NDLI: Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

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A study of the distribution of aluminium in human placental tissues based on alkaline solubilization with determination by electrothermal atomic absorption spectrometry

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Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

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Metallomics Vol - 2 Issue - 8

Metallomics Vol - 2 Issue - 7

Metallomics Vol - 2 Issue - 6

Metallomics Vol - 2 Issue - 5

Metallomics Vol - 2 Issue - 4

Metallomics Vol - 2 Issue - 3

Metallomics Vol - 2 Issue - 2

Metallomics Vol -1

Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

Content Provider	Royal Society of Chemistry (RSC)
Author	Blanco-González, E. Sanz-Medel, A. Rubio, A. Mudarra Montes-Bayón, M.
Copyright Year	2010
Abstract	A sample preparation strategy for the determination of the Fe-containing enzyme catalase (CAT) by Fe specific monitoring in human erythrocytes has been optimized. For this purpose, the combined use of elemental mass spectrometry (via inductively coupled plasma, ICP-MS), molecular mass spectrometry (via MALDI-TOF) and enzymatic activity measurements has been required. The procedure involved haemoglobin precipitation from cell lysate with a solution of ethanol–chloroform and preconcentration of the supernatant by using a Speed-Vac concentrator. Catalase recoveries of about 88 ± 15% could be measured by monitoring the protein enzymatic activity before and after precipitation. Further fractionation of Fe-containing proteins from the preconcentrated extract was achieved by size exclusion chromatography (Superdex 200) with a mobile phase of ammonium acetate (0.05 M, pH 7.4) coupled to ICP-MS (Fe monitoring) and UV/VIS detection (specific absorption of the heme-group at 408 nm). A second dimensional chromatography of the CAT-positive activity fraction was carried out by anion-exchange chromatography (Mono Q 5/50) using for elution a linear gradient of ammonium acetate (0–0.750 M in 15 min). This second step revealed a single Fe-containing species in the chromatogram and permitted the unambiguous characterization of the CAT in such fractions by MALDI-TOF. Column recoveries were evaluated and were quantitative, in terms of Fe bound to protein and CAT activity.
Starting Page	638
Ending Page	645
Page Count	8
File Format	HTM / HTML PDF
ISSN	17565901
Volume Number	2
Issue Number	9
Journal	Metallomics
DOI	10.1039/c0mt00003e
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Ammonium Enzyme Red blood cell Monaural Protein Chromatography Inductively coupled plasma Catalase Ionization Hemoglobin PH Anion-exchange chromatography Vac Size-exclusion chromatography Mass spectrometry
Content Type	Text
Resource Type	Article
Subject	Chemistry Medicine Metals and Alloys Biochemistry Biomaterials Biophysics

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Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

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