pressure cycling technology - a new paradigm of biological ...alexander lazarev, ph.d. analytical...
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Pressure Cycling Technology - a New Paradigm of Biological
Sample Preparation
Alexander Lazarev, Ph.D.
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Analytical Arms Race
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Utility of the accurate mass measurement (the Moore’s law of Mass Spectrometry)
2006
A.G. Marshall, et. Al. Anal. Chem. 74, 252-259A (2002)
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Sample preparation!
Well-defined experimental goal andwell-prepared sample are the foundation of
success.
? ?
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Genomic and proteomic samples:
• Biological fluids (serum, sputum, urine, etc.)
• Tissue cultures
• Tissues
• Other: (Bacteria, mycobacteria, fungi, spores, viral particles, etc.)
• Mixed matrices (e.g. pathogens in blood or plant tissue)
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Two alternatives in proteomic biomarker studies:
• Blind approach:Serum/Plasma
Fractionation/Enrichment Differential Display
Biomarker CandidatesIsolation and Characterisation
Immunodetection MethodsValdation
• Guided approach:Tissue, Proximal Fluids
Differential DisplayBiomarker Candidates
Isolation and Characterization Immunodetection Methods
Validation in Blood/Urine…
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Current extraction methods
•Mortar & Pestle or Dounce homogenizer (glass on glass)•Potter-Elvenhjem homogenizer (teflon on glass)•Enzymatic Digestion•Polytron shearing homogenizers•Blenders•Bead Beating•Sonication•Repeated Freeze/Thaw cycles•French Press (≤ 2000 PSI)
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A definition of “Cell Debris”?
• “[DNAse I] is responsible for clearing up cellular debris and does so by chopping up long strands of DNA into short pieces to facilitate their disposal.” BMJ 320:1495, 2000
• “Whole cell and cell-debris polysaccharide”United States Patent 6602997
• “CDR ( Cell Debris Remover ) Aids in protein purification through initial clean-up of cell lysates; removes unwanted suspended, colloidal and soluble matter , leaving target proteins in solutions.”
A description of the commercial reagent product.
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Introducing another “Omics” approach:
Cell-debriomics – the high-througputdissolution, separation, and analysis of all of the biomarkers the “other guys” have tossed!
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Ideal tissue and cell processor
• Disrupts lipid bilayer, 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 harsh extractions buffers• Compatible with a wide variety of extraction buffers• Uses chemically inert single use containers• Keeps samples enclosed during the processing• Provides precise temperate control• Capable of processing frozen samples• Processes samples with a throughput matching the analysis
steps• …
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What is PCT?
Pressure Cycling Technology
Cycles of hydrostatic pressure between ambient and ultra high levels,
which enables the precise thermodynamic control of
biomolecular interactions
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PCT Sample Preparation System
BarocyclerTM NEP3229
13 US patents4 EU patents1 AU patent
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PULSE™ Tube(Pressure Used to Lyse Samples for Extraction)
• Single-Use• Versatile, works with:
- Standard and custom reagents - Various sample types- Range of sample sizes
• Convenient• Efficient• Safe: closed tube, sample fully-contained
Specially designed multi-functional tube
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“Bubble Collapse” mechanism
Rapid pressure pulses create cavitation analogous to sonication. However, PCT distributes energy uniformely throughout the sample in a precisely controlled fashion. Cavitation velocities can exceed 300 m/sec.
Calculated Cavitation Velocity
050
100150200250300350
0 100 200 300 400 500Pressure (MPa)
Max
. Col
laps
ing
Vel
ocity
(m/s
ec)
0.01 um 0.1 um0.5 um 1 um10 um
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Cell disruption – cycle profiles
0
5000
10000
15000
20000
25000
30000
35000
40000
0 10 20 30 40 50 60 70
8 cycles50 sec ea.
50 cycles8 sec ea.
S. cerevisiaedisrupted in in PBS
Cycle duration has minimal effect on extraction efficiency,as compared to the number of cycles
0
5000
10000
15000
20000
25000
30000
35000
40000
0 50 100 150 200 250 300
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Cryogenic PCT
http://www.lsbu.ac.uk/water/phase.html
241.3 MPa
Ih Hexagonal ice 0.93g/cm 3
III Ice-three (teragonal) 1.14g/cm3
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Pressure threshold
• PCT allows to selectively disrupt membrane structures based on their size.
