View
31
Download
0
Category
Preview:
DESCRIPTION
Salamanca, 16-17 March, 2010. ProteoRed Multicentric Experiment 5 2D electrophoresis analysis. Samples to be analyzed . Centers involved. - PowerPoint PPT Presentation
Citation preview
ProteoRed Multicentric Experiment 5 2D electrophoresis analysis
Salamanca, 16-17 March, 2010
A mixture of around 150 E. Coli proteins (identical in each sample). This mixture has been prepared by fractionation of the cytoplasmatic proteome of E.Coli. It contains soluble proteins, of a wide range of pI and Mw.
Four spiked mammalian proteins:
CYC_HORSE (Cytochrome C, Mw 12362), added at the ~ 1 pmol/ 1 mg total protein level.MYG_HORSE (Apomyoglobin, Mw 16952), at ~ 200 fmol / 1 mg total proteinALDOA_RABIT (Aldolase, Mw 39212), at ~ 25 fmol / 1 mg total proteinALBU_BOVIN (Serum albumin, Mw 66430), at ~ 1 fmol / 1 mg total protein These four proteins have been spiked in different amounts in samples A and B, with ratios ranging from 1.5:1 to 5:1 between the two samples.
Results available from HUVH, CIB, IIB-UAB and CIMA.
Samples to be analyzed. Centers involved
Experimental procedure
• Data (MIAPE) are available from HUVH and CIMA.
• Sample preparation and application:o Sample buffer: 7M urea, 2M thiourea, 4% CHAPS, 120/40 mM DTT; ampholites pH
range 3-10 (1%) or 3-11 (2%); 1,2% destreak used in HUVH
• First dimension:o Sample loading: cup loading, 150 g HUVH and 9 g CIMA.o IPG strips pH range 3-10 or 3-11 NL.
• Second dimension:o 12,5% acrylamide gels (30%/0.8% or 40%/2.6% acrylamide/bis-acrylamide)
• Inter dimension treatment: No differences.
• Image capture:o Typhoon 9400 or trio with similar settings.
• Image analysis:o Same Spots v 3.3 or Decyder v 6.5. Automated mode.
Gel N. 72051 Gel N.72052
Gel N. 72053
Gel images. Group 1
Group 1 (n=3) Group 2 (n=4) Group 3Detected spots 873 1096 881Matched spots 750 1096 881
False positives Group 1 Group 2 Group2 Group 3 Group 4Number 2 16 45 3 11Fold 1.5 1.5 1.5 1.4 1.3p-value 1.4E-4 5.0E-5 1.0E-3
E. coli non spiked proteins Group 1 Group 2 Group 3 Group 4Avg ratio 1.027 1.49 1,01SD deviation 0.311 0.91 0,28%CV 30 60.81 28
Analysis of the results
Group 1 Group 2 Group 4 Expected
ratio B/A p-value SD ratio B/A p-value SD ratio B/A p-value SD
CytC 1,73 0,0089 1,5 8,00E-03 1,35 0,02 0,07 1,5
MYG 0,35 (2) 3,30E-06 0,077 0,37 (5) 7,46E-04 0,031 1,72 (5) 0,02 0,18 0,38
ALDOA 0,56 (5) 2,10E-05 0,025 0,59 (2) 7,60E-04 0,006 1,36 (5) 0,03 0,05 0,5
BSA 2,5 (2) 8,50E-04 0,214 1 (4) 0,35 0,05 5
Quantification of the spiked proteins
Group 1
Group 3Group 4
Group 2
Representative gel images
493T-test 2.10E-05Ratio -1.73
492T-test 7.70E-06Ratio -1.73
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
-0.14
BA BA B
Sta
ndar
dize
d Lo
g A
bund
ance
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
-0.14
858 T-test 1.40e-06Ratio -2.82
857 T-test 3.30e-06Ratio -2.93
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.25
0.2
0.15
0.1
0.05
0
-0.05
-0.1
-0.15
-0.2
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.25
0.2
0.15
0.1
0.05
0
-0.05
-0.1
-0.15
-0.2
-0.25
500 T-test 5.20E-05Ratio -1.8
372T-test 0.00062Ratio -1.79
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
-0.14
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
-0.14
-0.16
-0.18
-0.2
-0.22
-0.24
-0.26
-0.28
489 T-test 9.80E-06Ratio -1.77
495 T-test 1.60E-05Ratio -1.77
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
-0.14
-0.16
-0.18
BA BA B
Sta
ndar
dize
d Lo
g A
bund
ance
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
362 T-test 0.00014Ratio -1.69
926 T-test 0.0089Ratio -1.73
BA BA B
Sta
ndar
dize
d Lo
g A
bund
ance
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
-0.08
-0.1
-0.12
-0.14
-0.16
-0.18
BA BA B
Stan
dard
ized
Log
Abu
ndan
ce
0.3
0.28
0.26
0.24
0.22
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
0 0
0.052 0.112
0.1 0.208
0.18 0.366
0.3 0.665
0.4 0.873
ug/ul BSA A 570
X ug/ul en 14 ul
A 0.082 0.04078014 0.41 5.7
B 0.05 0.02623204 0.26 3.7
deberia haber unos 30 ug (aprox a 2 ug/ul)
Cuantificacion Cleanup Proteored muestras para Dige
Recommended