Solutions for Insoluble Problems: Exploring the Synergy of Hydrostatic Pressure and Chemistry for Biological

Sample Preparation

Alexander Lazarev, Ph.D.

Analytical Arms Race

Well-defined experimental goal and well-prepared

sample are the foundation of success.

Sample Preparation

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?

• 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.

• …

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)

Extraction 100 mg tissue: 1200 μL of solvent

centrifugation

Exchange solvent if necessary

supernatant pellet

resuspend in appropriate buffer50 μL for

protein assay250 μL for

2DGE50 μL for

SDS PAGE200 μL for

dot blot

50 μL forprotein assay

250 μL for2DGE

50 μL forSDS PAGE

20 μL fordot blot

no reducingagent

reductionalkylation

ultrafiltration

DTTreduction

no reductionno detergent

PRIMARY ANALYSIS

2nd Extraction

centrifugation pellet

Supernatant*

* exchange solvent if necessary

SECONDARY ANALYSIS

Multi-stage extraction approach employing orthogonal methods

etc.

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!

“Cycles of hydrostatic pressure between ambient

and ultra high levels,

which allow for the precise control of

biomolecular interactions”

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3:56:10 3:56:53 3:57:36 3:58:19 3:59:02 3:59:46

Pressure Cycling Technology (PCT):

16,000 PSI

PCT Sample Preparation System

BarocyclerTM NEP3229

13 US patents 4 EU patents 1 AU patent

PULSE Tube: disposable sample container

Pressure Used to Lyse Samples for Extraction

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

Incr

easin

g Pr

essu

re

P

Effect of High Pressure on Protein Activity

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20

40

60

80

100

0 100 200 300 400

Pressure (MPa)

LDH AST

ALT

Amylase

LipaseAlk P’ase

Act

ivity

(%

of

Unt

reat

ed C

ontr

ol)

Inactivation of Viruses by PCT

Log

Viru

s T

iter

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

0 100 200 300 400 500 600

HSV-1

PRV

PPV

HIV-1

Pressure, MPa

Inactivation of B. subtilis by PCT

No PCT-treatment After PCT-treatment

Thermodynamic impact on biological membrane structure

Pressure-induced interdigitationof lipid bilayers in an ester-ester linked HPPC bilayer: HP DSC data.

Pressure cycling at 33 ºC

Interdigitated bilayer

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

Lipid bilayer

MembraneProtein

Pressure Compresses Lipids Beyond Equilibrium

Hydrostatic Pressure Applied

Rapid De-pressurization Causes Membranes and Micelles to Disintegrate

Hydrostatic Pressure Rapidly Released

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

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

• 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

PCT applications

Human/Animal Tissue

Plant Tissue

Fungi

Microorganisms

Virus Cultured Cells

EnvironmentalSamples

Forensic Samples

Food Samples

Insects

ProteinPurification

Gene Expression

DNA and RNAPurification

RT-PCRqPCR

Protein Refolding

Immuno-diagnostics

Food Safety

Forensic Analysis

PathogenInactivation

HomogenizationExtraction

Metabolomics DMPK

Environmental Analysis

DNA extraction for forensic analysis

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pct6

0minR

T

pct6

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T

pct6

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T

pct6

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T

pct6

0minR

T

pct6

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T

pct6

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T

pct6

0minR

T

Sample PCT treated

DN

A C

on

c. o

f P

CR

Pro

du

ct (

nm

ol/

L)

N = 9

Mean = 7.8

STD = 5.7

PCT releases DNA from bone without a pulverization step

Test for Tick-Borne Pathogens

Ixodes ScapularisDNA

Borrelia burgdorferi DNA

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

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A y

eild

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g/g

)

Samples

DNA Yeild of Yogurt by OD (10092006)

Series1 3.4 6.3 5.7 11.4 0.0

1 2 3 4 5

Real-time PCR on total bacterial 16s DNA amplification

00.5

11.5

22.5

33.5

44.5

5

PC

T

PC

T

PC

T

PC

T

No

nP

CT

(ug/g) 0.5g 0.25g 0.125g 0.0625g 0.5g

Mixed Berries

Prune

Gene expression profiling

PCT +MW 1 2 3

PCT +MW 1 2 3

A. B. PCT C. “+” controlPCT +MW 1 2 3

PCT +MW 1 2 3

A. B. PCT C. “+” control

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

Escherichia coli lysis by PCT or bead mill

BEAD MILL(1,800 oscillations min-1, 3X 30 seconds)

Total spot volume: 5751701Number 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 treatment is practically unable to disrupt Frankia diazovesicles.PCT treatment of a French Press pellet produces vesicle protein extract.

Diazovesicles

method protein (mg/mL)

negative control 0.293 ± .058sonication 0.279 ± .092PCT, 20 cycles 0.411 ± .010

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

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 pressureIPG pH 4.5-6.5, Second dimension: 6-15% precast gels

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

Young Control

Young Lead

Medium Control

Medium Lead

Old Control

Old Lead

Stratum corneum – human skin cells collected on adhesive tape

PCRPCT Non-PCT - +

Proteins mtDNA

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

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

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

Protein yield from ostrich bone

protein a

method (mg)

negative control 0.327 ± 0.008PCT 1 b 0.336 ± 0.004PCT 2 c 0.187 ± 0.052total PCT 0.522 ± 0.055

a from 345 ± 15 mg initial bone mass

b 80 cycles

c additional 80 cycles following replacement with fresh ProteoSOLVE IEF Reagent.

70% INORGANIC

hydroxyapatite calcium phosphate calcium carbonate calcium fluoride citrate

30% ORGANIC

Mineral composition of cortical bone

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

demineralizationsolution PCT extracts

Protein extraction from cortical bone

10X

Isolation of Protein from Various Plant Tissues

Strelitzia reginae Inflorescence

Comparison to a centrifugal homogenizer

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:

• 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.

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 RobinsonRosalind Rosenthal

• Jennifer Isbister• James Willett• Emmanuel Petricoin• Lance Liotta• Valerie Calvert

HSPH

Alexander Ivanov