Mass Spectrometry a Multi-purpose Tool in Cancer Research
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1 Mass Spectrometry a Multi-purpose Tool in Cancer Research Stephen Barnes,PhD Director, Center for Nutrient-Gene Interaction in Cancer Prevention Director, Mass Spectrometry Shared Facility
2 Cancer Research and Mass Spectrometry Protein target identification and modifications Proteome imaging of tissues Dr. Pierre Chaurand SELDI analysis of body fluid proteomes Dr. David Chhieng
3 Synopsis of this talk How did mass spec emerge? What can mass spec (and proteomics) bring to your research? Time to change the research paradigm?
4 Direction of NIH research for the past 50 years Metabolism Lipid chemistry Protein chemistry Molecular Biology
5 Where did the interest in mass spectrometry come from? Fundamental experiments on the nature of the matter Lord Rutherford In the early 20th century, discovery of isotopes of the elements led to atomic weapons (U 235 /U 238 ) Ernst Chain Niels Bohr Ben Gurion Linus Pauling Post-World War II, generation of isotopes of hydrogen, carbon, nitrogen, phosphorus and sulfur created biomedical research as we know it today David Rittenberg
6 Accelerator MS in PRIME Lab Dr. David Elmore next to 10 MV accelerator
7 What is mass spectrometry? Compounds have to be converted to ions NaCl Na + + Cl - and then analyzed in the gas phase ph 1 ph 6 ph 10 Charge on a protein at different phs
8 What can mass spectrometry measure in 2003? Small molecules metabolomics DNA and Oligonucleotides Peptides and proteins How can we evaporate peptides and proteins?
9 Nobel Laureates in Chemistry John Fenn Koichi Tanaka "for the development of methods for identification and structure analyses of biological macromolecules" and "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules"
10 Electrospray Ionization (ESI) nebulizing gas sample solution +HV N 2 curtain gas Mass Analyzer Atmospheric pressure Solvent evaporation 2. Coulombic repulsion Vacuum [M + nh] n+
11 NanoElectrospray 5 µm
12 ESI spectrum of bacterially expressed protein A A56/B A48/B TOF MS ES+ 552 A: ±12.89 B: ±18.34 C: ±0.94 % C Each ion is [M+nH] n+ A A For 50+ charge state of a 50 kda protein, m/z = [50, ]/50 = 1, m/z Courtesy of Mindan Sfakianos
13 MaxEnt deconvolution of ESI-MS data , Da E. coli GRoEL % 50, Da 6xHis-tag BAT 15, Da 0 mass Courtesy of Mindan Sfakianos
14 How can ESI-MS of proteins help you? Establish that an expressed or isolated protein is authentic C. elegans proteins expressed in bacteria Measure the post-translational changes to protein caused by biochemical or chemical modifications e.g., changes in the mol wt of antibodies (Ahmad Safavy - attachment of radiometal complexing agents) Glycation of antibodies in diabetes
15 Unexpected peptide from a bacterially expressed protein 100 y y MELEMIQLTATPVSALVDEPVHIR y y y % b y1 y2 b b y5 y3 b b y y7 y8 y10 y9 y11 0 mass y y y y y
16 N-terminal extension revealed Antisense 3 -CATCTCGAGCTCCATGGT-5 Reversed Sense 5 -TGGTACCTCGAGCTCTAC-3 -hbat 5 -ACCATGGAGCTCGAGATG-3 -hbat 2nd start site hbat start site Xho1 site where hbat was inserted
17 MALDI - shining the light on your research A focused laser beam, either in the UV or infrared ranges, can evaporate compounds from the solid phase The resulting ions are injected into a tube (1-2 m in length), accelerated and allowed to drift towards a detector. Their time-of-flight is proportional to their (MW) 1/2
