Poster presented at the 8th Annual HUPO World Congress

Optimisation of protein extraction from tissue
Susanne Schwonbeck, Nathanaël Lemonnier, Ivo G. Gut
CEA/Institut de Génomique/Centre National de Génotypage,
2, rue Gaston Crémieux, 91000 Evry, France. [email protected]
Introduction
Extraction 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 prefractionated 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 were
tested in order to obtain MALDI-TOF MS compatible protein solutions.
Experimental
Workflow
Cell Culture
Centrifugation
Extraction
culture media removed
with lysis buffer
Cell Pellet
Cell lysate
n
io
t
a
g
u
f
entri
C
supernatant =
soluble proteins
Methods
Rat tissues and lymphoblastoid cell lines were used to extract proteins and peptides. Buffers containing detergents, salts and chaotropes were tested as well as mechanical disruption 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 fragment composition (SDS PAGE) and were either applied to magnetic beads for
fractionation (Bruker Daltonic) or directly applied to MALDI-TOF MS (Ultraflex
II, Bruker Daltonics) after mixing with alpha-Cyano-4-hydroxycinnamic acid.
BCA Assay
WCX beads fractionation
SDS-PAGE
4500,0
4000,0
3500,0
3000,0
2500,0
2000,0
1500,0
1000,0
500,0
0,0
72
359
372
678
1275
1438
MALDI-MS
Buffer Tests
Lysis Buffer
Tris HCl 50mM ± Ammonium Phosphate 150mM pH 7,5
Detergents
CHAPS 1%
614.88 Da
NP-40 1%
ca. 680 Da
MEGA-10 1%
349.46 Da
Sodium Deoxycholate 4% 414.55 Da
Test of Extraction Methods
Targeted ultrasound
Mechanical disruption/glass beads
Covaris E210, KBiosciences
TissueLyser, Qiagen
Mechanical disruption
Vortex
zwitterionic
non-ionic
non-ionic
anionic
Additives
Any combination with:
± Protein Cocktail Inhibitor
± chaotropes : Urea 7M / Thiourea 2M
± reducing agent : Dithiothreitol 50mM
Results
Detergents Test
Test of Extraction Methods
3500
3000
1
2500
1
M
2
no det.
3
CHAPS
4
NP-40
5
6
7
MEGA10 NaDeoxych. M
M
2000
2
3
4
Cell Culture Cells
Cova
TL
Vortex
5
Cova
6
7
Rat Liver
TL
Vortex
1500
1000
188
500
188
0
kDa
900
kDa
800
ug/ml
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.
38
700
600
500
400
38
300
200
100
17
ug/ml
C ells
R at Liver (1/20)
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.
3
3
SDS-PAGE of protein extracts with four different detergents 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 and
Sodium Deoxycholate.
1
2
no det. M
0
17
3
4
5
6
7
8
9
10
+ PIC + Urea + DTT + PIC + Urea + PIC + PIC
+ Urea + DTT + DTT + Urea
+ DTT
SDS-PAGE of protein extracts with three different methods
on Cells and Rat liver. Silver Staining. M: Marker; Cova: Covaris E210 ultrasonication extraction; TL: TissueLyser mechanical extraction; Vortex: simple vortex extraction. Lysis
buffer: Tris HCl 50 mM + Ammonium Phosphate 150 mM.
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 + Ammonium Phosphate 150 mM; blue: CHAPS; yellow : NP-40; purple: MEGA-10; dark green: Sodium Deoxycholate. Software: Bruker’s ClinProTools 2.2.
Additives
188
kDa
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.
38
Conclusion
17
3
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.
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 methods. Peaks obtained with MALDI-TOF MS cover a wide range.
Applications
Extraction 1
basic buffer
Extraction 2 of pellet
with detergent
MALDI-TOF MS analysis
CHAPS and NP-40 are the detergents of choice. MEGA10 and sodium deoxycholate 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.
3D Principal Component Analysis of protein extracts of different cell lines.
red: 72; green: 359; blue: 372; yellow: 678; purple: 1275; dark green: 1438.
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.
Acknowledgements
Gel like view on protein extracts from different cell lines.
red: 72; green: 359; blue: 372; yellow: 678; purple: 1275; dark green: 1438.
Gel like view on protein extracts. red: extraction 1 from cells, supernatant =
cytosolic fraction; green: extraction 2 from extraction 1 pellet = further extraction from membranes.
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.