Solutions for Insoluble Problems: Exploring the S

Solutions for Insoluble Problems: Exploring the Synergy of Hydrostatic Pressure and Chemistry for Biological Sample Preparation Alexander Lazarev, Ph.D. : Solutions for Insoluble Problems: Exploring the Synergy of Hydrostatic Pressure and Chemistry for Biological Sample Preparation Alexander Lazarev, Ph.D.

Slide2 : Analytical Arms Race

Sample Preparation : Well-defined experimental goal and well-prepared sample are the foundation of success. Sample Preparation

Slide4 : Organelles: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth ER (9) mitochondria (10) vacuole (11) cytoplasm (12) lysosome (13) centrioles Cells contain very few molecules in solution!

Ideal tissue and cell processor? : Ideal tissue and cell processor? Disrupts lipid bilayer and molecular complexes, but not covalent bonds (proteins, DNA, RNA, etc.) Distributes energy uniformly throughout the sample Facilitates partitioning of lipids, proteins and nucleic acid Does not depend on aggressive extractions buffers Yet, compatible with a wide variety of extraction buffers Prevents sample cross-contamination Keeps samples enclosed during the processing Provides precise temperate control Capable of processing frozen samples directly Processes samples with a throughput matching the downstream analysis. …

Slide6 : Conventional cell disruption methods Mortar & pestle or Dounce homogenizer (glass on glass) Potter-Elvenhjem homogenizer (Teflon on glass) Enzymatic Digestion Polytron shearing homogenizers Blenders Bead mills Sonication Repeated freeze/thaw cycles French press (≤ 2000 PSI)

Slide7 : Extraction 100 mg tissue: 1200 μL of solvent centrifugation Exchange solvent if necessary supernatant pellet resuspend in appropriate buffer 50 μL for protein assay 250 μL for 2DGE 50 μL for SDS PAGE 20 μL for dot blot PRIMARY ANALYSIS 2nd Extraction centrifugation pellet Supernatant* * exchange solvent if necessary SECONDARY ANALYSIS Multi-stage extraction approach employing orthogonal methods etc.

Understanding Hydrostatic Pressure : Understanding Hydrostatic Pressure U.S. Navy Bathyscaphe Trieste (1958-1963) Marianas Trench: 38,713 ft (11,800m) deep 16,000 PSI (120MPa) Significant portion of the Global Biosphere is subjected to high hydrostatic pressure!

Slide9 : “Cycles of hydrostatic pressure between ambient and ultra high levels, which allow for the precise control of biomolecular interactions” Pressure Cycling Technology (PCT): 16,000 PSI

Slide10 : PCT Sample Preparation System BarocyclerTM NEP3229 13 US patents 4 EU patents 1 AU patent

PULSE Tube: disposable sample container : PULSE Tube: disposable sample container Pressure Used to Lyse Samples for Extraction

Slide12 : Hierarchy of Pressure Effects Denaturation of Nucleic Acids Denaturation of Proteins (monomeric) Disassociation of Complex Structures (multimeric) Disruption of Viruses Killing of Cells, Bacteria, Fungi Increasing Pressure DP

Slide13 : Effect of High Pressure on Protein Activity LDH AST ALT Amylase Lipase Alk P’ase Activity (% of Untreated Control)

Slide14 : Inactivation of Viruses by PCT Log Virus Titer 1 10 100 1,000 10,000 100,000 1,000,000 10,000,000 0 100 200 300 400 500 600

Inactivation of B. subtilis by PCT : Inactivation of B. subtilis by PCT No PCT-treatment After PCT-treatment

Thermodynamic impact on biological membrane structure : Thermodynamic impact on biological membrane structure Pressure-induced interdigitation of lipid bilayers in an ester-ester linked HPPC bilayer: HP DSC data. Ichimori H. et al., 1999; in: Advances in High Pressure Bioscience and Biotechnology, Horst Ludwig (Ed.), Proceedings of the Intl. HPBB Conference, Heidelberg, 1998.

