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Published on: September 9, 2013
Membrane Protein Analyses Using Alkylated Trihydroxyacetophenone (ATHAP) as a MALDI Matrix
Yuko Fukuyama1,2, Chihiro Nakajima1, Shunsuke Izumi3
1Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation , 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
Abstract:
Membrane proteins containing hydrophobic regions have been difficult to analyze using MALDI-MS, probably due to the use of conventional matrices with a low affinity for hydrophobic peptides. Recently, we reported 1-(2,4,6-trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone (ATHAP)) as a matrix for hydrophobic peptides. In this study, ATHAP was applied to analyze membrane proteins containing transmembrane domains. As a result, we detected intact molecular ions for bacteriorhodopsin (BR) containing seven transmembrane domains that are difficult to detect using 2,4,6-trihydroxyacetophenone or sinapinic acid, by using ATHAP. In addition, we detected digest ions containing all seven transmembrane domains that are difficult to detect using α-cyano-4-hydroxycinnamic acid (CHCA), by using ATHAP. Moreover, ions for hydrophobic digests containing a single transmembrane domain for cadherin 1 (CDH1), fibroblast growth factor receptor 4 (FGFR4), epithelial cell adhesion molecule (EPCAM) recombinant proteins, and human epidermal growth factor receptor type 2 (HER2) were detected with higher sensitivity using ATHAP than with CHCA, confirming that ATHAP improved the membrane protein analyses, especially for hydrophobic regions such as transmembrane domains.
Insights
A new matrix, alkylated trihydroxyacetophenone (ATHAP), enhances the analysis of hydrophobic membrane proteins using MALDI-MS. ATHAP improves the detection of intact proteins and peptides, especially those with transmembrane domains.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Membrane proteins present analytical challenges in Mass Spectrometry due to hydrophobic regions.
- Conventional matrices often exhibit low affinity for hydrophobic peptides, hindering analysis.
Purpose of the Study:
- To evaluate the efficacy of alkylated trihydroxyacetophenone (ATHAP) as a matrix for analyzing membrane proteins with hydrophobic transmembrane domains using MALDI-MS.
- To compare ATHAP's performance against conventional matrices for hydrophobic peptide analysis.
Main Methods:
- Application of ATHAP matrix for MALDI-MS analysis of membrane proteins.
- Analysis of intact bacteriorhodopsin (BR) and its tryptic digests.
- Analysis of hydrophobic digests from cadherin 1 (CDH1), FGFR4, EPCAM, and HER2 recombinant proteins.
Main Results:
- ATHAP successfully detected intact molecular ions for bacteriorhodopsin (BR), including its seven transmembrane domains.
- ATHAP enabled detection of BR digest ions containing all seven transmembrane domains, which were difficult to detect with other matrices.
- ATHAP demonstrated higher sensitivity than CHCA for detecting hydrophobic digests with single transmembrane domains from CDH1, FGFR4, EPCAM, and HER2.
Conclusions:
- ATHAP is a superior matrix for MALDI-MS analysis of membrane proteins, particularly for hydrophobic regions like transmembrane domains.
- ATHAP significantly improves the detection sensitivity and coverage of hydrophobic peptides compared to conventional matrices.
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