1. No category

Pressure Cycling Technology:
An Innovative Approach to Sample Preparation in Proteomics
Gary Smejkal
Senior Applications Scientist
Pressure BioSciences
Proteomics and Small Molecules Applications Laboratory
Woburn, MA
Local Proteomics Seminar Series
Wednesday, August 2, 2006
Bascom Palmer Eye Institute
University of Miami
Miami, FL
A multi-disciplinary approach to Sample Preparation
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)
Ideal tissue and cell processor specifications
•
•
•
•
•
•
•
•
•
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
Employs chemically inert disposable 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
Introducing a new -omics approach in the “-Omics Era”
CELL DEBRIOMICS
“… the high-throughput dissolution, separation, and analysis of all of the
biomarkers the ‘other guys’ have tossed.”
Alexander Lazarev
CHI 6th Sample Preparation Summit
Some definitions 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.”
Description of a commercial reagent product
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
PCT Sample Preparation System
13 US patents
4 EU patents
1 AU patent
Barocycler
TM
NEP3229
PULSE™ Tube
(Pressure Used to Lyse Samples for Extraction)
Specially designed multi-functional tube
Single-Use
• Versatile, works with:
- Standard and custom reagents
- Various sample types
- Range of sample sizes
• Convenient
• Efficient
• Safe: closed tube, sample fully-contained
•
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
Stratum corneum (human skin cells) collected on adhesive tape
Proteins
mtDNA
PCT
PCR
Non-PCT - +
Comparison of PCT and enzymatic lysis of Rhodopseudomonas palustris
7M urea, 2M thiourea,
25 mM C7BzO
(4.1 mg/mL)
1.1 mS/cm
UF
0.2 mS/cm
lysozyme/benzonase
(3.5 mg/mL)
17.4 mS/cm
UF
0.2 mS/cm
Comparison of PCT, sonication, and grinding of murine liver:
Distribution of proteins in 2D gels
sonicator
1,739 spots
PCT
2,126 spots
ground glass
1,853 spots
10 cycles of 20s at 35,000 PSI/20s at atmospheric pressure
IPG pH 4.5-6.5; Second dimension 6-15% gels
Comparison of PCT, sonication, and grinding of murine liver:
Enlarged high molecular weight regions of 2D gels
Comparison of PCT, sonication, and grinding of murine liver:
Enlarged low molecular weight regions of 2D gels
sonicator
PCT
Under certain conditions sonic probe may be subjected to extensive cavitation,
introducing metal ions into the sample.
Proteolytic fragments are formed due to reactivation of metalloproteases.
ground glass
2DGE of rat liver lysates: Comparison PCT and Polytron homogenization
Protein spots not found in Polytron lysates
2DGE of rat liver lysates: Comparison PCT and Polytron homogenization
2DGE of rat liver lysates: Comparison PCT and Polytron homogenization
2DGE of rat liver lysates: Comparison PCT and Polytron homogenization
Comparison of proteins extracted from murine adipose tissues
using PCT or pulverization under liquid nitrogen
Applications of PCT to Microproteomics:
Proteins extracted from sub-milligram quantities of tissue
(e.g. needle biopsy) by PCT for 2DGE
0.5 to 1 mg of rat liver tissue processed in standard PULSE Tubes using silanized glass beads for volume
displacement. Sample volume was 0.5 mL. Gel loads were normalized to 100µg of protein per gel.
Specific challenges in sample preparation:
The proteomics of Daphnia pulex and related species
• Small freshwater crustacean (1 mm)
• Body mass is 70% chitin exoskeleton.
• Rapidly changes phenotype in response to
environmental changes.
• Parthenogenic. Clonal reproduction switches to sexual
reproduction when environmental conditions
deteriorate.
• Good environmental indicators.
• Diapausal. Ephippia over 300 years old have produced
viable Daphnia.
• Can be use to monitor environmental changes over
decades, possibly centuries.
Changes in Daphnia phenotype in response to predation
Increasingly higher MW proteins recovered by iterative
pressure cycling of Daphnia pulex exoskeletons
chitin
poly(N-acetyl-1,4-β-D-glucopyranosamine)
Specific challenges in sample preparation:
Breaking the tough cuticle of Caenhoribditis elegans
• Tough exterior cuticle makes the nematode
resilient to lysis and impedes proteomic and
glycoproteomic analyses.
• Cuticle is comprised largely of chitin and of
a type of collagen that is particularly rich in
N- and C-terminal cysteines, most of which are
involved in disulfide linkages.
• Lysis scheme inspired by earlier chemists who
developed the “permanent wave” in which the
cuticle of human hair is first softened in a
chemical process that reduces protein
disulfides.
