27. 11. 2014 1 Karel Klepárník Ústav analytické chemie Akademie věd České republiky Veveří 97, 602 00 Brno, klep@iach.cz From single cell to single molecule analysis 1 1956* Ústav analytické chemie AVČR Veveří 97 Brno www.iach.cz Oddělení bioanalytické instrumentace 2 Size 5 ‐ 10 m  Volume ~ 500 fl  Total mass ~ 500 pg Proteins: Only 10% of the cellular mass form about 2 fmol of 10 000 expressed proteins  A typical mammalian cell contains from 100 to 500 pg of protein.  Cellular receptors > 1000 molecules; 60 ms life‐time of complex molecule entering cell ‐ receptor   2000 glycans per cell Various signaling enzymes 103 – 105 molecules Structural proteins 108 molecules  DNA:  nucleus: 20 % of cell mass DNA ~5 pg (MCF 7 cells) Typical eucaryotic somatic cell 3 27. 11. 2014 2 Important molecules - analytes:  at maximum concentrations  already separated  at biologically relevant positions Cells as Test Tubes Single cell or single molecule analyses provide:  space – time correlation of cellular organization and dynamics  no ensemble averaging  absolute sensitivity – no separation needed  transition paths observable only in single cell/molecule  distribution of behaviors – static or dynamic heterogeneity. Tumor heterogeneity is one of the major causes of cancer drug resistance. 4 Methods of single cell analysis Imaging methods  Optical microscopies (far-, near-field) low  Correlative light and electron microscopy throughput  Mass spectrometry imaging Detection methods nm, ps  Laser induced fluorescence (LIF) resolution  Total internal reflection fluorescence (TIRF) absolute  Surface enhanced Raman scattering (SERS) sensitivity  Nonlinear optical spectroscopy  Foerster resonance energy transfer (FRET) high  Chemi- and bioluminescence detection throughput Separation methods hundreds HPLC, CE, IEF, on-line multidimensional zmol inj. separations with MS detection and identification tens pM conc. 5 Mass Spectrometry 6 27. 11. 2014 3 Mass Spectrometry Imaging Matrix assisted laser desorption and ionization (MALDI) Secondary ion mass spectrometry (SIMS) MS imaging spatial resolution <1 µm at ambient pressures, with very high mass accuracy and mass resolution. Subcellular resolution for many kinds of cells. Spatial resolution 5 - 30 µm - for generating enough signal per spot 7 Single cell proteomics 8 J. Michael Ramsey Minnie N. Goldby Distinguished Professor of Chemistry Department of Chemistry The University of North Carolina at Chapel Hill Chapel Hill, NC USA 9 27. 11. 2014 4 Mellors, J. S., Jorabchi, K., Smith, L. M., Ramsey, J. M., Anal Chem 2010, 82, 967-973. Integrated Microfluidic Device for Automated Single Cell Analysis Using CE Separation and ESI-MS cells EOF pump buffer buffer 4.7 cm 100 nm nanojunction 5.5 kV 75 x10μm 15% polyamine coating PolyE-323 300 V/cm 12 red blood cells per minute – cell lysis in high E Heme group, α and β subunits of hemoglobin 50:50 methanol/water + 2% acetic acid (155 µS) A baseline A B C B single cell C multiple cells Single erythrocyte: 450 amoles of hemoglobin → 1.8 fmoles of 2α 2β subunits + 1 heme group (carbonic anhydrase I ∼7 amol/cell not detected) pH 7 260 mM sucrose 15 mM NH2Ac 1.3 mS 10 Prof. Norman Dovichi Grace Rupley Professor of Chemistry and Biochemistry, University of Notre Dame Notre Dame, IN 11 Heat‐map of the CZE analysis of 100 ng of an  E. coli tryptic digest. Precursor ion  electropherograms were generated for all  identified peptides in the digest.  Comparison of peptide IDs from E. coli protein digests Digest mass UPLC-ESI-MS/MS CZE‐ESI‐MS/MS  100 ng 1 875 1 377 1 ng 342 627 Zhu, G. J., Sun, L. L., Yan, X. J., Dovichi, N. J., Anal Chem 2013, 85, 2569-2573. Single-Shot Proteomics Using CZE-ESI-MS/MS Production of More than 1 250 Escherichia coli Peptide Identifications in 50 min 12 27. 11. 2014 5 2D CE-MS system with immobilized enzyme reactor Li, Y. H., Wojcik, R., Dovichi, N. J., J. Chromatogr. A 2011, 1218, 2007–2011. 