ProteoScan Cancer Lysate Arrays. QC and Validation Data

Size: px

Start display at page:

Download "ProteoScan Cancer Lysate Arrays. QC and Validation Data"

Christian Edwards
5 years ago
Views:

1 ProteoScan Cancer Lysate Arrays QC and Validation Data

2 4.5 mm Barcode Cancer Lysate Array Layout PA02 60 mm B1 C2 K3 Ly1 O2 P3 Mel1 Lv2 St3 B2 C3 L1 Ly2 O3 Pr1 Mel2 Lv3 B3 K1 L2 Ly3 P1 Pr2 Mel3 St1 C1 K2 L3 O1 P2 Pr3 Lv1 St2 4.5 mm Sub-arrays B1-B3 Breast (B) C1-C3 Colon (C) K1-K3 Kidney (K) L1-L3 Lung (L) Ly1-Ly3 Lymphoma (Ly) O1-O3 Ovary (O) P1-P3 Pancreas (P) Pr1-Pr3 Prostate (Pr) Mel1-Mel3 Melanoma (Mel) Lv1-Lv3 Liver (Lv) St1-St3 Stomach (St)

3 BSA Backgroun d controls Cy3, Cy5 location controls Layout - Sub Arrays X1 and X2 PA Case Case 2 Case 3 BSA-Cy3 BSA-Cy BSA-Cy3-Cy5 Case 4 Case 5 Case 6 BSA (ug/ml) Buffer Case 7 Case 8 Case 9 Lysate (ug/ml) Empty Case 10 Case 11 Case Case 13 Case 14 Case 15

4 BSA Backgroun d controls Cy3, Cy5 location controls Layout - Sub Arrays X3 PA Case Case 32 Case 33 HEK293T lysate mg/ml Gene specific over expression Lysate 0.5X-0.004X Case 34 Case 35 Case 36 IgG Mix Positive control (Hu, Rab and Mouse) ug/ml Case 37 Case HEK293T Cell lysate Purified ß-actin ug/ml Lysate (ug/ml) Case 39 Case X 0.1X Over expressed Lysate 0.5X 0.1X X 0.1X BSA (ug/ml) BSA-Cy3-Cy BSA-Cy5 BSA-Cy3 ß-Actin IgG Mix Positive control Buffer Empty

5 Sypro-ruby Protein Staining PA02

6 Detection of β-actin PA02 ProteoScan Cancer Lysate Array was probed with 1:200 dilution of anti-β-actin monoclonal antibody followed by DyLight 649 conjugated goat anti-mouse IgG at 1:400 dilution. The array was scanned with GenePix Pro 4A reader with Cy5 setting

Detection of TP53 PA02 ProteoScan Cancer Lysate Array was probed with 1: dilution of anti-tp53 rabbit antibody and detected using TSA enhanced flouremetric method.

7 Detection of TP53 PA02 ProteoScan Cancer Lysate Array was probed with 1: dilution of anti-tp53 rabbit antibody and detected using TSA enhanced flouremetric method. HRP-conjugated goat anti-rabbit IgG at 1:1,000 dilution was used followed by Alexa Flour 647 tyramide at 1:50 dilution. The array was scanned with GenePix Pro 4A reader with Cy5 setting

8 Detection of TP53 PA02 The ProteoScan Cancer Lysate Array 1.0 was probed with 1:150 dilution of anti TP53 rabbit antibody and detected by the Colorimetric TSA enhanced method.