• PCT allows control of protein-ligand interactions
• PCT allows control of nucleic acid hybridization and enzymatic activity
• PCT can be combined with affinity purification
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PCT process compared to traditional extraction processes
Technology advantages
• Recovery – comparable or better than with traditional methods, no shearing molecules
• Reproducibility – reduces sample-to-sample variability
• Convenience - hands-off sample homogenization and extraction, no labor-intensive steps
• Speed – as fast as 3 min per sample
• Throughput - up to 3 samples in parallel, potential for more
• Safety - Fully contained sampleprocessing, chain of custody with disposable tubes that eliminates the danger of cross-contamination
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Technology advantages
• PCT combines the elements of most competing technologies in one powerful package
• Compatible with the chemical disruption buffers• Provides capacity to use mechanical grinding action and shearing
via PULSE Tube design modifications• PULSE Tubes accommodate beads• Can be performed in cryogenic conditions• Carries repeatedly a process of a French Press / Pressure Bomb
• Additionally:• no exposure to metal parts – preserves fragile redox-active
chemical species and prevents activation of metalloproteases• compatible with a wide variety of organic solvents
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Pressure Biosciences, Inc.www.pressurebiosciences.com
NASDAQ: PBIO
Irvine, CAManufacturing
Partner
San Diego, CASales Office
Woburn, MAProteomics and Small Molecule
ApplicationsLaboratory
Seatlle, WAEngineering
Design Group
Gaithersburg, MDDNA, RNA and
Forensics ApplicationsLaboratory
West Bridgewater, MACorporate HQ
Distribution/SupportPartner in Japan
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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
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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 brainPCT 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
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DNA extraction for forensics analysis
20.1
7.3
10.8
5.4
19.8
6.58.4
9.9
0
5
10
15
20
25
pct6
0minR
Tpc
t60m
inRT
pct6
0minR
Tpc
t60m
inRT
pct6
0minR
Tpc
t60m
inRT
pct6
0minR
Tpc
t60m
inRT
pct6
0minR
T
Sample PCT treated
DN
A C
onc.
of P
CR
Pro
duct
(nm
ol/L
)N = 9
Mean = 7.8
STD = 5.7
PCT releases DNA from bone without a pulverization step
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Q buffer L buffer _M PCT PCT Bead Neg PCT PCT Bead Neg
DNA released from Bacillus sp. spores forrapid detection of bioterror agents
PCT releases DNA from spores in a safer closed syst em than current methods
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Inactivation of B. subtilis by PCT
No treatment After PCT treatment
PCT can be applied to selectively kill bacteria, spores or viruses.
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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%)
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Comparison of spot volumes in two-dimensional gels ofEscherichia coli comparing PCT and bead mill lysates
X=YMean spot density
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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
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High Molecular Weight Region:
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sonicator PCT ground glass tissue grinder
Proteolytic fragments
Low Molecular Weight Region:
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Heat generation during disruption
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Stratum corneum – human skin cells collected on adhesive tape
PCRPCT Non-PCT - +
Proteins mtDNA
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Increasingly higher MW proteins recoveredby iterative pressure cycling of Daphnia pulex
chitin
poly(N-acetyl-1,4-β-D-glucopyranosamine)
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Caenorabditis elegans extraction
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0.04245.936140.0PCTanther
0.003 ± 0.0010.64 ± 0.19198.1. ± 2.3negative control
0.004 ± 0.0010.73 ± 0.03199.4 ± 4.1BioMasher
0.005 ± 0.0011.16 ± 0.03218.3 ± 12.8PCTsepal (blue)
0.003 ± 0.0010.57 ± 0.00201.2 ± 7.5negative control
0.012 ± 0.0012.40 ± 0.22206.1 ± 7.2PCTsepal (orange)
0.014 ± 0.0010.73 ± 0.0153.7 ± 2.5BioMasher
0.030 ± 0.0012.08 ± 0.6070.0 ± 19.9PCTstamen
0.011 ± 0.001 2.41 ± 0.17213.5 ± 10.1negative control
0.011 ± 0.0012.21 ± 0.06197.2 ± 4.4BioMasher
0.014 ± 0.001 2.98 ± 0.21208.9 ± 2.5PCTspathe
efficiencyyield (mg)mass (mg)methodtissue
proteintissueextraction
Isolation of protein from various components of the Strelitzia reginaeinflorescence by PCT or a centrifugal homogenizer
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Direct MALDI-TOFprofiling of plant extracts
Echinacea purpurea leaf, seed, and root extracted with a Barocycler. R. K. Harris, et al., Midwest Research Institute, K ansas City, MO
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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.
• Barocycler provides several advantages over conventional tissue homogenization methods, including reproducibility, safety, convenience, speed, automation and precise control over the process .
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PCT Application-specific advantages
• Life Science Research (biochemistry, molecular biology)– most convenient, fast method– controlled process provides reproducibility
– improved throughput, ability to process tough tissues, disposable tubes prevent cross-contamination
• Forensic– ability to process bone and other tough materials,
– disposable tubes, convenient sample collection/processing format, chain of custody
• Biodefense and clinical– safe sample collection/processing format, in-tube pathogen inactivation,– speed & reproducibility, validated methods
• Agricultural– ability to homogenize plant material,
– field sample collection and transportation
– reproducibility & convenience
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• Elena Chernokalskaya
• Sunny Tam• Douglas Hinerfeld
• Vernon Reinhold• Dibya Himali• Andrew Hanneman
Acknowledgements:
• Nathan Lawrence• Gary Smejkal• Chunqin Li• Jim Behnke• Ric Schumacher• Feng Tao
Frank Witzmann
Myra Robinson
Midwest Research Institute
• R.K. Harris• L.A. Ford• S.A. Schwartz• J.R. Guthrie• D.E. Gray