1st capillary: separation of protein mixture for 8 min Replaceable enzymatic microreactor (trypsin immobilized on magnetic particles captured by a pair of narrow magnets at the capillary outlet) on-line protein digestion in the reactor during the separation of peptides for 1 min 2nd capillary: portions of created peptides are periodically introduced via the first interface into the second capillary for separation Sheath-flow electrospray transfer of peptides via the second interface into a emitter. 2D separation requires roughly 30min to digest and separate 30 fractions. 13 Integrated CZE−ESI-MS/MS System with an Immobilized Trypsin Microreactor for Online Digestion and Analysis of Picogram Amounts of RAW 264.7 Cell Lysate Sun, L. L., Zhu, G. J., Dovichi, N. J., Anal Chem 2013, 85, 4187-4194. Triplicate analysis of RAW 264.7 cell lysates: 3 ng 7 ± 2 protein groups identified 300 pg 2 ± 1 protein groups identified (relatively highly abundant proteins) MM: 5.7 - 58.8 kDa; pI: 4.5 - 11.0 Protein amounts analyzed correspond to the protein content in three cells Microreactor-CZE-ESI-MS/MS Triplicate extracted ion electropherograms of  PMFIVNTNVPR peptide from the macrophage  migration inhibitory factor Electrokinetically pumped sheath‐flow  cuvette nanospray interface, i.d. 8 µm Uncoated capillary: 34 cm, 2 cm reactor, 3 min digestion time, Buffer: 5 mM NH4HCO3 (pH 8.0) LTQ‐Orbitrap Velos 50% (v/v) metOH 0.05% (v/v) FA 14 Single cell metabolomics 15 27. 11. 2014 6 Jonathan V. Sweedler Department of Chemistry University of Illinois Urbana, IL Director of the Biotechnology Center Associated with the Beckman Institute, Biotechnology Center, Neuroscience Program and Bioengineering Program Molecular gates 200 nm 15 nm 16 Culture and stimulation of a neuronal network. A) Schematic of the device (8 x 8 x 2 mm). Blue - channels. Red - pressure channels. B) Image of an ink solution in the stimulation channels with both valves closed. C) Peptidergic culture of bag cell neurons of Aplysia californica after 1 day in vitro. Microfluidic Device for the Selective Chemical Stimulation of Neurons and Characterization of Peptide Release with MALDI MS 500 μm 100 μm Croushore, C. A., Supharoek, S. A., Lee, C. Y., Jakmunee, J., Sweedler, J. V., Anal Chem 2012, 84, 9446-9452. Series of representative mass spectra of bag cell neuron peptide release following KCl stimulation. Identified peptides from the bag cell neurons (βBCP, m/z 728.4; αBCP1−7,m/z 922.5; αBCP, m/z 1122.6;AP, m/z 2959.5; pELH30−43, m/z 1471.8; δBCP1−36, m/z 4022.7; δBCP, m/z 4406.9) Exocytosis of neurotransmitters from a neuron through the depolarization of the cell membrane 17 Custom-designed coaxial sheath-flow CE-ESI interface hyphenated to MS. Single-neuron analysis by CE-ESI-MS Nemes, P., Knolhoff, A. M., Rubakhin, S. S., Sweedler, J. V., Analytical Chemistry 2011, 83, 6810-6817. sample buffer fused‐silica separation   capillary 18 27. 11. 2014 7 Hierarchical clustering of affected metabolites based on their participation in pathways containing differentially expressed genes. Pathways (rows) were clustered against metabolites (columns) monitored by CE−ESI‐MS. Yellow blocks indicate that a metabolite is involved in the corresponding pathway. A total of 18 distinct metabolites were identified in the mouse hippocampus. Knolhoff, A. M., Nautiyal, K. M., Nemes, P., Kalachikov, S., Morozova, I., Silver, R., Sweedler, J. V., Anal Chem 2013, 85, 3136-3143. Combining Small-Volume Metabolomic and Transcriptomic Approaches for Assessing Brain Chemistry Determination of chemical changes in mouse central nervous system.  Mice with profound deficits in spatial learning, memory, and neurogenesis. Metabolites extracted from the dissected left caudal hippocampus of a volume of 6 nL, hydrodynamically injected into a separation capillary  (40 μm i.d., 90 cm length) filled with 1% FA as a BGE. A coaxial sheath flow of 50% methanol with 0.1% (v/v) FA at a rate of 750 nL/min was  used in CE‐MS interface.  