Transcript Slide 1

Solutions for Insoluble Problems:
Exploring the Synergy of Hydrostatic
Pressure and Chemistry for Biological
Sample Preparation
Alexander Lazarev, Ph.D.
Analytical Arms Race
Sample Preparation
Well-defined experimental goal and well-prepared
sample are the foundation of success.
Cells contain very few molecules in solution!
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
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
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Keeps samples enclosed during the processing
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Provides precise temperate control
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Capable of processing frozen samples directly
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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)
Multi-stage extraction approach
employing orthogonal methods
Extraction 100 mg tissue: 1200 μL of solvent
supernatant
centrifugation
50 μL for 250 μL for 50 μL for 200 μL for
protein assay 2DGE
SDS PAGE dot blot
pellet
resuspend in appropriate
buffer
2nd Extraction
Exchange solvent if necessary
centrifugation
pellet
etc.
Supernatant*
no reducing
agent
reduction
DTT
no reduction
alkylation reduction no detergent
ultrafiltration
50 μL for 250 μL for 50 μL for
protein assay 2DGE
SDS PAGE
20 μL for
dot blot
* exchange solvent if necessary
PRIMARY ANALYSIS
SECONDARY ANALYSIS
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!
Pressure Cycling Technology (PCT):
35000
30000
25000
16,000 PSI
20000
15000
10000
5000
0
3:56:10
-5000
3:56:53
3:57:36
3:58:19
3:59:02
3:59:46
“Cycles of hydrostatic pressure between ambient
and ultra high levels,
which allow for the precise control of
biomolecular interactions”
PCT Sample Preparation System
13 US patents
4 EU patents
1 AU patent
BarocyclerTM NEP3229
PULSE Tube:
disposable sample container
Pressure Used to Lyse Samples for Extraction
Hierarchy of Pressure Effects
Denaturation of Nucleic Acids
Denaturation of Proteins (monomeric)
DP
Disassociation of Complex Structures
(multimeric)
Disruption of Viruses
Killing of Cells, Bacteria, Fungi
Effect of High Pressure on Protein Activity
Amylase
100
Lipase
Alk P’ase
80
ALT
60
LDH
AST
40
20
0
0
100
200
300
Pressure (MPa)
400
Inactivation of Viruses by PCT
10,000,000
Log Virus Titer
1,000,000
PPV
100,000
HIV-1
10,000
1,000
PRV
100
HSV-1
10
1
0
100
200
300
Pressure, MPa
400
500
600
Inactivation of B. subtilis by PCT
No PCT-treatment
After PCT-treatment
Thermodynamic impact on biological
membrane structure
Pressure-induced interdigitation
of lipid bilayers in an ester-ester
linked HPPC bilayer: HP DSC data.
Interdigitated bilayer
Pressure cycling at 33 ºC
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
Membrane
Protein
Pressure Compresses Lipids Beyond
Equilibrium
Hydrostatic Pressure
Applied
Rapid De-pressurization Causes Membranes
and Micelles to Disintegrate
Hydrostatic Pressure
Rapidly Released
Cryogenic PCT
241.3 MPa
Ih Hexagonal ice 0.93g/cm3
III Ice-three (teragonal) 1.14g/cm3
http://www.lsbu.ac.uk/water/phase.html
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
Environmental
Samples
Virus
Insects
Cultured Cells
Forensic
Samples
Food
Samples
Microorganisms
Homogenization
Extraction
Metabolomics
DMPK
Protein
Purification
DNA and RNA
Purification
Gene
Expression
RT-PCR
qPCR
Protein
Refolding
Immunodiagnostics
Food
Safety
Forensic
Analysis
Pathogen
Inactivation
Environmental
Analysis
DNA extraction for forensic analysis
25
19.8
20
15
10.8
10
6.5
5.4
5
9.9
8.4
7.3
2
0.1
Sample PCT treated
N=9
Mean = 7.8
STD = 5.7
in
RT
in
RT
pc
t6
0m
pc
t6
0m
in
RT
in
RT
pc
t6
0m
pc
t6
0m
in
RT
in
