can lead to artifacts in the reconstructed data, which may interfere with the accurate quantification of the different forms.

This poster presents data evaluating a number of different methods for the determination of levels of modified hemoglobin. These include protein reconstruction, selected reaction monitoring (SRM) of protein charge states, and multireaction monitoring (MRM) evaluation of the specific modified peptides. Using SRM screening, a number of different charge states have been monitored, which allows for shifts in the protein envelope due to differences in buffer composition or due to variations in the protein concentration. This showed a low degree of specificity when compared to the use of protein reconstruction. The use of peptide MRM with subsequent MS/MS provided the best specificity, removing any ambiguity in the data. This also provided the best route for automation using a liquid chromatography system and the Analyst Software 1.4.2 quantification module. This allows a fully automated workflow utilizing tools that provide features that assist users with good laboratory practices compliance.

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