質量分析法を用いたタンパク質定量・翻訳後修飾 プロファイ …type...
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麻布大学 研究推進・支援本部麻 布 大 学 研 究 推 進・支 援 本 部
〒252-5201 神奈川県相模原市中央区淵野辺1-17-71 TEL:042-754-7111(ext.438) FAX:042-850-2511E-mail: [email protected] URL: http://www.azabu-u.ac.jp/sgk/ 担当：寺本，角野，根本
Simultaneous method for quantification of protein and profiling of post-translational modification
質量分析法を用いたタンパク質定量・翻訳後修飾プロファイル同時解析技術
上家 潤一 講師
麻布大学 獣医学部獣医学科 病理学研究室
Junichi Kamiie / LecturerSchool of Veterinary Medicine, Department of Veterinary Medicine
Our Absolute quantitative assay with stableisotope labeled protein (AQUA protein) methodmake it possible to examine simultaneously1 )Protein expression level with a high
degree of accuracy,2)Post translational modification (PTM) sites,3)PTM rates at each sitein biological samples.
LC-MS/MS analysis
Detected PTM of nephrinType (Swiss-prot) Modification%
peptide1 Glycosylation 96%peptide2 Glycosylation 99%peptide3 unknown 46%peptide4 Phosphorylation 51%
peptide 1peptide 2
peptide 3
peptide 4
Sequence Cover: 40%Average: 1.3±0.1 fmol/glomerulus
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2 NontreatedAlkalin Phosphatase
Out line of AQUA protein method : 1) stable isotope labeled full lengthprotein is synthesized by E.coli expression system. 2) Biological samplespiked with labeled protein as internal reference is digested by trypsin.3) Tryptic peptides derived from endogenous and reference protein aremeasured by LC-MS/MS. Quantitative values are calculated from thepeak area ratios.
Trypsin digestion
Data analysis
Principle of PTM analysis with AQUA protein method: PTM site isidentified by lower quantitative value compared with average value. PTMrate can be calculated as difference of quantitative values between targetpeptide and average.
Quantification of nephrin in renal glomeruli :200 fmol of stableisotope labeled nephrin was spiked into 50 of isolated glomerulifrom rat kidney . After trypsin digestion, mixture was analized byLC-MS/MS. 38 peptide (40% of total sequence) derived fromnephrin could be quantified. Average of quantitative value was1.3 ±0.1fmol/glomerulus. 4 peptides showed lower values withsignificant difference between total average.
Isolated glomeruli
Modification amounts of 4 peptides showed lower values werecalculated as differences between average of quantitative values.Modification rates (%) against average value are shown in table.Petide3 has been reported as no PTM site in database (swiss-prot), although peptide1,2 and 4 have been reported.
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1 2Conventional method
AQUA protein
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Evaluation of AQUA protein results :A. Comparison ofquantitative values between conventional LC-MS/MS methodand AQUA protein method. B. Verification of phosphorylation.Phosphorylation of peptide 3 and 4 were verified by recovery ofquantitative values with alkaline phosphatase treatment.*P>0.01.
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Case StudyQuantification of nephrin in renal glomeruli
Now, you are ready to analyze all proteins with AQUA protein method!
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