期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 405, 期 25, 页码 8073-8081出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-013-7056-1
关键词
Amino acid analysis; Protein quantification; Post-translational modifications; Absolute quantification; Protein hydrolysis; Proteomics
资金
- Ministry of Health, Labor and Welfare of Japan
- Chemical Genomics Research Project, RIKEN Advanced Science Institute (ASI)
- RIKEN ASI
We developed an ultra-sensitive method of amino acid analysis (AAA) for the absolute quantification of less than 100 ng of proteins, in solution or on polyvinylidene difluoride (PVDF) membranes using an oxygen-free chamber for protein hydrolysis. We used a pre-label method with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for fluorescence detection, ion-pair chromatography with a reversed-phase column, and an ultra-high-pressure high-performance liquid chromatography. We optimized both handling- and instrument-dependent factors for accurate quantification and showed that the least amount of proteins to quantify was determined by handling accuracy rather than instrumental limit for quantification which was 0.6 fmol/amino acid. As a new evaluation method for the handling accuracy, we adopted the protein identification by the obtained amino acid compositions by AAA and the Swiss-Prot database search without the restriction of species. As a result, the least amount of starting material for AAA was 16 ng (0.24 pmol) for a solution of bovine serum albumin (BSA), 33 ng (0.50 pmol) for BSA on a PVDF membrane, and 44 ng (0.15 pmol) for thyroglobulin on a PVDF membrane. These results demonstrate that the ultra-sensitive AAA developed in this study is feasible for absolute quantification of biological significant protein.
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