Novel time-resolved fluorophores for protein detection

Transcription

1 Novel time-resolved fluorophores for protein detection Dr. Thole Züchner Head of Ultrasensitive Protein Detection Unit Institute of Bioanalytical Chemistry Center of Biotechnology and Biomedicine Leipzig University Wolfen, May 31st 2012

2 Protein detection Changes on the level of proteins as cause/consequence of nearly all existing diseases (infections diseases, cancer, Alzheimer, heart diseases ) Schlüsseltechnologie für Key technology for Pharm Biotech Diagnosti Pharma a Biotech Diagnostics k important: specific detection

3 Protein detection most important methods capillary electrophoresis 2D-PAGE 1D-PAGE Protein chips ELISA HPLC/ UPLC antibody/ protein x staining / dyes x mass spec visualization

4 Protein staining: state-of-the-art stain detection limit molar range Coomassie brilliant blue ~50ng fmol-pmol conventional fluorophores ~1-20ng fmol-pmol silver staining ~1ng fmol method detection limit molar range mass spec (FT-ICR) fg amol mass spec (Orbitrap) fg amol capillary electrophoresis ag zmol mol picomol mol femtomol mol attomol mol zeptomol

5 Complexity of the proteome f(x) # of proteins different posttranslational modifications known: more than 300 (phosphorylation, acetylation etc.) -at multiple sites of AT LEAST 150,000 proteins -can be more than one modification per protein 0,25 0,2 50µg 50,000 different proteins? 0,15 0,1 0,05 average protein amount: 1 ng Protein concentration x

6 Another challenge in Proteomics f(x) # of proteins Huge range of protein expression levels of at least seven orders of magnitude (see e.g.: Liebler et al., Nat Rev Drug Discov. 2005, 4(5):410-20) can be as little as 1-10 receptors per cell 100pmol 100zmol 0,25 0,2 9 orders of magnitude! 0,15 0,1 0, Protein concentration x

7 Protein detection: state-of-the-art Signal intensity Signal intensity , ,0001 0,001 0,01 0, ng protein , ,0001 0,001 0,01 0, ng protein bad linear ranges prevent protein expression changes to be detected

8 Problem Autofluorescence of membranes/gels

9 Time-resolved fluorescence Fluorescence intensity Time-res. fluor. [EuL]H Background fluorescence Measure- ment time excitation

10 [EuL]H Stokes-shift Excitation (360nm) Emission (616nm) 1 3 0, Stokes shift: 256nm I , , , 0 N M Anal. Chem. 2009, 81:

11 [EuL]H / carboxyfluorescein dot blot Anal. Chem. 2009, 81:

12 Quantification Anal. Chem. 2009, 81:

13 Labeled lysozyme on PVDF membranes Anal. Chem. 2009, 81:

14 SDS-PAGE with labeled BSA protein BSA Phosphorylase B Ovalbumin detection limit SDS-PAGE 1.5fmol 7.5fmol 15fmol Anal. Chem. 2009, 81:

15 Signal linearity linear over a range of 5 orders of magnitude (100amol 0.1nmol) Anal. Chem. 2009, 81:

16 Comparison sensitivity model calculation (200pg total protein/cell, 7,500 different proteins/cell, average occurrence of every protein) # of cells needed for detection of one single kind of protein Silver staining 37,500 [EuL]H 250

17 Comparison linearity # of cells needed for detection of one single kind of protein Silver staining 37,500 [EuL]H 250 silver Coomassie [EuL]H cell #: 100 1,000 10, ,000 1,000,000 10,000, ,000,000

18 Fast, homogeneous assay Existing assays: HTRF assays (10 180min) Turbidimetry/Nephelometry (>10min) Homogeneous EIA (~30min) - Peptides instead of proteins: faster displacement Kreisig T, Hoffmann R, Zuchner T. (2011) Anal Chem 83,

19 RET: spectral overlap Fluorescein & BHQ-10 Kreisig T, Hoffmann R, Zuchner T. (2011) Anal Chem 83,

20 Quenching efficiency acceptors Q-value BHQ-10: Quenching of up to 45 % TAMRA: quenches only up to 30 % TAMRA as a fluorophore contributes to the background fluorescence 50% Q - Value I unquenched I quenched I unquenched 40% 30% 20% TAMRA labeled antibody 10% BHQ-10 labeled antibody 0% 0 0,1 0,2 0,3 0,4 Concentration mab [µmol/l] Kreisig T, Hoffmann R, Zuchner T. (2011) Anal Chem 83,

21 relative fluorescence intensity rel. fluorescence intensity Assay response with analyte low-affinity peptide native peptide Control without antibody Sample Background (no analyte) analyte concentration (µmol/l) Time (sec) Detection limit: 0,05µmol/L Total assay time: 90 sec Linear range: 0.1-1µmol/L Kreisig T, Hoffmann R, Zuchner T. (2011) Anal Chem 83,

22 Sample compatibility Real life samples like blood, saliva or urine contain a lot of different proteins E. coli cell lysate (0.1 g/l) mimics these conditions Kreisig T, Hoffmann R, Zuchner T. (2011) Anal Chem 83,

23 Working area

24 Acknowledgement Team Karin Büttner Partners Industry Kristin Dobslaff Thomas Kreisig Stefanie Langanke Constance Nürnberger Kristina Paskhaver Agneta Prasse Peter Simeonov Thomas Zauner Kristin Zscharnack Research Groups Ralf Hoffmann, Leipzig, Germany Norbert Sträter, Leipzig, Germany Patrik Brundin, Lund, Sweden Ron Wetzel, Pittsburgh, USA Regino Perez-Polo, Galveston, USA Financial support

25 Contact phone: +49 (0)