GENOMICS PROTEOMICS GENOME 372
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This 16 page Class Notes was uploaded by Rupert Davis on Wednesday September 9, 2015. The Class Notes belongs to GENOME 372 at University of Washington taught by J. Bruce in Fall. Since its upload, it has received 35 views. For similar materials see /class/192402/genome-372-university-of-washington in Genome Sciences at University of Washington.
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Date Created: 09/09/15
Genomic amp Pmteumicx r nal Exam smdy Qumiuns m mmmwmg ltv m m w a H mGaHusgalus mm mm ymprea output or Kentucky yrea cncoz Ho 97gt a 4m Vanna Vanna roman a 2a on em m mu m m m m 2 a c eavagesquot 3 y mtEractmgpannErSDprSV 4 W Thegmupsfnund swgn cant dwffErEncEsntharrEsu ts Whmh strE accuraE and Wm 5 Mn 9 9 VH4 mm and Exp am ts swgm cancE succEsswE y scan 5H WEE mnsv WW7 RF amplitude m bEgm u rEmammgmassEs m yuur spE rum m E76 311 5 Whmh Emma and stSI hke ythenen mm 52mm NamEtwn mamas prub Ems m w D ssmmnmdammmgpmspmmam mmapmmmmmm 11 F N F W 4 F U1 F Gquot F I F 00 F4 k0 N C In the phosphopeptide heat maps below describe which set is acidophilic and why q 4 3 K2ltHmwcmzzrxummmmangt N2ltAmwomzxrwumnmhangt A c n E F G H I x L M N p 9 R s 1 v w Y A group interested in developing vaccines against specific virus used a genome engineering approach They created a mutant strain of the virus whose genome encoded the same proteins as the wild type strain but did so with a significantly lower codon adaptation index CAI What effect do you think this would have on transcription rates On translation rates On the replication rate of the virus Can this be an effective strategy to develop vaccines Tandem mass spectrometry is a critical tool in proteomics Describe two different ways in which tandem MS is currently employed in proteomics research Describe how the yeast two hybrid method works what the data indicates and the strengths and weaknesses of this approach In the YZH paper by Uetz et al two different methods were used to acquire data Describe how each method works and the benefits of each Compare and contrast the challenges of genomic and proteomic measurements including in your answer the specific challenges that are unique to each Describe how specificity of detection is achieved in genomics and proteomics research methods Describe why 2D gels are used in proteomics research and how quantitative measurements can be achieved Indicate which of the following 4 peptides can be uniquely identified by mass spectrometry methods discussed in class and which cannot and describe why YHLIFK YIHFLK ILFHYK YHILFK Below is an example of a MASCOT search result Indicate what the green shaded area and the bars present in this area represent Number OF H 1 ts 21 Quadrupole mass spectrometers utilize mass selective stability What does this mean and why does this 75 Probability Based 100 House Score make the quadupole a unique mass spectrometer 22 Why do you think cytoplasmic proteins were the most commonly observed proteins in all fractions from the MUDPIT paper 23 Here is a representative list from GPM results a Based on the spectral counting method list the proteins in order of abundance high to low b The expectation score and protein coverage are two ways to gauge confidence in protein identification List the lowest ranked protein in each case and explain why they might not be the same ie the protein with the least confidence identification based on protein coverage may not be the one with the least confident expectation score loge39 logy 39 Total Mr Accession 3278 759 4255 30 77 268 YDRO5OC gmeB131866 p ro39tein TPI1 Triose phosphate isomerase 2696 806 4884 25 162 368 YOLO86C gmeB 653436 homoll3l protein ADH1 Alcohol dehydrogenase 2594 780 3747 24 84 468 YGR254W gmeB 5344063 h omolOll protein EN01 Enolasel 2038 673 3659 20 34 613 YBR196C gmeB 1541980 protein PGI1 Glycolytic enzyme phosphoglucose isomerase 1426 732 395 1 3 469 H YHR174W gmeB 24234161 homollll p39 rotein EN02 Enolase ll 1360 677 1724 13 26 537 YFRO53C gmeB1 351674 protein HXK1 Hexokinase isoenzyme 1 24 An SILAC experiment comparing the protein abundances in two types of cells Cisplatin resistant C and normal cells N was done and the results clustered as below 4 different experimental conditions were used with either cell type being grown in either heavy H or light L conditions State conclusions that can be made about the genes 6PGD and DDX5 and support your conclusions with evidence from the graph mom ramAw up H 1 r 1 N U39I N 00 N U U In comprehensive proteomic analyses of several organisms identified proteins