RT
pc
t6
0m
pc
t6
0m
in
RT
in
RT
pc
t6
0m
pc
t6
0m
in
RT
0
pc
t6
0m
DNA Conc. of PCR Product (nmol/L)
PCT releases DNA from bone without a pulverization step
Test for Tick-Borne Pathogens
Ixodes
Scapularis
DNA
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
Real-time PCR on total bacterial 16s DNA amplification
DNA Yeild of Yogurt by OD
(10092006)
DNA yeild (ug/g)
14.0
12.0
10.0
8.0
6.0
4.0
2.0
2
3
4
5
3.4
6.3
5.7
11.4
0.0
Sam ples
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Mixed Berries
0.5g
0.25g
0.125g
0.0625g
NonPCT
PCT
(ug/g)
PCT
Prune
PCT
Series1
1
PCT
0.0
0.5g
Gene expression profiling
PCT releases high quality RNA for microarray analysis
A.
MW
PCT
1
2
+
3
B. PCT
Sample: Rat brain
PCT condition: 4°C, 5 x 1 min cycles, 35 kpsi
RNA extraction buffer: 1.1 ml 4M GTC/1% NP40
C. “+” control
Escherichia coli lysis by PCT or bead mill
PCT
(35,000 psi, 5X 20 seconds)
Total spot volume: 6569661 (+14.2%)
Number of spots detected: 801 (+5.4%)
BEAD MILL
(1,800 oscillations min-1, 3X 30 seconds)
Total spot volume: 5751701
Number of spots detected: 760
French Press followed by PCT: extraction of
proteins from Frankia sp.
Diazovesicles
method
negative control
sonication
PCT, 20 cycles
protein (mg/mL)
0.293 ± .058
0.279 ± .092
0.411 ± .010
French Press treatment is practically unable to disrupt Frankia diazovesicles.
PCT treatment of a French Press pellet produces vesicle protein extract.
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
Analysis of mouse liver lysates by 2DGE:
Comparison of PCT, sonication, and ground glass tissue grinder
sonicator lysate
PCT lysate
1,739 spots
2,126 spots
ground glass
tissue grinder lysate
1,853 spots
10 cycles of 20/20s at 35,000 PSI/atmospheric pressure
IPG pH 4.5-6.5, Second dimension: 6-15% precast gels
Caenorhabditis elegans extraction by
various methods
Freeze-thaw
Bead Beater, 4x20s
20x
20x
Sonication 3x20s
PCT – 5 cycles
20x
40x
T=65ºC!
C. elegans as a proteomic model of Pb2+ toxicity
Young Control
Medium Control
Old Control
Young Lead
Medium Lead
Old Lead
Stratum corneum – human skin cells
collected on adhesive tape
PCT
Proteins
PCR
Non-PCT - +
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
method
negative control
PCT 1 b
PCT 2 c
total PCT
protein a
(mg)
0.327 ± 0.008
0.336 ± 0.004
0.187 ± 0.052
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.
Mineral composition of cortical bone
70% INORGANIC
hydroxyapatite
calcium phosphate
calcium carbonate
calcium fluoride
citrate
30% ORGANIC
Protein extraction from cortical bone
demineralization
solution
MW
FA
HAc
10X
HCl
PCT extracts
1
2
3
“no acid”
control
10X
1DGE of ostrich bone following acid demineralization,
PCT, and Norgen column for removal of Ca and PO4
Isolation of Protein from Various Plant Tissues
Comparison to a centrifugal homogenizer
Strelitzia reginae
Inflorescence
Chloroplast Isolation from Spinacia oleracea
Spinach leaves
Isolation of chloroplast
fraction using conventional
centrifuge

Filter and size exclusion of
intact chloroplasts
Organelle Identification

De-veined and minced
leaves processed in
0.05M phosphate
buffer, pH 7.3 +
sucrose
Chloroplasts

PCT
10s:10s:30cycles
Supernatant from
PULSE tubes placed
into fresh tubes
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.
Acknowledgements:
Frank Witzmann
Myra Robinson
Rosalind Rosenthal
Ric Schumacher
Nathan Lawrence
Gary Smejkal
Chunqin Li
Jim Behnke
Feng Tao
Vera Gross
Ilyana Romanovsky
Ada Kwan
• Vernon Reinhold
• Dibya Himali
• Andrew Hanneman
• Sue Chase
HSPH
Alexander Ivanov
Elena Chernokalskaya
Sunny Tam
Douglas Hinerfeld
• Jennifer Isbister
• James Willett
• Emmanuel Petricoin
• Lance Liotta
• Valerie Calvert