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”
-5000
0
5000
10000
15000
20000
25000
30000
35000
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
0
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
20.1
7.3
10.8
5.4
19.8
6.58.4
9.9
0
5
10
15
20
25
pct6
0minR
T
pct6
0minR
T
pct6
0minR
T
pct6
0minR
T
pct6
0minR
T
pct6
0minR
T
pct6
0minR
T
pct6
0minR
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
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
DN
A y
eild
(u
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