• “Giving the worms a perm.”
• Type of reducing agent, not only concentration,
effects lysis efficiency. (TBP oxidizes in
minutes.)
Breaking the tough cuticle of Caenhoribditis elegans
freeze-thaw
20X
bead beater, 4 x 20s
20X
sonication, 3 x 20s
PCT, 20 cycles
T = 65º C
20X
40X
Heat generation during disruptive methods
such as sonication and bead beating
Problems associated with cooling samples on ice
to offset heat generated during cell disruption
7M urea
2M thiourea
2% CHAPS
Breaking the tough cuticle of Caenhoribditis elegans
using PCT and an optimized lysis reagent
“Giving the worms a perm”
Protein Expression Analysis of C. elegans mutation accumulation lines
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
Comparison of spot volumes in two-dimensional gels of
Escherichia coli comparing PCT and bead mill lysates
mean spot density
X=Y
Application of PCT in multidimensional proteomics:
Delving into the subliminal layers of complex proteomes
Multi-Compartment Electrolyzer (MCE)
• Seven chamber MCE produces partitions
samples into five pI intervals using six different
isoelectric membranes.
• Narrow range pI fractions are produced
(0.5 pI units)
2DGE
925 ± 10 spots
unfractionated cell lysate
IPG pH 3-10
MCE pH 5-6.5 fraction
unconcentrated
IPG pH 3-10
MCE pH 5-6.5 fraction
unconcentrated
IPG pH 3-10
1048 ± 9 spots
MCE pH 5-6.5 fraction
concentrated 2X
IPG pH 4-7
unfractionated cell lysate
IPG pH 3-10
619 ± 13
MCE pH 6.5-8 fraction
concentrated 2X
IPG pH 5-8
unfractionated cell lysate
IPG pH 3-10
240 ± 11
MCE pH 8-11 fraction
concentrated 10X
IPG pH 8-11
unfractionated cell lysate
IPG pH 3-10
410 ± 5
MCE pH 3-5 fraction
concentrated 10X
IPG pH 3-5
Summary of E. coli proteins detected in fractionated
and unfractionated cell lysates
MCE fraction
concentration
number of
spots detected
unfractionated cell lysate
-
925 ± 10
pH 3-5 fraction
pH 5-6.5 fraction
pH 6.5-8 fraction
pH 8-11 fraction
10X
2X
2X
10X
410 ± 5
1048 ± 9
619 ± 13
240 ± 11
total of MCE fractions
-
2317 ± 38
MCE pH 3-4.5 fraction
concentrated 70X
IPG pH 3-5
2
1
3
5
4
PCT isolates proteins from multilammelar vesicles
in Frankia bacterium, while French press does not.
v
m
2DGE of proteins isolated from multilammelar vesicles
using PCT and ProteoSOLVE S Lysis Reagent
Following acetone precipitation and concentration of vesicle
Proteins. IPG pH 4-7. 8-18% polyacrylamide gradient gel.
Isolation of protein from various
components of the Strelitzia reginae
inflorescence by PCT or a centrifugal
homogenizer
Direct MALDI-TOF profiling of plant extracts
Echinacea purpurea leaf, seed, and root
extracted with a Barocycler.
Harris, R.K. et al., Midwest Research Institute, Kansas City, MO.
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 .
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
Pressure Biosciences, Inc.
www.pressurebiosciences.com
NASDAQ: PBIO
Seattle, WA
Engineering
Design Group
Irvine, CA
Manufacturing
Partner
Distribution/Support
Partner in Japan
San Diego, CA
Sales Office
West Bridgewater, MA
Corporate HQ
Woburn, MA
Proteomics and
Small Molecule
Applications
Laboratory
Gaithersburg, MD
DNA, RNA and
Forensics
Applications
Laboratory
Acknowledgements:
• Richard Schumacher
• Nathan Lawrence
• Chunqin Li
• Jim Behnke
• Feng Tao
• Sunny Tam
• Douglas Hinerfeld
• Frank Witzmann
Midwest
Research
Institute
• Myra Robinson
• Elena Chernokalskaya
• Kelley Thomas
• Vernon Reinhold
• Deena Small
• Dibya Himali
• Andrew Hanneman
• R.K. Harris
• L.A. Ford
• S.A. Schwartz
• J.R. Guthrie
• D.E. Gray
•
Sample Program: “Send us your samples”
alazarev@pressurebiosciences.com
gsmejkal@pressurebiosciences.com
•
Purchase/Leasing Options
•
For More Information:
jlanuza@pressurebiosciences.com
nlawrence@pressurebiosciences.com
(508) 580-1818
www.pressurebiosciences.com