9 Colon Normal Lung Normal Lymphoma Normal Ovary Normal Proatate Normal Colon Cancer Lung Cancer Lymphoma Cancer Ovarrian Cancer Prostate Cancer Breast Normal Kidney Normal Liver Normal Melanoma Normal Pancreas Normal Stomach Normal Breast Cancer Kidney Cancer Liver Cancer Melanoma Cancer Pancreas Cancer Stomach Cancer TP53 in Normal and Cancer Tissues 1, , , , , , < >4 ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti TP53 antibody (1:) and detected with the TSA enhanced method. A cancer differential expression index was created by dividing the readout for each cancer sample with the median from all normal samples of the same tissue. The results are presented as heat map format for all 4 protein concentrations (4 columns) Particular cancer types with higher and lower expression (compare to normal) are easily detected. Results can be further analyzed for individual cancers and samples

10 Colon Normal Lung Normal Lymphoma Normal Ovary Normal Proatate Normal Colon Cancer Lung Cancer Lymphoma Cancer Ovarrian Cancer Prostate Cancer Breast Normal Kidney Normal Liver Normal Melanoma Normal Pancreas Normal Stomach Normal Breast Cancer Kidney Cancer Liver Cancer Melanoma Cancer Pancreas Cancer Stomach Cancer Survivin in Normal and Cancer Tissues 1, , , , , , > < ProteoScan Cancer Lysate Array was probed with rabbit anti survivin (BIRC5) antibody (1:) and detected with the TSA enhanced method. A cancer differential expression index was created by dividing the readout for each cancer sample with the median from all normal samples of the same tissue. The results are presented as heat map format for all 4 protein concentrations (4 columns) Particular cancer types with higher and lower expression (compare to normal) are easily detected. Results can be further analyzed for individual cancers and samples

11 Colon Normal Lung Normal Lymphoma Normal Ovary Normal Proatate Normal Colon Cancer Lung Cancer Lymphoma Cancer Ovarrian Cancer Prostate Cancer Breast Normal Kidney Normal Liver Normal Melanoma Normal Pancreas Normal Stomach Normal Breast Cancer Kidney Cancer Liver Cancer Melanoma Cancer Pancreas Cancer Stomach Cancer Phospho-C Myc in Normal and Cancer Tissues 1, , , , , , > < ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti Phospho c-myc (pt58/s62) monoclonal antibody (1:) and detected with the TSA enhanced method. A cancer differential expression index was created by dividing the readout for each cancer sample with the median from all normal samples of the same tissue. The results are presented as heat map format for all 4 protein concentrations (4 columns) Particular cancer types with higher and lower expression (compare to normal) are easily detected. Results can be further analyzed for individual cancers and samples

and curve fitting was performed with Microvigene TM software and the RPPA module

12 Quantitative Data Analysis Linear SuperCurve To derive one specific expression value for each cancer sample across all 4 dilutions in replicates, array reading and curve fitting was performed with Microvigene TM software and the RPPA module from VigeneTech Inc. The expression level value is represented by the Y0 value for each curve.

13 Colon Normal Lung Normal Lymphoma Normal Ovary Normal Prostate Normal Colon Cancer Lung Cancer Lymphoma Cancer Ovarrian Cancer Prostate Cancer Breast Normal Kidney Normal Liver Normal Melanoma Normal Pancreas Normal Stomach Normal Breast Cancer Kidney Cancer Liver Cancer Melanoma Cancer Pancreas Cancer Stomach Cancer HER2 in Normal and Cancer Tissues (Heat map view before curve fitting) 1, , , , , , < > < >4 ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti- HER2-ERBB2 antibody (1:50) and detected with the TSA enhanced method. A cancer differential expression index was created by dividing the readout for each cancer sample with the median from all normal samples of the same tissue. The results are presented as heat map format for all 4 protein concentrations (4 columns). Particular cancer types with higher and lower expression (compare to normal) are easily detected. Results can be further analyzed for individual cancers and samples

