Absolute quantification of low abundance proteins by shotgun proteomics Dr. Stefanie Wienkoop www.proteomefactory.com In cooperation with: Max-Planck-Institut für Molekulare Pflanzenphysiologie Stable isotope labelled peptides: Biopolymers, Ulm, Germany
Introduction Multiple Reaction Monitoring (MRM) General procedure Complex samples and in solution digestion Complex samples and in gel digestion => MASS WESTERN
General procedure: 1. Signature Peptide 2. Synthesis (Standard peptides) 3. Tuning (SRM or MRM) 4. Analyses
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, 643-65
RT: 19.58-9.1 SM: 15G 1 95 9 mol4 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 Tim e (m in) RT: 19.6-9. SM: 15G 56.8 NL: 6.22E6 1 9 8 fmol4 7 6 5 4 3 2 1 56.6 NL: 6.58E4 1 9 8 fmol4 7 6 5 4 3 2 1 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 Tim e (m in) NL: 1.7E7 TIC F: + p SRM MS Contol_sta ndard_8f TIC F: + p SRM ms2 576.6@-26. [ 2.65-923.75] MS Contol_standard_8 TIC F: + p SRM ms2 573.1@-26. [ 2.65-916.75] MS Contol_standard_8 RT: 19.6-9. SM: 15G 38.5 NL: 6.73E7 1 TIC F: + p SRM ms2 9 369.8@-17. [ 142.65-568.55] MS 8 Contol_standard_8 fmol4 7 6 5 4 3 2 1 38.8 NL: 6.74E4 1 TIC F: + p SRM ms2 9 366.3@-17. [ 142.65-561.55] MS 8 Contol_standard_8 fmol4 7 6 5 4 3 2 1 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 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
in solution vs in gel digestion Advantage: proteins of different sizes (physiological pathway) => faster Disadvantage: less sensitive
Complex sample and in solution digestion -Pathway Analysis-
R T : 1. - 17. LTQ: sequential precursor isolation and fragmentation 1 95 9 SM: 15G N L : 2.66E6 Base Peak M S IC IS W T2AQ U A _ 1 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 Tim e (m in) dependent MSMS standard analysis Method according to: Venable JD et al. (24) Nature Methods Wienkoop and Weckwerth 26, JExB
TSQ vs. LTQ TSQ higher sensitivity for targeted analyses using MRM. Absolute quantification.
Purity Control 1 9 8 7 6 5 Native ion trace SRM 489.17@19.15-484.45 NL: 9.11E1 4 3 2 1 1 9 8 7 Stable isotope labelled standard ion trace SRM 49.5@19.15-485.45 54.51 NL: 1.59E4 6 5 4 3 2 1 5 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 Time (min)
Sample without standard Sample with standard 1 9 A RT: 52.88 MA: 451347 NL: 1.2E5 Base Peak F: + c SRM ms2 489.17@-15. [ 19.15-484.45] MS 1 9 B RT: 54.75 MA: 4569138 NL: 1.45E5 Base Peak F: + c SRM ms2 489.17@-15. [ 19.15-484.45] MS 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 fmol 8 7 Native ion 6 trace 5 Native ion trace 4 3 2 1 NL: 6.83E3 RT: 54.92 Base Peak F: + c SRM MA: 3754279 ms2 49.5@-15. [ 1 19.15-485.45] MS 9 8 7 ion trace 6 Standard fmol ion 5 trace 4 Native ion trace NL: 9.58E5 Base Peak F: + c SRM ms2 49.5@-15. [ 19.15-485.45] MS Standard ion trace 5 5 fmol 3 3 2 2 1 1 3 4 5 6 7 8 Time (min) 3 4 5 6 7 8 Time (min)
Automated protein pathway assignment: MAPMAN Usadel et al. 25, PlantPhys
Complex sample and in gel digestion MASS WESTERN
Mass Western vs. Western Blot Isoforms (specificity)? Quantification?
Traditional Western Blot Analysis C C T T C C T T Coomassie gel Western blot E. Larrainzar, Uni Navarra, Spain
(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:.1-5.2 SM : 15G 1 8 6 Isoform 1 23.1 RT: Standard ion trace NL: 1.57E5 TIC F: + p S R M m s2 521.7@ - 1 8. [ 721.95-851.75] M S 5 p m o l_ b RT:. - 5.2 SM : 15G 1 8 6 Isoform 2 22.96 RT: Standard ion trace NL: 2.58E5 TIC F: + p SRM m s2 491.7@ - 17. [ 191.65-791.65] M S 5 p m o l_ b 4 4 2 2 1 8 6 4 2 1 8 6 23.1 Native ion trace NL: 2.1E7 TIC F: + p S R M m s2 518.2@ - 1 8. [ 721.95-851.75] M S 5 p m o l_ b 23.24 RT: NL: 1.4E5 1 Isoform 4 TIC F: + p S R M m s2 527.2@ - 8 Isoform 3 1 8. [ 732.95-862.75] 6 M S 5 p m o l_ b Standard ion trace 1 8 6 4 2 22.95 25.46 RT: Native ion trace Standard ion trace NL: 3.17E4 TIC F: + p SRM m s2 488.2@ - 17. [ 184.65-791.65] M S 5 p m o l_ b NL: 2.15E6 TIC F: + p SRM m s2 54.2@ - 18. [ 191.65-888.85] M S 5 p m o l_ b 4 4 2 2 1 8 6 23.24 Native ion trace NL: 7.8E4 TIC F: + p S R M m s2 523.8@ - 1 8. [ 732.95-862.75] M S 5 p m o l_ b 1 8 6 32.24 32.21 Native ion trace NL: 6.94E2 TIC F: + p SRM m s2 536.8@ - 18. [ 184.65-888.85] M S 5 p m o l_ b 4 4 2 2 5 1 15 2 25 3 35 4 45 5 Time (min) 5 1 15 2 25 3 35 4 45 5 Time (min)
Detection of low abundance isoforms (in gel) Isoform 1 12 pmol Isoform 2 22 fmol Isoform 3 nd Isoform 4 171 fmol
Isoform Identification isoform identification and quantification of corresponding western blot signals isoform identification responsible for enzyme activities
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.
Proteomics Services @ Proteome Factory www.proteomefactory.com 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
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