represent only 50 80 of the potential proteins encoded in the genome Suggest reasons for this Name two different methods for protein fragmentation enzymatic physical via collisionskinetic Compare and contrast the advantages of ESI vs MALDI What are the advantages and disadvantages of label free methods for protein quantification What s the difference between monoisotopic and average masses Why are most mass spectra generated under vacuum The following representation of a gel is of proteins pulled down using co immunoprecipitation co IP with the protein p53 The molecular weight ladder is in lane 1 and the result of the co IP experiment in lane 2 Describe the control experiment that needs to be done to show the specific interaction of protein A with p53 and draw the gel result in lane 3 1 2 3 A B C Liquid chromatography mass spectrometry LC MS is a common technique for the analysis of peptides in mixtures These data are three dimensional with one dimension of intensity and two dimensions of separation What are the two dimensions of separation You are searching for a biomarker for ovarian cancer that occurs in approximately 2 per 2500 people You have determined that a new protein that you have discovered has a sensitivity of 100 and a specificity of 98 In a random population of 10000 patients how many positives and how many false positives will your biomarker detect YeastinoiM PS112703 RT 47 06 AV 1 NL 150E5 T lTMS C NSl d Full m52 554 82Cld35 00 140 0071120 00 100 91 62 95 90 45 92 85 80 75 70 65 60 68248 55 50 45 58336 40 35 30 25 50549 20 314 27 427 39 546 55 795 66 962 77 15E 199 20 399 42 891132 10 17123 65551 55 l 21822 25919 34176 l 7291 71938 82580 980 69 0 11 ll ll mlll lil m ll llillnll lln 11 ll ll lli il lliln ml l l l n i m l mm 1118781 200 300 400 500 600 700 800 9 0 1000 1100 3439 mZ The spectrum above is from the peptide VVDLVEHVAK Label the peaks corresponding to the y6 ion the b3 ion and the a4 ion 35 You have an interesting protein related to heart disease in mice Careful analysis of the mRNA sequence shows the mutation to be a 3 amino acid deletion in the middle ofthe protein sequence You decide to confirm this by peptide fragmentation analysis in a mass spectrometer However after database searching on your data using the mouse reference proteome you only find peptides upstream or downstream of your region of interest Why did your approach fail 36 How does targeted proteomics differ from shotgun proteomics that uses data dependent acquisition U l What are MALDI and ESI used for and how do they differ DJ 00 Name three advantages and three disadvantages of protein analysis using the shotgun mass spectrometry approach over the gel based approach 39 What is the mass ofthis peptide LNVEASAPQTR What is the 2 mz of this peptide 40 What are the two low mass ions that are diagnostic of the yl ions for a tryptic peptide 41 Choose from the list or Antibody Pulerown BollomrUp shotgun Mass Spectrometry Targeted Proteomics ICA ToprDown Mass Spectrometry AntibodyArray Proteinrinteraclion Array Western Blot a u L L y use and why b J39 regions ofthe protein ifsome ombinalions Ul L 39 limiting factor which experimental method should you use and why generated for each protein and each antibodyreoognizes a different epitope Vour colleague would like to know which antibody is the most speci c for each of the 20 proteins Which experimental method should you use and why 42 Name the F 39 J the following spectrum 1 DD 937 47 MH 11834 an Eu a m E 50 459 45 2 g 4m 7m 37 m an 459 31 70 37 2 247 14 a 725 26 lg 45 333 r 39 in gig 13 D 33909 103739 352 3Bi 479 as i5 99 597 21 774 49 920 37 U iiiiiii iiiiiiiiiiiii iiiiiiii39iiiiiiiiiii 200 3am 400 Sun 500 mu sun emu mun mm 43 39 39 I 39 nieionowingi iuuri experiment what are they what are the advantages ottnis setup SCX RP Mass r scx Strong cation exchange RP Reverse Phase 44 Why are two antibodies useci in Western blotting 45 what is peak capacity Explain your answer in terms of SDSPAGE 4n unproteins 47 weigiu ui uie quotproteinquot in tanueni ma spectrum Humarmprdu nmixiurem 325473345 RT 48 D7VEUH AV 14 NL gizga r ims ci c iinsiwu iriu mo am 2511 90 154596 ED 951m 70 E 5 77162 5 103096 g an 1374 E 1 40 723117 c 112462 30 123701 20 mun m mafzm mm 4 i ii iii ii i i ii plain 130771 i i i i i i i i i 400 sun sun iuuu i200 1400 i600 1300 2000 m 48 i L A iIILquc u answer 5 mm anmkumamm mm mm mm xv mm mm mm mm mam W ID 1 Yvo 49 mman a m in 5 squot c IAII w w n r 50 From the MudP T majorm of detected protems7 MSrbased pmreumms h 1 000 m I msbased proleomics TAP GFP Number 51 proteins amwbwm m 00003050 announce 3 o a a a m