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Comparing the efficiencies of hydrazide labels in the study of protein carbonylation in human serum albumin

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Abstract

In this work, we establish a methodology for comparing the efficiencies of different hydrazide labels for detecting protein carbonyls. We have chosen acrolein-modified human serum albumin as a model. This system provides a convenient means of reproducibly generating carbonylated protein. Five hydrazide-based labels were tested. Three carry a biotin affinity tag, and the others are simple fatty acid hydrazides. For the biotin-based labels, the yield of the labeling reaction varies considerably, and the most commonly used label, biotin hydrazide, gives the lowest yield. The total tandem mass spectrometry (MS/MS) spectrum counts of modified peptides are similar for all of the biotin-based tags, indicating that factors beyond the labeling efficiency are important in determining the effectiveness of the label. In addition, there is a large variation in the number of spectra obtained for specific, modified peptides depending on the nature of the labeling group. This variation implies that the relative detectability of a particular modification site is highly dependent on the tagging reagent, and more importantly, titration schemes aimed at identifying the most reactive site based on its threshold concentration will be biased by the choice of tagging reagent. The fatty acid hydrazides are somewhat more effective than the biotin-based hydrazides in generating identifiable MS/MS spectra but offer no opportunity for enrichment. For the biotin-based tags, avidin affinity chromatography was used with the tryptic digests, and each tag led to similar enrichment levels.

Spectrum counts for HSA peptides modified by acrolein and labeled with different hydrazide tags

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Acknowledgments

We would like to thank Dr. Simpson for his assistance in the implementation of the DNPH protocol. We acknowledge the support of the Virginia Commonwealth University and the National Institute of Health (1R01AG034167).

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  1. Department of Chemistry, Virginia Commonwealth University, 1001 West Main St., P.O. Box 842006, Richmond, VA, 23284-2006, USA

    Zafer Ugur, Chelsea M. Coffey & Scott Gronert

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  1. Zafer Ugur
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  2. Chelsea M. Coffey
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  3. Scott Gronert
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Correspondence to Scott Gronert.

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Ugur, Z., Coffey, C.M. & Gronert, S. Comparing the efficiencies of hydrazide labels in the study of protein carbonylation in human serum albumin. Anal Bioanal Chem 404, 1399–1411 (2012). https://doi.org/10.1007/s00216-012-6235-9

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  • DOI: https://doi.org/10.1007/s00216-012-6235-9

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