metabolites pathways 19 Patch clamp electrophysiology and CE−MS metabolomics for single cell characterization of brain cells (rat thalamus) Aerts, J. T., Louis, K. R., Crandall, S. R., Govindaiah, G., Cox, C. L., Sweedler, J. V., Anal Chem 2014, 86, 3203-3208. Ex vivo whole cell patch clamp as a sampling approach. Intracellular material collected by applying additional negative pressure to puncture a passageway into the cell membrane and withdraw a small volume of 3 pL of the cytoplasm contents into the patch clamp pipet for a small-volume assay using CE-MS. Combination of whole-cell patch clamp with single cell cytoplasm metabolomics. Physiological activity of neurons and astrocytes correlated with their neurochemical state and quantitate changes in the cellular metabolome. Striking cell to cell heterogeneity in the brain. ornithine glycine serine tryptophan glutamine tyrosine prolinenonburst firing TRN neuron electrophysiological recording photomicrograph LIF extracted ion electropherogram microTOF or a maXis 4G Qq‐ToF (Bruker Daltonics, Billerica)  positive ion mode  capillary length 65−70 cm, voltage 14−16 kV sample injection volume of ∼28 nL 20 Schematic of the probe ESI setup. Microscopic image of the sampling procedure. Monolayer A. cepa cells placed on a glass slice. The dark cells alive, the light ones dead. Coupling with MS: DC high voltage (+2.5 kV for the positive mode and −2.6 kV for the negative mode) applied to the probe. Assistant solvents directly sprayed onto the tip of the probe to generate electrospray. Single Cell Analysis with Probe ESI-Mass Spectrometry: Detection of Metabolites at Cellular and Subcellular Levels Gong, X. Y., Zhao, Y. Y., Cai, S. Q., Fu, S. J., Yang, C. D., Zhang, S. C., Zhang, X. R., Anal Chem 2014, 86, 3809-3816. 21 27. 11. 2014 8 Optical methods 22 Membrane proteins CD58-Cy3 (green) ICAM-1-Cy5 (red) in a glass-bound planar phospholipid bilayer under two PMA/ionomycin-treated Jurkat cells. Single molecule imaging Total Internal Reflection Fluorescence Microscopy (TIRF) prism glycerol slide sample objective immersion oil 23 Surface-enhanced Raman scattering (SERS, TERS) Label-free method 1) Molecule in contact with colloid 2) Irradiation 3) Surface plasmon 4) Excitation 5) Raman scattering 6) Surface plasmon 7) SERS >1012 enhancement Single molecule spectroscopy colloid molecule irradiation SPR 24 27. 11. 2014 9 -100 0 100 200 300 400 500 600 700 0 500 1000 1500 2000 2500 Raman shift [cm-1] Suspension of Ag NP (50 – 100 nm) concentration [mg/ml] 0 0.05 0.2 0.4 Rhodamine 6G Epifluorescence microscope, objective 50x (0.95 NA); He-Ne laser, 632.8 nm, 15 mW; spectrograph Shamrock SR-303i, diffraction grating 600 l/mm blazed for 500 nm; back-illuminated CCD camera iDus, Andor temperature –90°C; exposure time 0.5 s Přikryl, J., Klepárník, K., Foret, F., Journal of ChromatographyA, 1226, 43 – 47, 2012. Qualitative analysis by SERS 25 Bioluminescence detection of caspase 3 - protease relevant in apoptosis Interest of biologists, pharmacologists and physicians:  Failure of apoptosis is one of the main contributions to tumor development, neurodegenerative and autoimmune diseases.  Potential target for new drugs.  New functions in cellular differentiation Molecular mass ~ 33 000 Da Caspase 3 - play a central role in the execution-phase of apoptosis - increased amount of caspase indicates the pointof-no-return in apoptosis - protease recognizing tetra-peptide sequence Asp-Glu-Val-Asp↓Gly 26 Bioluminescent determination of caspase 3 activity caspase 3 + ATP + O2 hν luciferase Mg2+ amino luciferin Z-DEVD-luciferin Advantages:  one step mixing system  no washing steps  no immobilized agents  convenient kinetics  free aminoluciferin excluded D – aspartic acid E – glutamic acid V – valine E. Adamová, M. Lišková, E. Matalová, K. Klepárník Anal Bioanal Chem (2014) 406, 5389–5394 I. Chlastakova, M. Liskova, J. Kudelova, L. Dubska, K. Kleparnik, E. Matalova, Cellular and Developmental Biology, 48, 2012, 545-549. Lišková M., Klepárník K., Matalová E., Hegrová J., Přikryl J., Švandová E., Foret F. Electrophoresis, 34, 2013, 1772-1777 27. 11. 2014 10 Chemiluminescence detection PMT CL chamber assembly with PMT glass vial - 5 µl  Limit of caspase 3 bioluminescent detection is ~ 0.2 of its content in apoptotic cell, i.e. < 1 fg.  Contents of caspase 3 in individual apoptotic cells vary from ~ 50 to 250 thousand molecules. 