"Analysis Of Dda Data Works I Want To Briefly Give You A Sense Of Generally How You'Ll Analyze Your D Da Data Although If You'Re Staying For The MexicoĂS On Summer School Later In The Week You'Ll Get This In A Lot More Detail The First Step In Identifying Your Peptides Or The First Step In The Analysis Is To Identify Your Peptides From Your Ms/Ms Spectra And This Is A Process Called Peptide Spectral Matching And There'S Really Three Ways That People Do This The First Way Is A Database Search And This Is Where You Compare Your Experimental Spectra To All Theoretical Spectra Predicted From A Database Of Possible Peptides Based On The Genomic Sequence So A Database Search Requires That You'Ve Also Have Some Genomic Reference Sequence For That Organism The Second Way Is Called A Spectral Library Search And This Is Where You Compare Your Experimental Vectra To A Library Of All Spectra That You'Ve Identified Previously Probably From Another Database Search And The Last Method Is De Novo And This Is Where You Look At The Fragment Spectra And Try To Piece Together What The Amino Acid Sequence Should Be And After Each Of These Different Options I'Ve Listed A Few Different Software Tools That Perform Each Of These Types Of Analysis Now For Any Of These Peptide Identification Methods We Need A Way To Assign Statistics That We'Ve Assigned The Right Peptide To Our Spectra And We Do This With Something Called The Target Decoy Approach And That Means That You Search For Real Peptides That You Expect Should Be In Your Sample But You Also Search For Shuffled Or Reversed Fake Peptide Sequences Which Should Not Be There And Those Are Your Decoys This Allows You To Determine How Often Your Search Finds The Wrong Answers Which Is Called Your False Discovery Rate Or Fdr And The Way This Works Is You Determine Distributions Of Your Target Decode Or Your Target Hits In Green And Your Decoy Hits In Red And That Allows You To Set A Score Threshold Above Which You'Ll Accept Any Peptide Id At A Known Proportion Of Decoy Hits Often We Use 1% Fdr After You'Ve Identified Your Peptides And Assign Statistics To Those Identifications You Probably Need To Infer Proteins So Although In Bottom-Up Proteomics Were Actually Looking At Peptides We Need Statistically Rigorous Ways To Determine What Proteins We Found And It'S Important To Note That You Must Compute Your Protein And Peptide Fdr Separately So A 1% Peptide Fdr Will Almost Always Correspond To A Higher Protein Fdr And There'S Many Different Programs That Will Do This For You A Couple Examples Are Protein Profit And Mayu And There'S Actually Many Different Tools Well The Final Step Is To Quantify Your Peptides And Proteins And I'Ll Talk About That More On The Next Slide But I Want To Make The Point That There'S Many Different Tools That We'Ll Do Each Of These Steps That Are Developed Academically Or Commercially One Really Great Tool Is Max Font Because It Will Do All Of These Steps For You It Puts Everything Together But I'Ve Also Given You A Citation For An Example Of A Different Route That I Published Earlier This Year"
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