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In the Laboratory
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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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Helen Cleary Stynes and Araceli Layo
The Biotechnology Centre for Applied Research and Training, School of Biological Sciences and Applied Chemistry, Seneca College of Applied Arts and Technology, Toronto, ON M3J 3M6, Canada
Richard W. Smith
Agilent Technologies, Mississauga, ON L4W 5M2, Canada
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February 2004
Vol. 81 No. 2
p. 266
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| Abstract |
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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.
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| Supplement |
Student materials, teacher instructions, and protein database and ChemSW results are available.
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 Contents |
JCE2004p0266W.doc (Microsoft Word)
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| Download |
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| More Information |
Citation |
Stynes, Helen Cleary; Layo, Araceli; Smith, Richard W. J. Chem. Educ. 2004 81 266.
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Keywords |
Analytical Chemistry; Biochemistry; Biotechnology; Chromatography; Instrumental Methods; Laboratory Instruction; Mass Spectrometry; Proteins / Peptides; UV-Vis Spectroscopy
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History |
Created:
Last Updated: |
January 5, 2004
February 25, 2005
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| Home > JCE Print > Journal of Chemical Education > Issues >
2004
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February
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266
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