title: Examination of an absolute quantity of less than a hundred nanograms of proteins by amino acid analysis.
abstract: 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.
authors.affiliation: Biomolecular Characterization Team, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan, aki@riken.jp.
authors.forename: Akiko
authors.initials: A
authors.lastname: Masuda
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D000634:Aminoquinolines / Q000737:chemistry; D000818:Animals; D002219:Carbamates / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005453:Fluorescence; D006868:Hydrolysis; D057230:Limit of Detection; D008567:Membranes, Artificial; D011145:Polyvinyls / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D013954:Thyroglobulin / Q000032:analysis; D005465:Fluorocarbon Polymers
chemical_list: D000596:Amino Acids; D000634:Aminoquinolines; D002219:Carbamates; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012710:Serum Albumin, Bovine; D013954:Thyroglobulin; C078178:6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers