development of essential sample …...development of essential sample preparation techniques in...
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DEVELOPMENT OF ESSENTIAL SAMPLE PREPARATION TECHNIQUES IN
PROTEOMICS USING ULTRA-HIGH PRESSURE
Alexander R. Ivanov
HSPH Proteomics ResourceDepartment of Genetics and Complex Diseases
Harvard School of Public Healthwww.hsph.harvard.edu/proteomics
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Sample Preparation:
-Proteolytic DigestionIn-solutionIn-gelOn-membrane
-Cell LysisPressureOrganic solvents
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Protein Mix
Proteolytic enzymes: trypsin, Lys-C
Dissolution additives: (HFIP, urea, methanol)
Reduction reagent/ concentration:DTT vs. TCEP
Tested variables in optimization of digestion
LC-MS/MS analysis and database searching (Scaffold, Protein Prophet, Peptide Prophet)
Time of digestion
Conventional (In Incubator)
Pressure-Assisted, PCT (In Barocycler)
Reduction environment:temperature and pressure
Workflow for Optimization of Digestion Protocol
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ProtocolDissolution Additives:
Digestion conditions
In Incubator (37˚C) In PCT (45˚C)
Shor
than
d pr
otoc
ol
nam
eBr
ief
desc
ript
ion
HFI
P
MeO
H
Ure
a
1-tr
ypsi
n al
iquo
t, 1
2-hr
s,
2-tr
ypsi
n ad
ditio
ns,
40-h
rs1-
Lys-
C, 1
-tr
ypsi
n, 4
0-hr
s1-
tryp
sin
aliq
uot,
1-
hr 2-tr
ypsi
n,
2-hr
1-Ly
s-C,
1-
tryp
sin,
2-
hr
2-tr
ypsi
n,
4-hr
2-tr
ypsi
n,
8-hr
I-A_ Standard protocol xI-C_ Standard in PCT xI-D Standard (small volume) xII-A Lys-C xII-C Lys-C in PCT xIII-A HFIP (Hexafluoroisopropanol) x xIII-C HFIP +PCT x xIV-A MeOH x xIV-C MeOH +PCT x xV-A Urea x xV-C Urea +PCT x xVI-A DTT for Reduction xVI-C DTT for Reduction in PCT xVII-A Urea/HFIP x x xVII-C Urea/HFIP/PCT x x xI-A-1 Standard, only (1) 12-hr tryp xI-A-2 Standard (I-A) xI-C-1 Only 1 PCT digest xI-C-2 Standard in PCT (I-C) xI-C-4 4 PCT digests xI-C-8 8 PCT digests xI-A* Standard, only (1) 12-hr tryp xI-AC* I-A, digestion in PCT xI-C* I-C, 1 tryp digest xVI-A50 VI-A with 50mM DTT xVI-C50 VI-C with 50mM DTT x
Protocol Variables
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0
20
40
60
80
100
120
140
Conv
entio
nal
PCT
Lys-
C
Lys-
C +
PCT
HFI
P
HFI
P +
PCT
MeO
H
MeO
H +
PCT
Ure
a
Ure
a +
PCT
0%
20%
40%
60%
80%
100%
120%
Conv
entio
nal
PCT
Lys-
C
Lys-
C +
PCT
HFI
P
HFI
P +
PCT
MeO
H
MeO
H +
PCT
Ure
a
Ure
a +
PCT
Uni
que
stan
dard
pep
tide
s
In-Solution Tryptic Digestion, 100 fmol/analysis
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In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances
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y = 1.207xR² = 0.936
0
50000
100000
150000
200000
250000
300000
350000
0 50000 100000 150000 200000 250000 300000
Average peptide abundance, PCT vs. conventional.
In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances
Run-to-run reproducibility (R2)
LC-MS Runs 1 and 2, R2
LC-MS Runs 2 and 3, R2
LC-MS Runs 1 and 3, R2
Mean R2 CV
Conventional Digestion 0.945 0.815 0.892 0.884 7.4%PCT Digestion 0.908 0.938 0.950 0.932 2.3%
Sample-to-sample reproducibility,(R2)
Samples 1 and 2, R2
Samples 2 and 3, R2
Samples 1 and 3, R2
Mean R2 CV
Conventional Digestion 0.862 0.918 0.883 0.888 3.2%PCT Digestion 0.966 0.944 0.939 0.950 1.5%
PCT
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-3
-2
-1
0
1
2
3RATIO (Conv/PCT)
KD
In-Solution Tryptic Digestion: Differential Peptide Recovery
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In-Solution Tryptic Digestion: Differential Peptide Recovery
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In-Solution Tryptic Digestion: Differential Peptide Recovery
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Higher throughput (20x fold)
Higher efficiency of proteolysis
Lower preanalytical variability
Better overall peptide recovery
Absence of PCT-induced in vitro peptide oxidation
Digestion specificity is not hampered by ultra-high pressure
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PCT-Assisted Cell Rupture
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Credit: R. Schlicher, R. Apkarian, and M. Baran, www.cchem.berkeley.edu, www.sciencephotolibrary.com
Conventional Cell/Tissue Rupture Approaches
Mechanical stress Ultrasound Osmosis
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Pressure Cycling – Assisted Lysis and Protein Extraction
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Pressure Cycling-Assisted and Organic Solvent –Assisted Cell Lysis
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Pressure Cycling-Assisted and Organic Solvent –Assisted Cell Lysis
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Pressure Cycling-Assisted and Organic Solvent –Assisted Cell Lysis
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Pressure Cycling-Assisted and Organic Solvent – Assisted Cell Lysis
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GO Term Enrichment Analysis. Cellular Localization.
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Conclusions
(1.) higher throughput;
(2.) higher efficiency;
(3.) superior reproducibility of enzymatic digestion;
(4.) more efficient cell lysis;
(5.) superior recovery of membrane, organelle, and complex forming proteins in comparison to the conventional protocols, as well as increased identification of proteins containing TMDs.
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Acknowledgements
Emily FreemanAlexander Lazarev
Vera GrossPBI
Funding:NIEHS, HSPH GCD Department,
CRDF, Harvard Catalyst