Optimisation of protein extraction from tissueSusanne Schwonbeck, Nathanaël Lemonnier, Ivo G. Gut

CEA/Institut de Génomique/Centre National de Génotypage, 2, rue Gaston Crémieux, 91000 Evry, France. schwonbeck@cng.fr

IntroductionExtraction of proteins and peptides from body fluids and tissues is a crucial step during sample preparation for proteomic profiling. Body fluids such as serum or plasma are pre-fractionated to break down sample complexity e.g. by liquid chromatography or magnetic bead extraction prior to mass spectrometry. Preparing proteins from tissue or cell culture prior to MALDI-TOF mass spectrometry is a much more demanding step regarding buffer composition and extraction conditions compared to the fractionation of serum or plasma.Here, we present the development of optimised conditions of protein extraction from rat tissue and cell cultures. Several different extraction buffers and disruption methods weretested in order to obtain MALDI-TOF MS compatible protein solutions.

MethodsRat tissues and lymphoblastoid cell lines were used to extract proteins and peptides. Buff-ers containing detergents, salts and chaotropes were tested as well as mechanical disrup-tion with glass beads (Qiagen TissueLyser), with a protein extraction kit (Allprep DNA/RNA/Protein kit, Qiagen) and targeted ultrasound (KBiosiences,Covaris). The extracts were tested for protein concentration (BCA assay), for frag-ment composition (SDS PAGE) and were either applied to magnetic beads for fractionation (Bruker Daltonic) or directly applied to MALDI-TOF MS (UltraflexII, Bruker Daltonics) after mixing with alpha-Cyano-4-hydroxycinnamic acid.

Cell Pellet

Mechanical disruption/glass beadsTissueLyser, Qiagen

Targeted ultrasoundCovaris E210, KBiosciences

WCX beads fractionation

MALDI-MS

Lysis Buffer

Detergents

Additives

Tris HCl 50mM ± Ammonium Phosphate 150mM pH 7,5

CHAPS 1% 614.88 Da zwitterionicNP-40 1% ca. 680 Da non-ionicMEGA-10 1% 349.46 Da non-ionicSodium Deoxycholate 4% 414.55 Da anionic

Any combination with:± Protein Cocktail Inhibitor± chaotropes : Urea 7M / Thiourea 2M± reducing agent : Dithiothreitol 50mM

Centrifugation

culture media removed

Cell lysate

supernatant =soluble proteins

Centrifugation

Mechanical disruptionVortex

SDS-PAGEBCA Assay

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72 359 372 678 1275 1438

Extractionwith lysis buffer

ExperimentalWorkflow

Cell Culture

Buffer Tests Test of Extraction Methods

Acknowledgements

ResultsDetergents Test

Additives

Applications

Extraction 1basic buffer

Extraction 2 of pelletwith detergent

Test of Extraction Methods

Conclusion

SDS-PAGE of protein extracts with four different deter-gents on Covaris E210. Coomassie Staining. M: Marker; no det.: extraction with basic buffer and salt (Tris HCl 50 mM + Ammonium Phosphate 150 mM) without detergent; lanes 3-6 respectively with CHAPS, NP-40, MEGA-10 andSodium Deoxycholate. Gel like view representing spectra obtained with MALDI-TOF MS analysis on protein extracts with

four different detergents. red: basic buffer Tris HCl 50 mM; light green: Tris HCl 50 mM + Ammo-nium Phosphate 150 mM; blue: CHAPS; yellow : NP-40; purple: MEGA-10; dark green: Sodium De-oxycholate. Software: Bruker’s ClinProTools 2.2.

SDS-PAGE of protein extracts with 8 different lysis buffers. Coomassie Staining. no det.: basic buffer + salt (Tris HCL 50 mM + Ammonium Phosphate 150 mM); M: Marker; no add.: basic buffer + salt + NP-40; lanes 4-10: basic buffer +salt + NP-40 + additives (PIC and/or Urea/Thiourea and/or DTT).

Gel like view on protein extracts with different lysis buffers. red: basic buffer + salt + CHAPS;green: same as red + PIC; blue: + Urea/Thiourea; yellow: + DTT.

Gel like view on protein extracts. red: extraction 1 from cells, supernatant = cytosolic fraction; green: extraction 2 from extraction 1 pellet = further ex-traction from membranes.

BCA Assay on protein extracts with three different methods on Cells and Rat liver. red: Covaris E210; green: TissueLyser; blue: Vortex. Lysis buffer: Tris HCl 50 mM + AmmoniumPhosphate 150 mM. Cell line 678.

Gel like view from extracts with three different methods on Cells. red: Covaris E210; green: TissueLyser; blue: Vortex. Lysis buffer: Tris HCl 50 mM + Ammonium Phosphate 150 mM. Cell line 678.

The optimisation of protein extractions aims to provide a suitable protein extract, which can be used for downstream MALDI-TOF MS analysis.

Complete extraction method tests showed that the targeted ultrasound (Covaris E210) is a very power ful disruption technique and more efficient than other meth- ods. Peaks obtained with MALDI-TOF MS cover a wide range.

CHAPS and NP-40 are the detergents of choice. MEGA10 and sodium deoxycho- late were not suitable. NP-40 gives a higher number of peaks than CHAPS, but peak resolution is less good.

Additives to the basic buffer provide an increase of peak number, e.g. chaotropes support solvation of proteins.

An analysis of different cell lines has been shown. Systematic differences can be observed. Further experiments are underway.

Although buffer additives help the extraction, sequential extraction might be the method of choice. Double extraction separates the cytosolic fraction from the lysate pellet proteins (debris, membranes). The resulting solutions are less complex and therefore better suited for analysis than a single fractionation.

MALDI-TOF MS analysis

3D Principal Component Analysis of protein extracts of different cell lines. red: 72; green: 359; blue: 372; yellow: 678; purple: 1275; dark green: 1438.

1 2 3 4 5 6 7M no det. CHAPS NP-40 MEGA10 NaDeoxych. M

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1 2 3 4 5 6 7 8 9 10no det. M + PIC + Urea + DTT + PIC + Urea + PIC + PIC + Urea + DTT + DTT + Urea + DTT

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1 2 3 4 5 6 7 Cell Culture Cells Rat LiverM Cova TL Vortex Cova TL Vortex

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3SDS-PAGE of protein extracts with three different methods on Cells and Rat liver. Silver Staining. M: Marker; Cova: Co-varis E210 ultrasonication extraction; TL: TissueLyser me-chanical extraction; Vortex: simple vortex extraction. Lysis buffer: Tris HCl 50 mM + Ammonium Phosphate 150 mM.

The work was supported by the EU FP6 Integrated Project GABRIEL.We would like to thank the EGEA cohort for giving us access to blood samples for the cultivation of lymphoblastoid cell lines.

Gel like view on protein extracts from different cell lines. red: 72; green: 359; blue: 372; yellow: 678; purple: 1275; dark green: 1438.

BCA Assay on protein extractions with four different detergents.red: basic buffer Tris HCl 50 mM; light green: Tris HCl 50 mM + Ammonium Phosphate 150 mM; blue: CHAPS; yellow: NP-40;purple: MEGA-10; dark green: Sodium Deoxycholate.

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