Pressure Cycling Acts Directly on Membranes : Pressure Cycling Acts Directly on Membranes Lipid bilayer Membrane Protein

Pressure Compresses Lipids Beyond Equilibrium : Pressure Compresses Lipids Beyond Equilibrium Hydrostatic Pressure Applied

Rapid De-pressurization Causes Membranes and Micelles to Disintegrate : Rapid De-pressurization Causes Membranes and Micelles to Disintegrate Hydrostatic Pressure Rapidly Released

Cryogenic PCT : Cryogenic PCT http://www.lsbu.ac.uk/water/phase.html 241.3 MPa Ih Hexagonal ice 0.93g/cm3 III Ice-three (teragonal) 1.14g/cm3

Heat generation during disruption : Heat generation during disruption

Slide22 : Effect of High Pressure on Nucleic Acids Dissociation of DNA and histones No shearing of covalent bonds Supercoiling of DNA under pressure is reported Hybridization is affected Inactivation of nuclease activity may be beneficial

Synergy of Chemistry and Physics : Synergy of Chemistry and Physics PCT allows to selectively disrupt membrane structures based on their size, compressibility, membrane fluidity. PCT allows control of protein-ligand interactions PCT allows control of nucleic acid hybridization and enzymatic activity PCT can be efficiently combined with affinity purification, chemical or osmotic lysis or freeze-thaw grinding. Hydrogels are shown to be hydrated and “opened up” using PCT

Slide24 : PCT applications Human/Animal Tissue Plant Tissue Fungi Microorganisms Virus Cultured Cells Environmental Samples Forensic Samples Food Samples Insects Protein Purification Gene Expression DNA and RNA Purification RT-PCR qPCR Protein Refolding Immuno- diagnostics Food Safety Forensic Analysis Pathogen Inactivation Homogenization Extraction Metabolomics DMPK Environmental Analysis

DNA extraction for forensic analysis : DNA extraction for forensic analysis N = 9 Mean = 7.8 STD = 5.7 PCT releases DNA from bone without a pulverization step

Test for Tick-Borne Pathogens : Test for Tick-Borne Pathogens Ixodes Scapularis DNA Borrelia burgdorferi DNA

Detection of fungal plant pathogens in soil and plant root samples. : Detection of fungal plant pathogens in soil and plant root samples. Using a novel extraction system that uses Pressure Cycling Technology (PCT), we have obtained Rhizoctonia solani DNA from lyophilized wheat roots that were recalcitrant to homogenization. PCT also improved the extraction of Rhizoctonia and Pythium DNA from agricultural soils up to 16-fold compared to a bead beating extraction method. Furthermore, reproducibility of the extraction was so reliable that pathogen quantification generally could be derived from a single rather than triplicate extractions. Okubara P. et al., 2007, in press

Quantitation of bacteria in yogurt : Quantitation of bacteria in yogurt Real-time PCR on total bacterial 16s DNA amplification

Gene expression profiling : Gene expression profiling Sample: Rat brain PCT condition: 4°C, 5 x 1 min cycles, 35 kpsi RNA extraction buffer: 1.1 ml 4M GTC/1% NP40 PCT releases high quality RNA for microarray analysis

Slide30 : Escherichia coli lysis by PCT or bead mill BEAD MILL (1,800 oscillations min-1, 3X 30 seconds) Total spot volume: 5751701 Number of spots detected: 760 PCT (35,000 psi, 5X 20 seconds) Total spot volume: 6569661 (+14.2%) Number of spots detected: 801 (+5.4%)

French Press followed by PCT: extraction of proteins from Frankia sp. : French Press followed by PCT: extraction of proteins from Frankia sp. French Press treatment is practically unable to disrupt Frankia diazovesicles. PCT treatment of a French Press pellet produces vesicle protein extract. method protein (mg/mL) negative control 0.293 ± .058 sonication 0.279 ± .092 PCT, 20 cycles 0.411 ± .010

Slide32 : Frankia hopanoids stabilize the vesicle membranes Pressure cycling does the reverse! Schematic: Eberhard Karls University, Tubingen

