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

Sample Preparation:

-Proteolytic DigestionIn-solutionIn-gelOn-membrane

-Cell LysisPressureOrganic solvents

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

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

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

In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances

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

-3

-2

-1

0

1

2

3RATIO (Conv/PCT)

KD

In-Solution Tryptic Digestion: Differential Peptide Recovery

In-Solution Tryptic Digestion: Differential Peptide Recovery

In-Solution Tryptic Digestion: Differential Peptide Recovery

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

PCT-Assisted Cell Rupture

Credit: R. Schlicher, R. Apkarian, and M. Baran, www.cchem.berkeley.edu, www.sciencephotolibrary.com

Conventional Cell/Tissue Rupture Approaches

Mechanical stress Ultrasound Osmosis

Pressure Cycling – Assisted Lysis and Protein Extraction

Pressure Cycling-Assisted and Organic Solvent –Assisted Cell Lysis

Pressure Cycling-Assisted and Organic Solvent –Assisted Cell Lysis

Pressure Cycling-Assisted and Organic Solvent –Assisted Cell Lysis

Pressure Cycling-Assisted and Organic Solvent – Assisted Cell Lysis

GO Term Enrichment Analysis. Cellular Localization.

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.

Acknowledgements

Emily FreemanAlexander Lazarev

Vera GrossPBI

Funding:NIEHS, HSPH GCD Department,

CRDF, Harvard Catalyst