Absolute quantification of low abundance proteins by shotgun proteomics

Transcription

1 Absolute quantification of low abundance proteins by shotgun proteomics Dr. Stefanie Wienkoop In cooperation with: Max-Planck-Institut für Molekulare Pflanzenphysiologie Stable isotope labelled peptides: Biopolymers, Ulm, Germany

2 Introduction Multiple Reaction Monitoring (MRM) General procedure Complex samples and in solution digestion Complex samples and in gel digestion => MASS WESTERN

3 General procedure: 1. Signature Peptide 2. Synthesis (Standard peptides) 3. Tuning (SRM or MRM) 4. Analyses

4 A practical approach B theoretical approach multidimensional fractionation FPLC digestion 2D HPLC SCX/RP analysis Protein sequence information from database theoretical digestion MS MS/MS peptide identification data base search signature peptide list labelling stable isotope labelled standard peptides Wienkoop et al. 24, RCM 18,

5 RT: SM: 15G mol Tim e (m in) RT: SM: 15G 56.8 NL: 6.22E fmol NL: 6.58E fmol Tim e (m in) NL: 1.7E7 TIC F: + p SRM MS Contol_sta ndard_8f TIC F: + p SRM ms @-26. [ ] MS Contol_standard_8 TIC F: + p SRM ms @-26. [ ] MS Contol_standard_8 RT: SM: 15G 38.5 NL: 6.73E7 1 TIC F: + p SRM ms @-17. [ ] MS 8 Contol_standard_8 fmol NL: 6.74E4 1 TIC F: + p SRM ms @-17. [ ] MS 8 Contol_standard_8 fmol Tim e (m in) Absolute quantification procedure + sample + labelled standard peptides TIC in gel digest: in solution MS MRM standard peptide x native peptide x standard peptide y native peptide y

6 in solution vs in gel digestion Advantage: proteins of different sizes (physiological pathway) => faster Disadvantage: less sensitive

7 Complex sample and in solution digestion -Pathway Analysis-

8 R T : LTQ: sequential precursor isolation and fragmentation SM: 15G N L : 2.66E6 Base Peak M S IC IS W T2AQ U A _ Tim e (m in) dependent MSMS standard analysis Method according to: Venable JD et al. (24) Nature Methods Wienkoop and Weckwerth 26, JExB

9 TSQ vs. LTQ TSQ higher sensitivity for targeted analyses using MRM. Absolute quantification.

10 Purity Control Native ion trace SRM @ NL: 9.11E Stable isotope labelled standard ion trace SRM 49.5@ NL: 1.59E Time (min)

11 Sample without standard Sample with standard 1 9 A RT: MA: NL: 1.2E5 Base Peak F: + c SRM ms @-15. [ ] MS 1 9 B RT: MA: NL: 1.45E5 Base Peak F: + c SRM ms @-15. [ ] MS fmol 8 7 Native ion 6 trace 5 Native ion trace NL: 6.83E3 RT: Base Peak F: + c SRM MA: ms2 49.5@-15. [ ] MS ion trace 6 Standard fmol ion 5 trace 4 Native ion trace NL: 9.58E5 Base Peak F: + c SRM ms2 49.5@-15. [ ] MS Standard ion trace 5 5 fmol Time (min) Time (min)

12 Automated protein pathway assignment: MAPMAN Usadel et al. 25, PlantPhys

13 Complex sample and in gel digestion MASS WESTERN

14 Mass Western vs. Western Blot Isoforms (specificity)? Quantification?

15 Traditional Western Blot Analysis C C T T C C T T Coomassie gel Western blot E. Larrainzar, Uni Navarra, Spain

16 (no original sequence) Isoform 1... GDVQYILDDVRXLFSEALSRIKKQGLDIIPRLQIITRLLTDEVGSTCGQRLFKVYGIEHC Isoform 2... GDVQYILDDVRXLFNEALRRIKQQGLDIKPRLQIITRLLTDEVGSTCGERLFKVYDIEHC Isoform 3... GDVQYILDDVRXLFEEALQKIELQGLNVKPQLQVVTRLITNEKGSTCNQELFPIIKIKHS Isoform 4 GDVQYILDDVRXLFNEALARIQKQGLDFTPRLQIVTRLITDEKGSTCNQRLFRVSGIDYT... MASS WESTERN Searching for Isoforms C:\Xcalibur\...\Gel1116\5pmol_b 11/4/26 2:12:19 AM 1626 RT: SM : 15G Isoform RT: Standard ion trace NL: 1.57E5 TIC F: + p S R M m s @ [ ] M S 5 p m o l_ b RT: SM : 15G Isoform RT: Standard ion trace NL: 2.58E5 TIC F: + p SRM m s @ [ ] M S 5 p m o l_ b Native ion trace NL: 2.1E7 TIC F: + p S R M m s @ [ ] M S 5 p m o l_ b RT: NL: 1.4E5 1 Isoform 4 TIC F: + p S R M m s @ - 8 Isoform [ ] 6 M S 5 p m o l_ b Standard ion trace RT: Native ion trace Standard ion trace NL: 3.17E4 TIC F: + p SRM m s @ [ ] M S 5 p m o l_ b NL: 2.15E6 TIC F: + p SRM m s2 54.2@ [ ] M S 5 p m o l_ b Native ion trace NL: 7.8E4 TIC F: + p S R M m s @ [ ] M S 5 p m o l_ b Native ion trace NL: 6.94E2 TIC F: + p SRM m s @ [ ] M S 5 p m o l_ b Time (min) Time (min)

17 Detection of low abundance isoforms (in gel) Isoform 1 12 pmol Isoform 2 22 fmol Isoform 3 nd Isoform fmol

18 Isoform Identification isoform identification and quantification of corresponding western blot signals isoform identification responsible for enzyme activities

19 Conclusion Stable isotope dilution technique is highly sensitive for targeted absolut quantification especially also for low abundance proteins. Detailed and complex pathway analyses are possible. Mass Western approach gives more detailed information than traditionel western blot analyses.

20 Proteomics Proteome Factory Analysis of all kind of protein samples by extreme high resolution 2DE - separation of up to 1, protein spots (4x3 cm 2DE) Target / Biomarker Identification Differential Proteomics Studies Plasma / CSF Proteomics Studies with depletion of high abundant proteins Pharmaco Proteomics Studies Immuno Proteomics Protein Separation / Western Blots

21 Thanks to Biopolymers, Ulm, Germany Dr. Christian Scheler Joel Louette Max-Planck-Institut für Molekulare Pflanzenphysiologie Estibaliz Larrainzar Ute Lehmann Dr. Wolfram Weckwerth Prof. Dr. Mark Stitt