14 Breast Normal Colon Normal Breast Cancer Colon Cancer SC SC SC SC SC SC Kidney Normal Lung Normal Kidney Cancer Lung Cancer Liver Normal Lymphoma Normal Liver Cancer Lymphoma Cancer Melanoma Normal Ovary Normal Melanoma Cancer Ovarian Cancer Pancreas Normal Prostate Normal Pancreas Cancer Prostate Cancer Stomach Normal Stomach Cancer HER2 in Normal and Cancer Tissues (Heat map view after curve fitting) < >12 ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti- HER2-ERBB2 antibody (1:50) and detected with the TSA enhanced method. Expression level were calculated using the SuperCurve curve fitting mode of the RPPA module from VigeneTech Inc. A cancer differential expression index was created by dividing the readout for each cancer sample with the median from all normal samples of the same tissue. The results are presented as heat map format after curve fitting (one column). A Curve fitting model is the preferred analysis method and provide a more accurate relative expression levels results

15 HER2 Expression in Breast Tissue (Y0 value) ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti HER2-ERBB2 as before. Results were calculated using the RPPA module from VigeneTech Inc and the expression level value (Y0) determined. 17 cancer samples and two normal samples have higher expression of HER2-ERBB2.

HER2 Expression Validated by IHC Data HER2 Expression in breast cancer and normal tissues ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti- ERBB2 as before.

16 HER2 Expression Validated by IHC Data HER2 Expression in breast cancer and normal tissues ProteoScan Cancer Lysate Array (PA02) was probed with rabbit anti- ERBB2 as before. Results were calculated as ratio of Y0 of each sample divided by the median of Y0 from all normal breast samples. For comparison with IHC data on record the data was converted to three expression levels. IHC expression scale: 1= Normal expression 2= Moderate over-expression 3= High over-expression RPPA expression converted to IHC scale: 0-5 fold = 1(Normal expression) fold = 2 (Moderate over-expression) > 20 fold = 3 (High over-expression) 21 of the 25 samples (84%) matched the IHC data. two samples had higher expression than detected by IHC and two samples had positive IHC not detected by our panel.

17 ,328 7,460 5,443 1, ,078 21,703 2,099 2,234 1,060 3,491 3, ,608 2, ,250 7,377 21,810 1, , , BC1 BC2 BC3 BC4 BC5 BC6 BC7 BC8 BC9 BC10 BC11 BC12 BC13 BC15 BC16 BC17 BC18 BC19 BC20 BC21 BC22 BC23 BC24 BC25 HEK293T BN1 ERBB2 BN2 BN3 BN4 BN5 Empty BN7 BN8 BN9 BN10 BN11 BN12 BN13 BN15 HER2 Expression in Breast Tissues Sample WB RPPA IHC (from File) Quantitative Results of ERBB2-Her2 expression measured with the ProteoScan Cancer Lysate Array (RPPA) were confirmed by Western blot analysis of breast samples (10 ug) and matches the IHC data from the donor pathology report.

1,760 3,263 1,709 1,103 834 3,967 2,644 1,009 2,835 2,072 4,220 4,142 15,771 2,830 15,282 5,217 6,607 7,053 6,279 1,406 3,754 4,398 820 1,397 731 510 889 721 622 816 454 1,620 1,612 649 541 958 746

18 1,760 3,263 1,709 1, ,967 2,644 1,009 2,835 2,072 4,220 4,142 15,771 2,830 15,282 5,217 6,607 7,053 6,279 1,406 3,754 4, , ,620 1, OC1 OC2 OC3 OC4 OC5 OC6 OC7 OC8 OC9 OC10 OC11 OC12 OC13 OC14 OC15 ND OC17 OC18 OC19 OC20 OC21 OC22 OC23 OC24 OC25 HEK293T ON1 ON2 ON3 ON4 ON5 ON6 ON7 ON8 ON15 ON10 ON11 ON12 ON13 ON14 ERBB2 HER2 Expression in Ovarian Tissues Sample WB RPPA Quantitative Results of ERBB2-Her2 expression measured with the ProteoScan Cancer Lysate Array (RPPA) were confirmed by Western blot analysis of ovarian samples (10 ug)