28  Huge absorption coefficient  Broad excitation spectra  Narrow emission spectra  Low photobleaching Quantum dot – donor in FRET sensor for confocal fluorescence caspase 3 microscopy FRET - Foerster resonance energy transfer - „Angstrom optics“ 29 Correlative luminescence and electron microscopy of QD labeled proteins in cells Transmembrane proteins: Fas receptor (CD95), molecular mass 48 kDa, and Fas ligand (CD178 - 40 kDa). Molecular probe: conjugate of monoclonal antibody with QD QD provides: dynamic image overview due to a bright luminescence (diffraction limit ~ 200 nm) nanometer precise position due to a high electron density 30 27. 11. 2014 11 Single molecule detection 31 Stretching dsDNA in Nanochannels • evaluation of size • chromatography or electrophoresis • detection of nucleotides consecutively cleaved by exonuclease 32 The Nobel Prize in Chemistry 2014 "for the development of super-resolved fluorescence microscopy" Janelia Research Campus,  Howard Hughes Medical  Institute, Ashburn, VA, USA Max Planck Institute for  Biophysical Chemistry,  Göttingen, Germany Stanford University,  Stanford, CA, USA William E. MoernerStefan W. HellEric Betzig 33 27. 11. 2014 12 Super-resolution Optical Microscopy Nanoscopy (resolution below diffraction limit) STED - stimulated emission depletion microscopy stimulated emission inactivate anuli around a point STORM - stochastic optical reconstruction random on and off states PALM – photoactivated localization microscopy photoswitchable fluorophores SOFI - super-resolution optical fluctuation imaging correlation function of fluctuations A sufficiently low percentage of the probes are activated to allow the image of each fluorescing molecule to be seen separately 34 Abbe‘s Diffraction limit 35 Point Spread Function Original object Diffraction disc Reconstructed area 36 27. 11. 2014 13 200 nm Stained object and its diffraction limited image 37 Parameters of image reconstruction 38 Stochastic optical reconstruction 39 27. 11. 2014 14 Most subcellular structures (such as actin fibers, intermediate filaments, microtubules, ribosomes, and transport vesicles) exhibit features much smaller than the diffraction limited size. Single molecule superresolution microscopy 40 Time-resolved confocal fluorescence microscope with unique single molecule sensitivity MicroTime 200 STED 33 000 EUR 41 3D SPRAIPAINT super-resolution images of the cell surface and CreS in Caulobacter 42 27. 11. 2014 15 Fluorescent saxitoxin lighting up single NaV channels on live PC12 cell 43 Tracking a Single Quantum Dot in a Living Cell with a DH-PSF 44 Application of micro- and nanotechnologies in analytical chemistry provides:  Methods with absolute sensitivity  Space resolution - 25 nanometers  Time resolution – ps (molecule – 6 GHz processor)  Single molecule detection in individual cells (targeted proteomics)  Label-free high-sensitivity methods  High throughput multiplex parallel analyses 45 27. 11. 2014 16 Next generation DNA sequencing 46 Helicos The HeliScope™ Sequencer 2 . 109 b/day 109 reads/run 25 – 55 bp read lengths Genome Sequencer FLX System 3 . 108 b/day 100 Mb/7.5 hour run 400 000 reads/7.5 hour 200 – 300 bp read lengths Solexa Illumina Genome Analyzer 6 . 108 b / day 3 . 109 b / 5 days run 50 . 106 oligo clusters 36 – 50 bp read lengths Parallel single molecule sequencing by synthesis 47 The HeliScope™ Sequencer http://helicosbio.com/ 48 27. 11. 2014 17 Single molecule real time sequencing (SMRTTM) Pacific Biosciences Next generation DNA sequencing DNA sequencing – DNA polymerase RNA sequencing – reverse transcriptase Codone-resolved translation elongation by single ribosomes Tens of nucleotide peaks in 1 sec Read length 1 – 15 kb 80 000 detection points 15 min/genome: 50 n/s * 80 000 points * 15 min * 60 s = 3.6 Gb DNA polymerase 529 processivity 20 kB – 400 b/s Some enzymes are not processive $ 100/genome 49 Pacific Biosciences  Single Molecule Real Time (SMRT™) DNA sequencing 50 PacBio RS instrument 51 27. 11. 2014 18 Single molecule real time sequencing 52 Pacific Biosciences Read Length 53 Pacific Biosciences Read Length 54 27. 11. 2014 19 DNA sequencing development 2001: Genome draft of 5 individuals in 9 months – more than billion $ 2014: Complete human genome in an hour – ~100 $ 55