2DGE of Purified Vesicle Fractions Isolated from Frankia EAN1pec : 2DGE of Purified Vesicle Fractions Isolated from Frankia EAN1pec

Slide34 : sonicator lysate 1,739 spots PCT lysate 2,126 spots ground glass tissue grinder lysate 1,853 spots Analysis of mouse liver lysates by 2DGE: Comparison of PCT, sonication, and ground glass tissue grinder 10 cycles of 20/20s at 35,000 PSI/atmospheric pressure IPG pH 4.5-6.5, Second dimension: 6-15% precast gels

Slide35 : Freeze-thaw 20x PCT – 5 cycles 40x Bead Beater, 4x20s 20x Sonication 3x20s 20x T=65ºC! Caenorhabditis elegans extraction by various methods

C. elegans as a proteomic model of Pb2+ toxicity : C. elegans as a proteomic model of Pb2+ toxicity Young Control Young Lead Medium Control Medium Lead Old Control Old Lead

Stratum corneum – human skin cells collected on adhesive tape : Stratum corneum – human skin cells collected on adhesive tape Proteins mtDNA

Problems with traditional methods of protein extraction from sample with high lipid content : Problems with traditional methods of protein extraction from sample with high lipid content Adipocytes may contain up to 70% lipids by weight Small amount of detergent (1-5%) is sequestered into micelles Membrane proteins are captured by micelles or remaining lipid phase Sonication and Polytron shearing promotes micelle formation French press treatment causes “frothing” Dounce homogenizers, bead beaters: sample loss on the surfaces

Slide39 : Murine adipose tissue proteins extracted using PCT or pulverization under liquid nitrogen

Slide40 : Murine adipose tissue extracted by PCT or pulverization under liquid nitrogen in RIPA buffer

Slide41 : Protein yield from ostrich bone protein a method (mg) negative control 0.327 ± 0.008 PCT 1 b 0.336 ± 0.004 PCT 2 c 0.187 ± 0.052 total PCT 0.522 ± 0.055

Slide42 : 70% INORGANIC hydroxyapatite calcium phosphate calcium carbonate calcium fluoride citrate 30% ORGANIC Mineral composition of cortical bone

Slide43 : 1DGE of ostrich bone following acid demineralization, PCT, and Norgen column for removal of Ca and PO4 MW FA HAc HCl 1 2 3 control “no acid” 10X demineralization solution PCT extracts Protein extraction from cortical bone 10X

Slide44 : Isolation of Protein from Various Plant Tissues Strelitzia reginae Inflorescence Comparison to a centrifugal homogenizer

Chloroplast Isolation from Spinacia oleracea : Chloroplast Isolation from Spinacia oleracea Spinach leaves De-veined and minced leaves processed in 0.05M phosphate buffer, pH 7.3 + sucrose PCT 10s:10s:30cycles Supernatant from PULSE tubes placed into fresh tubes     Isolation of chloroplast fraction using conventional centrifuge Filter and size exclusion of intact chloroplasts Organelle Identification Chloroplasts 100 μm NIGMS SBIR Grant R43 GM079059-01

Conclusions: : Conclusions: Cell and tissue disruption frequently present a bottleneck in biomarker analysis. Pressure cycling technology is applicable to a variety of applications, including initial steps of sample preparation for genomics and proteomics. PCT should be considered as an orthogonal extraction technique, not just homogenization or cell disruption method. Barocycler system provides several advantages over conventional extraction methods, including reproducibility, safety, convenience, speed, automation and precise control over the process.

Slide47 : Elena Chernokalskaya Sunny Tam Douglas Hinerfeld Vernon Reinhold Dibya Himali Andrew Hanneman Sue Chase Acknowledgements: Ric Schumacher Nathan Lawrence Gary Smejkal Chunqin Li Jim Behnke Feng Tao Vera Gross Ilyana Romanovsky Ada Kwan Frank Witzmann Myra Robinson Rosalind Rosenthal Jennifer Isbister James Willett Emmanuel Petricoin Lance Liotta Valerie Calvert HSPH Alexander Ivanov