Integrated Proteomic and Genomic Analysis of Gastric Cancer

Transcription

1 SUPPORTING INFORMATION PAGE S-1 Integrated Proteomic and Genomic Analysis of Gastric Cancer Patient Tissues Julia Fangfei Yan 1, Hoguen Kim 2, Seul-Ki Jeong 3, Hyoung-Joo Lee 3, Manveen K. Sethi 4, Ling Y. Lee 4, Ronald C. Beavis 5, Hogune Im 6, Michael P. Snyder 6, Matan Hofree 7, Trey Ideker 8, Shiaw-lin Wu 1, Young-Ki Paik 2,3, Susan Fanayan 9 and William S. Hancock 1* 1 Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, United States 2 Yonsei University College of Medicine, Yonsei University, 50-1 Yonsei-Ro, Seodaemun-gu, Seoul , Korea 3 Yonsei Proteome Research Center, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul , Korea 4 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia 5 Department of Biochemistry and Medical Genetics, Faculty of Health Sciences, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, Manitoba R3E 0J9, Canada 6 Department of Genetics, Stanford University, Stanford, California 94305, United States 7 Department of Computer Science and Engineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States 1

2 8 Program in Bioinformatics, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States 9 Department of Biomedical Sciences, Macquarie University, Sydney, New South Wales 2109, Australia Supplementary Table S-1. Clinical parameters of the study group which were selected. Part a. Scoring criteria from immunohistochemistry. Score IHC staining pattern ERBB2 overexpression assessment 0 no reactivity or membranous reactivity in <10% of tumor cells 1+ Faint/barely perceptible membranous reativity in > 10% of tumor cells; cells are reactive only in part of their membrane 2+ Weak to moderate complete, basolateral, or lateral membranous reactivity in >=10% of tumor cells Negative Negative Equivocal 3+ Strong complete, basolateral, or lateral membranous reactivity in 10% of tumor cells Positive 2

3 Part b. ERBB2+gastric cancer set Case # IHC staining FISH Age Sex T stage N stage Stage Microsatellite instability 1 positive positive 60 F T3 N3a IIIb stable dead 2 positive positive 67 M T3 N3a IIIb stable alive 3 positive positive 78 F T4a N3a IIIc low dead 4 positive positive 69 M T3 N3a IIIb stable alive 5 positive positive 64 M T4a N1 IIIa stable alive 6 positive positive 69 M T4a N2 IIIb stable alive 7 positive positive 59 M T4a N2 IIIb N/A alive 8 positive positive 75 M T4a N1 IIIa N/A alive survival (up to ) Part c. ERBB2- patient sample sets Case # IHC staining FISH Age Sex T stage N stage Stage Microsatellite instability survival (up to ) 1 positive negative 72 M T3 N3a IIIb low dead 2 positive negative 55 M T3 N3a IIIb stable alive 3 negative N/A 65 M T3 N2 IIIa N/A dead 4 negative N/A 72 F T4a N3a IIIc N/A dead 5 negative N/A 62 M T3 N3a IIIb N/A alive 6 negative N/A 75 M T3 N2 IIIa stable alive 7 negative N/A 54 M T3 N3a IIIb stable dead 8 negative N/A 64 M T3 N2 IIIa stable alive 9 negative N/A 69 M T3 N2 IIIa stable alive 3

4 Supplementary Figure S-1. SDS PAGE separation of extracted proteins from gastric cancer patient tissues. Extracted proteins from the selected gastric cancer patient tissues were separated by SDS-PAGE, followed by in-gel digestion and LC-MS/MS analysis. For ERBB2+ set, there are 8 sets (8 patients). For ERBB2- sample set, there are 9 sets (9 patients). Each set includes a tumor and a corresponding control sample, illustrated in two gel lanes, respectively. This gel image only shows a single set, tumor (left lane) and control (right lane). 4

5 Supplementary Figure and Table S-2. Example of MS/MS spectra of a unique peptide identified in ERBB2 and EGFR and identification of ERBB2. Supplementary Figure S-2a. An example of the MS/MS identification of a unique peptide for ERBB2 (a) and EGFR (b) Precursor m/z (exact mass measurement) and MS2 spectra are illustrated for these examples (major b and y ions are labeled for clarification). 5

6 Supplementary Table S-2b: A catalog of quality observed peptides for ERBB2_HUMAN in the gastric cancer sample sets together with corresponding GPMDB d. Peptide sequence FVVIQNEDLGP ASPLDSTFYR LLDIDETEYHA DGGK AVTSANIQEFAG CK NPQLCYQDTIL WK VLGSGAFGTVY K LGSQDLLNWC MQIAK VCYGLGMEHL R Charge state observed in GI cancer samples Rank in GI cancer samples Number of sets observed in GI cancer (spectral counts) (z=2) Rank in GPMDB Charge in GPMDB Observed in GPMDB z=1 z=2 z= (10) 3 2, (12) 9 2, (7) (4) (2) 1 1, (2) (1) 25 2, d GPMDB (The Global Proteome Machine Database): The rank in GPMDB is determined by the quality of the MS/MS results and the number of observations in different proteomic studies. 6

7 Supplementary Figure S-3. Violin plots summarize the histograms for the list of oncogenes. Each violin plot is basically a smoothed histogram turned on its side. The quantile normalized RPKM values are overlay for the cell lines on top of these. In this analysis TCGA, level 3, RNA-Seq data (RPKM normalized) for 58 gastric cancer tumor samples were downloaded from the TCGA data portal on Mar TCGA patient tumor genome wide expression measurements were merged with RPKM values for the set of examined cell lines. The joint data set of patient and cell line expression was then quantile normalized. We plot TCGA expression measurements of selected genes using violin plots (smoothed kernel based density estimates). RNA-Seq RPKM values for the cell line is indicated by symbols shown on top of the violin generated for each gene. 7

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10 d Supplementary Figure S-4. Illustration of ERBB family signaling and EGFR development pathways together with the corresponding proteomics and transcriptomic data: a. ERBB2 signaling; b. development EGFR signaling via small GTPases; c. development EGFR signaling; d. cytoskeleton remodeling integrin outside-in signaling. Pathways are from GeneGo database. Blue stars: oncogenes. Red stars: up-regulated in disease/tumor set in ERBB2+. Highlighted in yellow: tumor unique proteins. Blue brackets: colocalization. Red brackets: co-expression of genes in the same genomic region. 10