19 RFU RFU IgG Mix Positive Control IgG mix Results 3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 1,000, ,000 1,800,000 1,600,000 1,400,000 1,200,000 1,000, , , , , ug/ml IgG y = x R 2 = ug/ml IgG A mix of Human, Mouse and Rabbit IgG each at 0-10 ug/ml are spotted on each 3 rd sub-array. ProteoScan Cancer Lysate Array (PA02) was probed with 1:400 dilution of DyLight 649 conjugated goat anti- mouse IgG and scanned with GenePix Pro 4A reader with Cy5 setting. The IgG curve is linear from 0-5 ug/ml with outstanding repetition The results from the IgG positive control mix can be used to convert the array results to actual protein concentrations The graph present the average and SD of each concentration of spots from the entire array (33 each)

20 RFU Purified β-actin Control Purified Recombinant b-actin 3,000,000 2,500,000 2,000,000 1,500,000 1,000, ,000 y = x R 2 = ug/ml b-actin The β-actin curve is linear from 0-10 ug/ml with outstanding repetition The results from the β-actin control can be used to convert the array results to actual protein concentrations Purified recombinant β- actin at 0-10 ug / ml are spotted on each 3 rd subarray. ProteoScan Cancer Lysate Array (PA02) was probed with anti β-actin diluted 1:200 and detected with 1:400 dilution of DyLight 649 conjugated goat antimouse antibody. The graph present the average and SD of each concentration for spots from the entire array (33 each)

21 14.2 mm Label Assay Validation Array Layout PA01 51 mm 22.5 mm

22 BSA Backgroun d controls Cy3, Cy5 location controls Assay Validation Array Layout PA01 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 HEK293T lysate mg/ml TP53 over expression Lysate 0.5X-0.004X S13 S14 S13 S14 S15 S15 S16 S16 S17 S17 S18 S18 S19 S20 S21 S22 S23 S24 S19 S20 S21 S22 S23 S24 IgG Mix Positive control (Hu, Rab and Mouse) ug/ml S25 S26 S25 S26 S27 S27 S28 S28 S29 S29 S30 S30 S31 S32 S31 S HEK293T Cell lysate Tissue Lysate samples 1,000 ug/ml S25 S26 S33 S34 S33 S34 S33 S34 S27 S35 S35 S35 S28 S36 S36 S36 S29 S37 S37 S37 S30 S38 S38 S38 S31 S32 S39 S40 S39 S40 S39 S X 0.1X X Over expressed Lysate X 0.1X BSA (ug/ml) BSA-Cy3-Cy5 S41 S42 S43 S BSA-Cy5 BSA-Cy3 S41 S42 S43 S44 IgG Mix Positive control Buffer Empty S41 S42 S43 S44

23 Sypro-ruby Protein Staining PA01

Detection of TP53 PA01 The Assay Validation Array was probed with 1:200 dilution of anti TP53 rabbit antibody and detected using TSA enhanced flouremetric method.

24 Detection of TP53 PA01 The Assay Validation Array was probed with 1:200 dilution of anti TP53 rabbit antibody and detected using TSA enhanced flouremetric method. HRP-conjugated goat anti-rabbit IgG at 1:1,000 dilution was used followed by Alexa Flour 647 tyramide at 1:50 dilution. The array was scanned with GenePix Pro 4A reader with Cy5 setting

25 Detection of TP53 PA01 The ProteoScan Assay Validation Array was probed with 1:150 dilution of anti TP53 rabbit antibody and detected by the Colorimetric TSA enhanced method.

Detection of β-actin PA01 The ProteoScan Assay Validation Array was probed with 1:200 dilution of anti β-actin mouse monoclonal antibody followed by

26 Detection of β-actin PA01 The ProteoScan Assay Validation Array was probed with 1:200 dilution of anti β-actin mouse monoclonal antibody followed by DyLight 649 conjugated goat anti-mouse IgG at 1:400 dilution. TSA enhanced method was used. The array was scanned with GenePix Pro 4A reader with Cy5 setting

condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1%

FIGURE LEGENDS Supplementary Fig 1 (A) sumoylation pattern detected under denaturing condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1% SDS in the presence and absence