02hnov05p2

02hnov05p2 - Harvard-MIT Division of Health Sciences and...

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Harvard-MIT Division of Health Sciences and Technology HST.508: Genomics and Computational Biology Protein1: Last week's take home lessons • Protein interaction codes(s)? • Real world programming • Pharmacogenomics : SNPs • Chemical diversity : Nature/Chem/Design • Target proteins : structural genomics Folding, molecular mechanics & docking • Toxicity animal/clinical : cross-talk 1
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Protein2: Today's story & goals • Separation of proteins & peptides • Protein localization & complexes • Peptide identification (MS/MS) – Database searching & sequencing. • Protein quantitation – Absolute & relative • Protein modifications & crosslinking • Protein - metabolite quantitation 2
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Why purify? • Reduce one source of noise (in identification/quantitation) • Prepare materials for in vitro experiments (sufficient causes) • Discover biochemical properties 3
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(Protein) Purification Methods • Charge: ion-exchange chromatography, isoelectric focusing •S i z e : dialysis, gel-filtration chromatography, gel-electrophoresis, sedimentation velocity • Solubility: salting out • Hydrophobicity: Reverse phase chromatography • Specific binding: affinity chromatography • Complexes: Immune precipitation (± crosslinking) •D e n s i t y : sedimentation equilibrium 4
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Protein Separation by Gel Electrophoresis • Separated by mass : Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. – Sensitivity: 0.02ug protein with a silver stain. – Resolution: 2% mass difference. • Separated by isoelectric point (pI) : polyampholytes pH gradient gel. – Resolution: 0.01 pI. 5
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Comparison of predicted with observed protein properties (localization, postsynthetic modifications) E.coli See Link et al. 1997 Electrophoresis 18:1259-313 (Pub) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9298646&dopt=Abstract) 6
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Computationally checking proteomic data Property Basis of calculation Protein charge RKH YCDE ( N , C ), pKa, pH (Pub) (http://gcg.nhri.org.tw/isoelectric.html#algorithm) Protein mass Calibrate with knowns (complexes) Peptide mass Isotope sum (incl.modifications) Peptide LC aa composition linear regression Subcellular Hydrophobicity, motifs (Pub) Expression Codon Adaptation Index (CAI) 7
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Protein2: Today's story & goals • Separation of proteins & peptides • Protein localization & complexes • Peptide identification (MS/MS) – Database searching & sequencing. • Protein quantitation – Absolute & relative • Protein modifications & crosslinking • Protein - metabolite quantitation 8
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Cell fraction: Periplasm 2D gel: SDS mobility isoelectic pH See Link et al. 1997 Electrophoresis 18:1259-313 (Pub) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9298646&dopt=Abstract) 9
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Cell localization predictions TargetP: using N-terminal sequence discriminates mitochondrion, chloroplast, secretion, & "other" localizations with a success rate of 85%. (pub) (http://www.cbs.dtu.dk/services/TargetP/ ), (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10891285&dopt=Abstract) Gromiha 1999, Protein Eng 12:557-61. A simple method for predicting transmembrane alpha helices with better accuracy.
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This note was uploaded on 01/24/2010 for the course HST. 508 taught by Professor Dr.georgechurch during the Fall '02 term at MIT.

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02hnov05p2 - Harvard-MIT Division of Health Sciences and...

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