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