ai eq e i sy Molecules per cell quot 4quot 51 The SILAC studies by de Godoy et al on yeast included the above charts in their Nature publication Were SILAC data actual measured ratios used to prepare either graph above and ifso how g Protein Abundance Lqu comescell 5 o 5 1015 20 25 90 35 4a 45 5c 55 60 65 7o 75 so 55 so 95 I00 Frolelr Measured 52 The SRM data from Picotti et 0 resulted in the above graph Open circles indicate yeast proteins quantified by isotope abeled species Explain how this was done p m sxwamwmw unsaved u y aa CA mm m NEI and arwau daermmesven cm afNEI a reamvewzh m cu prmems szmsswe mad rEgmazmn amangzhe cu dassmemherssmd 2 Emma scarewwh pemme massrmgerpm demx camn mprmems 55 FartavrdawnWanamawhySESthmzthadafchmcg samvksmHznzdfmm Datmntsand ma thvvamntzm What mzthadwamdyau mm a uuamazmn 59 rzszamh as amvand m transmvtmmcs 5mm a n dumb 64 65 For a protein with MW 13215 the mz of the 12 charge state would be a 110225 b 110125 c 110025 d none ofthe above The NAPPA approach is beneficial because Proteins can be synthesized on the array Interacting proteins can be co synthesized DNA can be spotted on arrays A single antibody can be used to bind all proteins All of the above None of the above rhmew Indicate why Lys C esterification and ETD were each used in the phosphoproteome paper by Chi et al What amino acids were chosen to exploit in the SILAC paper and why Describe how false positive and false negative results each can result from TAP and yeast two hybrid studies What is activity based proteomics and how does it differ from targeted proteomics iTRAQ reagents consist of three primary components Describe each component and illustrate how the technique works What are proteotypic peptides PTPs and what utility do they present Why is SISCAPA a useful approach for biomarker studies Circle the letter above the appropriate dotted line to indicate where bond cleavage would occur to produce a b2 ion Put a square around the letter that indicates where bond cleavage would occur to produce a y1 ion If R1A R2G R3N and R4S what would be the masses of those ions 74 Why is trypsin the most commonly used enzyme in proteomics experiments 75 In a MudPIT experiment you identify five serum proteins that change in abundance in cancer patients undergoing chemotherapy You now would like to analyze 20 timecourse samples from 10 cancer patients ie 200 samples to evaluate the utility ofthese 5 proteins as diagnostic markers Assumingyou do not have antibodies available to these proteins which method belowwillyou use shotgun proteomics SRM methods 1 peptide mass fingerprint none of the above 7quot 995339 8 gt i ma M Penateral Prm enrmcs mm 7 u i74i2n quot 0 22 6o 76 From the paper by Perrot et al on the yeast reference proteome map describe the two dimensions of separations shown in the figure above and how previously unidentified protein identities were determined 77 As you observed in the peptide mass fingerprint lab decreasing the peptide mass tolerance Decreases specificity Increases specificity Decreases sensitivity c9651 Increases sensitivity 78 Running two MSMS experiments on the same sample improves the ability to detect proteins because a You are more confident when you identify a protein in both runs b Each run produces an independent set of fragments for each protein increasing sequence coverage and thus confidence in protein identification c Each run identifies an independent set of proteins so you can detect more proteins but not necessarily be more confident in the identification of any particular one 79 When peptides are fragmented for de novo sequencing using MSMS what is actually measured by the mass spectrometer Imagine that only b and y ions are produced a The residues that fall offofthe peptide b Both halves of each fragment c The half of a fragment that receives the charged proton 80 Why are low mass peptides usually not as specific as high mass peptides when mapped back to the potential protein from which they were derived a They could have come from multiple proteins b They are observed in the low mz space that is subject to a lot of noise c They have a lower probability of being generated during trypsin cleavage 81 Which terminus ofa peptide do a and c ions contain a N and C respectively b C and N respectively c Both contain the C terminus d Both contain the N terminus 82 Immonium ions are useful in de novo sequencing of peptides using MSMS because a They indicate which residue is on the C terminus of the peptide b They indicate the presence of certain residues in the peptide c They indicate the abundance of certain residues in the peptide