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LC–MS of Metmyoglobin at pH = 2. Separation and Characterization of Apomyoglobin and Heme by ESI–MS and UV–Vis
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Abstract
This article describes an experiment employing LC–MS where metmyoglobin is denatured to apomyoglobin and heme in a mobile phase gradient of acetonitrile H2O with 0.1% trifluoroacetic acid. The apomyoglobin is separated from the heme group by reversed-phase chromatography on a 300 Å pore size C18 column. The apomyoglobin elutes before the heme group, consistent with a hydrophilic protein and a hydrophobic heme group. The apomyoglobin and heme group are detected by UV–vis absorption and electrospray ionization mass spectrometry (ESI–MS). The curve-fitted protein mass spectra deconvolutes to an average molar mass of 16,949 ± 2 Da. The mass spectrum of the heme group is consistent with a gas-phase four-coordinate singly-charged iron(III) protoporphyrin IX complex. In this experiment students are introduced to (i) ESI–MS, (ii) calculating masses for proteins and small molecules, and (iii) isotopic patterns. For laboratories equipped only with a HPLC, this experiment introduces undergraduates to chromatography of proteins. Instructors may elect to have students carry out the experiment by HPLC using UV–vis detection and provide the mass spectrometry results for analysis as an exercise. The fragility of the prosthetic heme group, the oxygen carrying moiety of the protein, is of great interest to students.
Keywords (Audience):
Second-Year UndergraduateKeywords (Domain):
Analytical ChemistryKeywords (Pedagogy):
Hands-On Learning / ManipulativesKeywords (Subject):
BiotechnologyCiting Articles
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This article has been cited by 7 ACS Journal articles (5 most recent appear below).
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- Received: August 03, 2009
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