Symposium on Protein Fractionation
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1 Symposium on Protein Fractionation Proteomic Analysis of Organ-Specific Breast Cancer Metastasis Speaker: Emily I. Chen, Ph.D The Scripps Research Institute Department of Cell Biology
2 CANCER METASTASIS Modified from Welch D.R. et al. Journal of the National Cancer Institute, 91(16), 1999:
3 BREAST CANCER METASTASIS Brain Metastasisc Lymph Nodes Metastasis Liver Metastasis Parent Breast Cancer Cell Lung Metastasis Bone Metastasis
4 ULTIMATE GOAL OF THE STUDY Brain Metastasisc Protein Signature of Organ-Specific Metastasis Parent Bone Brain Parent Breast Cancer Cell Bone Metastasis
5 Characterization of Organ-Specific Breast Cancer Metastasis Step 1: Generation of organ-targeted cancer cells QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Brain Metastasis Heterogeneous Parent Breast Cancer Cells Tumor/Host Interaction QuickTime and TIFF (Uncompressed) are needed to see thi Bone Metastasis Step 2: Proteome analysis of organ-targeted cancer cells Fractionation of complex cell lysate + MudPIT (ProteomeLab PF 2D)
6 ProteomeLab PF 2D PROTEIN FRACTIONATION SYSTEM 1D Isoelectric focusing 2D reverse phase By Beckman Coulter
7 MUDPIT (Multidimensional Protein Identification Technology) 1.Sample preparation 3. Purification of peptides 5. MudPIT 2. Digestion Digested Peptides SCX RP 4.Preparation of column 2D Chromatographic Separation of Peptides Identify Proteins in Mixture Mass spectrometer
8 PROTEOME ANALYSIS OF CELL LYSATES 1D Protein Fractionation (1mg) Protein Precipitation Rapigest + Trypsin Digest 2D LC-MS/MS Analysis
9 Protein Fractionation by ProteomeLab PF 2D System 1 fraction 10 fractions ph Tracer AU Minutes
10 Protein Fractionation by ProteomeLab PF 2D System BCM2 Bone BCM2 Parent AU BCM2 Brain Minutes
11 PEPTIDE FILTERING CRITERIA +1 Xcorr Xcorr Xcorr 3.5 Minimum DeltCN: 0.08 Minimum Length: 7 a.a. Maximum Length: 100 a.a. BCM2 Parent BCM2 Bone BCM2 Brain Peptide IDs 26,577 26,739 29,222
12 PROTEIN IDENTIFICATION IN THREE METASTATIC BREAST CANCER CELL LINES Total Protein Identified: 3688 Parameter: minimum 2 peptides, half tryptic BCM-2 Parent BCM-2 Bone BCM-2 Brain
13 SPECIFICITY OF PROTEIN SEPARATION Percentage of protein IDs BCM2 Parent BCM2 Bone BCM2 Brain # of fractions More specific More abundant
14 SPECIFICITY OF PROTEIN SEPARATION BCM2 Parent ALL No Histones Single Elution F Fraction Number ph
15 Comparison Of Identified Proteins According To PI BCM2 Paren BCM2 Bone BCM2 Brain <4 4 <x< 5 5<x<6 6<x<7 7<x<8 8 <x<9 9 <x<10 10 <x<11 11 <x<12 >12 PI
16 Comparison Of Identified Proteins According To MW BCM2 Parent BCM2 Bone BCM2 Brain <10000 >10000 >20000 >30000 >40000 >50000 >60000 >70000 >80000 >90000 > > > MW (Da)
17 CELLULAR LOCALIZATION OF IDENTIFIED PROTEINS Total Protein Identified: % 56% 13% 12% cytoplasm nucleus membrane extracellular unknown 2%
18 Semi-Quantitative Analysis of Differential Proteome Expression Anal. Chem. 2004, 76, A Model for Random Sampling and Estimation of Relative Protein Abundance in Shotgun Proteomics Hongbin Liu, Rovshan G. Sadygov, and John R. Yates, III
19 Semi-Quantitative Protein Expression Analysis Based on Spectra Counts NP_ Aryl hydrocarbon receptor interacting protein;aryl hydrocarbon receptor-interacting protein; HBV-X associated protein [Homo sapiens] BCM2 Parent U Peptide Sequence Xcorr DeltCN SpeCount SeqCov * R.EGEIAQFLCDIK.H * K.EQPGSPEWIQLDK.Q * K.AHAAVWNAQEAQADFAK.V * K.VLELDPALAPVVSR.E total BCM2 Bone * Y.RTLHSDDEGTVLDDSR.A * K.VLELDPALAPVVSR.E total BCM2 Brain 13 peptides identified total
20 Aryl Hydrocarbon Receptor Interacting Protein H.AAVWNAQEAQADFAK.V
21 Semi-Quantitative Protein Expression Analysis Based on Spectra Counts gi ref NP_ hydroxyacyl dehydrogenase, subunit B; 3-ketoacyl-Coenzyme A thiolase [Homo sapiens] BCM2 Parent U Peptide Sequence Xcorr DeltCN SpeCount SeqCov * R.TPFLLSGTSYK.D * R.AALTGLLHR.T * K.AQDEGLLSDVVPFK.V * K.DQLLLGPTYATPK.V BCM2 Bone total * R.NVVVVDGVR.T * R.AALTGLLHR.T * R.LAAAFAVSR.L * R.LEQDEYALR.S * M.IVEAYPK total BCM2 Brain 16 peptides identified total Express in Brain/Neuron but not Breast
22 SUMMARY Aim 1: Fractionate complex mammalian cell lysate Analyze and obtain protein expression profiles Aim 2: Generate differential protein expression analysis Build on more specific hypotheses Aim 3: Biological interrogation
23 IMPORTANT ISSUES FOR PROTEIN FRACTIONATION Starting Material Sample Complexity High abundant proteins Protein Solubility Post-translation protein modifications
24 IMPORTANCE OF PROTEIN FRACTIONATION High Abundance Medium Abundance Low Abundance Protein Fractionation Reduce Sample Complexity Increase Protein Identification
25 ACKNOWLEDGEMENT The Scripps Research Institute Department of Cell Biology John R. Yates III, Professor Johannes Hewel, Ph.D Department of Molecular & Experimental Medicine Brunhilde Felding-Harbermann, Associate Professor
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