18 Matrix-Assisted Laser Desorption Ionization (MALDI) Short laser pulse Flight tube and drift region to measure the time-of-flight (TOF) detector Accelerating pulse
19 Protein analysis 2003 Image analysis Robotic spot picking Desalting Ziptip MALDI plate Destaining. drying and trypsinolysis 2500 Mass (m/z) 3000
20 Tryptic mass fingerprint of a porin from Drosophila YQLDDDASVR MAPPSYSDLG KQARDIFSKG YNFGLWKLDL KTKTSSGIEF NTAGHSNQES 51 GKVFGSLETK YKVKDYGLTL TEKWNTDNTL FTEVAVQDQL LEGLKLSLEG 101 NFAPQSGNKN GKFKVAYGHE NVKADSDVNI DLKGPLINAS AVLGYQGWLA 151 GYQTAFDTQQ SKLTTNNFAL GYTTKDFVLH TAVNDGQEFS GSIFQRTSDK 201 LDVGVQLSWA SGTSNTKFAI GAKYQLDDDA SVRAKVNNAS QVGLGYQQKL 251 RDGVTLTLST LVDGKNFNAG GHKIGVGLEL EA 70 % Intensity LTTNNFALGYTTK LSLEGNFAPQSGNK TSSGIEFNTAGHSNQESGK trypsin autolysis product Mass (m/z)
21 Other Applications of Mass Spec in Cancer Research antibody streptavidin Mapping the protein:protein network by antibody or affinity isolation glutathione biotin Reproducibly sub-dividing the proteome in meaningful ways - cell-to-cell (see Pierre Chaurand s talk this afternoon) or within cells (see Huang-Ge Zhang s work on the composition of the exosome) GST Multiprotein complex
22 Emerging or emerged mass spec methods in proteomics TOF-TOF MUDPIT H/D exchange LC-MRM-MS FT-ICR-MS High speed peptide sequencing - some limitations because of light sensitivity Automated proteomics based on 2D-LC tandem mass spectrometry An alternative to NMR for the study of protein structure in solution An old method revamped for quantitation in proteomics An exotic method whose time has come
23 Detection in the FT-ICR cell
24 Bovine Serum Albumin (66 kda) 4.7 T Act. Shielded Magnet ESI: BSA m/z m/z Bruker Daltonics
25 Sequencing O-GlcNAc peptides by ECD FT-ICR-MS Casein kinase II - AGGSTPVSSANMMSG [Abs. Int. * ] c V S S A N M [M+2H] c8 fragment with sugar attached c 7 c c c c 10 c c m/z R1 O R2 O R3 H 3 N C H C N H C H C N H C H COOH b ion cleavage c ion cleavage
26 The future of NIH research lipidomics proteomics metabolomics genomics
27 Integration for life extension in yeasts Caloric restriction increases yeast life span - dependent on the SIR-2 gene (a sirtuin) - a NAD + -dependent deacetylase - calorie restriction increases NAD + Human equivalent SIRT1 - deacetylates p53 (used K382) Sinclair et al. screened a library of compounds - quercetin and piceatannol emerged as activators of deacetylase OH OH OH HO HO OH piceatannol OH resveratrol Resveratrol increased life span of the yeast by 70% in the absence of caloric restriction, decreased p53 K382 acetylation, and reduced the frequency of repetitive DNA recombination
28 Conclusions Mass spectrometry and proteomics are good partners (aided by the genome effort and computers) MALDI identification of proteins from 1D- and 2D-gels is routine (and available in the Cancer Center - see Mass spectrometry can probe protein:protein interactions and protein structure Mass spectrometry is poised for the question of quantitation in proteomics, the prelude to integration of data from several dimensions (and consistent with the roadmap espoused by NIH Director, Dr. Zerhouni)
29 Acknowledgments Marion Kirk Tracy D Alessandro Landon Wilson Amanda Foxwell Ray Moore II Erin Shonsey (Lori Coward) Sam Wang (Scott Isbell) Jeevan Prasain Ramu Vempati Junlong Shao Helen Kim Jessy Deshane Heath McCorkle Todd Sanderson (Tivanka De Silva) Support from NCI P30 CA13148 NCI U54 CA NCCAM P50 AT00477 NIDDK R01 DK46390 David Rittenberg
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