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Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up
U Bergmann1, R Ahrends, B Neumann
1Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany.
Analytical Chemistry
|June 5, 2012
Summary
Metal-coded affinity tags (MeCAT) enable absolute protein quantification. Two MeCAT methods accurately measured aprotinin in complex samples, offering precise protein amount determination for proteomics research.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biotechnology
Background:
- Absolute protein quantification is crucial for advancing proteomics beyond comparative studies.
- Current methods for determining exact protein amounts are limited.
- Metal-coded affinity tags (MeCAT) offer a novel approach by integrating ICP-MS with proteomics.
Purpose of the Study:
- To present and validate two independent MeCAT methodologies for the absolute quantification of a specific protein.
- To determine the absolute amount of recombinantly expressed aprotinin within a host cell protein background.
Main Methods:
- Utilized two MeCAT strategies: a bottom-up approach labeling primary amino groups on peptides and a top-down approach labeling cysteine residues on proteins.
- Employed inductively coupled plasma mass spectrometry (ICP-MS) for precise elemental analysis.
- Used flow injection analysis (FIA)-ICP-MS for external quantification of synthetic peptides and dissolved gel spots.
Main Results:
- The enzymatic assay determined 8.3 and 9.8 ng/μg of fusion protein for two batches.
- Top-down MeCAT analysis yielded 4.0 and 5.4 ng/μg of aprotinin.
- Bottom-up MeCAT analysis resulted in 14.5 and 15.9 ng/μg of aprotinin.
Conclusions:
- MeCAT methodologies provide accurate and reliable absolute quantification of proteins.
- These methods are effective in determining protein amounts even within complex biological matrices.
- MeCAT technology enhances quantitative proteomics by enabling precise measurement of protein concentrations.
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