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Advanced Library Generation. Tobias Knaute / Johannes Zerweck Peptide Arrays JPT Peptide Technologies, Berlin. What´s the Problem?. The peptide library generation for determining antibody / antigen interactions gets complex if many proteins are involved
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Advanced Library Generation Tobias Knaute / Johannes Zerweck Peptide Arrays JPT Peptide Technologies, Berlin
What´sthe Problem? • The peptidelibrarygenerationfordeterminingantibody / antigeninteractionsgetscomplexifmanyproteinsareinvolved • Post-translationalmodificationsincreasecomplexityevenmore • Numberofpeptidesis limited due to experimental setup (max. 7000 peptides per experiment) • Problem: Howtogeneratethemostefficientlibraryconcerningcoverageandcontent
Conventional MethodFixed Overlap C N Overlap = Length - Offset Generation of overlapping peptides based on primary structure MGARASVLSGGELDR RASVLSGGELDRWEK VLSGGELDRWEKIRL GGELDRWEKIRLRPG LDRWEKIRLRPGGKK ... MGARASVLSGGELDR RASVLSGGELDRWEK VLSGGELDRWEKIRL GGELDRWEKIRLRPG LDRWEKIRLRPGGKK ... • PRO: • Easy to understand & implement • Sufficientfor simple screeningsandepitopemappings • CON: • Insufficientformanyhomologoussequences • Couldleadtobloatedpeptidelists • Coverage not adjustable
AdvancedMethodDynamicalOverlap • Algorithmenabling optimal coverageofproteinsequencesbypeptidelibraries • Task 1: find optimumbetweennumberofpeptidesandcoverage • Task 2: generatepeptide-score accordingtofrequencyofmotifs (e.g. in otherhomologoussequences) • Task 3: abilitytoinjectfixedsequences (e.g. frompreviousprojects) • Task 4: abilitytoadjustcoverageand/ornumberofpeptidestospecificneeds
Example Protein 1 Protein 2 Task: - 2 homologoussequences - makepeptidescan - peptidelength 15 - maximumoverlap11 - makesureeveryresidueiscoveredbyat least 3 peptides
Example - Convential Scan Convential scan Fixed overlap 15/11 Peptides of protein 1 Peptides Protein 1 Protein 2
Example - Convential Scan Convential scan Fixed overlap 15/11 Peptides of protein 2 Peptides Protein 1 Protein 2
Example - Convential Scan Convential scan Fixed overlap 15/11 All individual peptides, mapped to protein 1 Several peptides from protein 2 fit into sequence of protein 1 Number of peptides covering specific amino acid in protein sequence amino acid coverage Peptides 4 5 6 7 8 7 7 7 5 4 Protein 1 Protein 2 Redundancy introduced by homology.
Example - DynamicalOverlap All peptides, mapped to protein 1 Redundant peptides are rejected by algorithm overlap 8 Amino acid coverage is optimized Peptides 3 3 4 4 4 4 4 4 3 Protein 1 Protein 2 Overlap adjustable by coverage constraint.
Case Study: HIV1 ENV gp16024 Sequencesof Group M Subtype B Task: Minimum coverage: 3 peptides/residue Length: 15 Max. overlap: 11 highly conserved region highly variable region 3283 peptides needed for traditional method 2303 peptidesneeded for advanced method
Case Study: HIV1 ENV gp160ResidueCoverageMap Conventional 3283 peptides mean density: 6.7 peptides/residue Advanced 2303 peptides meandensity: 3.7 peptides/residue
Summary • Algorithmtobuild optimal librariesdeveloped • Coverage / numberofpeptidescanbeadjusted • Elimination of redundant motifs • Addressesdiversity, bestsuitedforproteome-widesequencealignments in complexlibraries • ApplicabletoPepStarTMmicroarrays, PepTrackTM Libraries, PepMixTMpeptidepools ... • Based on theinputofproteinsequences, JPT will develop an optimizedlibraryforseroprofilingor immune monitoringexperiments
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