LTQ Orbitrap Shotgun proteomics Large scale proteomics by orbitrap technology. Thermo fisher scientific K.K SID C&MS Application OMICS Team
TMT 2
Proteome Cell and Tissue Protein Peptide Extraction Protease Bodily Fluids Sample Database Search Mass spectrometer 3
LC-MS/MSn m/z0.2 LTQ Relative Abundance 100 80 60 40 20 588.3 588.3 589.3 589.3 590.3 590.3 VAQDR 588.3 VAKDR 588.3 0 587.0 587.5 588.0 588.5 589.0 589.5 590.0 590.5 591.0 591.5 592.0 m/z 3ppm ~m/z1000.000 m/z0.003~ LTQ Orbitrap Relative Abundance 100 80 60 40 20 588.3464 VAQDR 588.3100 588.3100 589.3127 589.3491 VAKDR 588.3464 0 587.0 587.5 588.0 588.5 589.0 589.5 590.0 590.5 591.0 591.5 592.0 m/z 4
Proteome etc. In-gel digest In-Solution digest SILAC, TMT etc. 5
Thermo Scientific / Mass Spec Sample Handbook Cell and Tissue Cell Lysis and Protein Extruction Bodily Fluids High Abundant Protein Removal Pre-fractionation & Enrichment Gel Separation In-Gel Digestion Sample Clean up Peptide Enrichment Peptide Clean up Mass Spectrometry In-Solution Digestion 6 Mass Spec Sample Handbook
Cell and Tissue Bodily Fluids In-Solution Digestion 7
Cell and Tissue Bodily Fluids In-Solution Digestion 8
LC-MS/MS LC-ESI-MS/MS Intensity Chromatogram m/z R.T. / MS spectrum K N A G MS/MS spectrum 9
LC-ESI-MS/MS MS/MS 10
LC-ESI-MS/MS HPLC-ESI MALDI-MS or MS/MS MALDI 11
5 ppm Orbitrap Quality Albumin False positive Albumin 2 Da Unit MS system 12
2 Da False positive Albumin 0.1 Da False positive Albumin 100 ppm False positive Albumin 5 ppm Orbitrap Quality Albumin 13
False Discovery Rate ( FDR ) Protein Database Database search results ( ) Data file Decoy Database Decoy database Search results Criteria Criteria FDR<5% FDR = Nature Methods 2, 667-675 (2005) Published online: 23 August 2005; doi:10.1038/nmeth785 Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations Decoy database Search results Database search results 14
LC-MS/MSn Database search results ( ) Decoy database Search results LTQ Criteria FDR<5% Database search results ( ) LTQ Orbitrap Decoy database Search results Criteria FDR<5% 15
LTQ Orbitrap E.coli 100ng Database search results ( ) Decoy database Search results LTQ Criteria FDR<5% LTQ Orbitrap 16
Pierce Biotechnology is Part of Thermo Scientific TMT (Tandem Mass Tag) TMT reagent (229Da) 126-131 103-97 Reporter Balance N-term and K 126 Da ペプチドレベルでラベル化 N-末端 / Kのアミノ基に結合 多群間での比較定量が一度の分析結 果から解析可能 同定はMS/MSから 定量もMS/MSス ペクトルから解析 129 Da 127 Da 130 Da 128 Da 17 131 Da
アイソバリックタグ試薬を用いた手法 ラベル化試薬 18
TMT Cleavable Linker Mass Reporter Mass Normalizer Reactive Group 0 100 90 MS/MS spectra 130.1409 129.1374 130 TMT126 (229Da) 80 70 60 50 40 30 20 10 0 126 126.1281 127 127.1309 128 128.1341 131.1382 126 127 128 129 130 131 132 m/z 131 126 103 TMT127 (229Da) 127 102 19
TMT exp. ratio 1:2:4:6:8:3 obs. ratio1:2:4:7:8:3 126-131 Reporter 127.1309 + 1.5 ppm 126.1281 + 2.9 ppm 128.1341 + 2.6 ppm 129.1374-3.0 ppm 130.1409 + 1.9 ppm 131.1382 + 1.2 ppm Relative Abundance 20 100 50 130.1409 y 1 175.1188 230.1703 b 1 393.2339 b 2 506.3185 126 127 128 129 130 131 132 b 4 798.4240 a b 1 3 669.3823 365.2380 y a a 3 2 4 b 5 359.2400 478.3249 y a 770.4313 3 2 911.5057 b 6 1139.6388 246.1567 641.3881 a 5 883.5048 982.5510 1156.6582 126~131 103~97 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z m/z
TMT LC-ESI-MS/MS 126~131 126~131 126~131 126~131 21 MS/MS