1. ProteoRed Multicentric Experiment 5 2D electrophoresis analysis Salamanca, 16-17 March, 2010

2. 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 protein ALDOA_RABIT (Aldolase, Mw 39212), at ~ 25 fmol / 1 mg total protein ALBU_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

3. 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.

4. Gel N. 72051 Gel N.72052 Gel N. 72053 Gel images. Group 1

5. Group 1 (n=3)Group 2 (n=4)Group 3 Detected spots8731096881 Matched spots7501096881 False positives Group 1Group 2 Group 3Group 4 Number21645311 Fold1.5 1.41.3 p-value1.4E-45.0E-51.0E-3 E. coli non spiked proteins Group 1Group 2Group 3Group 4 Avg ratio1.0271.491,01 SD deviation0.3110.910,28 %CV3060.8128 Analysis of the results

6. Group 1 Group 2 Group 4 Expected ratio B/Ap-valueSDratio B/Ap-valueSDratio B/Ap-valueSD CytC1,730,0089 1,58,00E-03 1,350,020,071,5 MYG0,35 (2)3,30E-060,0770,37 (5)7,46E-040,0311,72 (5)0,020,180,38 ALDOA0,56 (5)2,10E-050,0250,59 (2)7,60E-040,0061,36 (5)0,030,050,5 BSA 2,5 (2)8,50E-040,2141 (4)0,350,055 Quantification of the spiked proteins

7. Group 1 Group 3 Group 4 Group 2 Representative gel images

8. 493 T-test 2.10E-05 Ratio -1.73 492 T-test 7.70E-06 Ratio -1.73

9. 858 T-test 1.40e-06 Ratio -2.82 857 T-test 3.30e-06 Ratio -2.93

10. 500 T-test 5.20E-05 Ratio -1.8 372 T-test 0.00062 Ratio -1.79

11. 489 T-test 9.80E-06 Ratio -1.77 495 T-test 1.60E-05 Ratio -1.77

12. 362 T-test 0.00014 Ratio -1.69 926 T-test 0.0089 Ratio -1.73

13. 00 0.0520.112 0.10.208 0.180.366 0.30.665 0.40.873 ug/ul BSAA 570 Xug/ulen 14 ul A0.0820.040780140.415.7 B0.050.026232040.263.7 deberia haber unos 30 ug (aprox a 2 ug/ul) Cuantificacion Cleanup Proteored muestras para Dige