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2012 11 15 Slide 2 Cell Recognition Protein Folding Reproduction Immunity Cell Adhesion Glycosylation Biological Functions of Glycosylation Slide 3 Analytical question 1. Trypsin Recognition of glycosylated peptides 2. PNGase Recognition of glycosite Lectins specific recognition Glyco-motif recognition Anti-body specific recognition Boron Acid, HC/HA specific recognition MS analysis of glycopeptides AA-NXS/T-AA MS analysis of glycopeptides A B Slide 4 Slide 5 Stand Glycoprotein Interpretation Construction of relational network to select correct peaks and reduce false positive results. Focus on constant breakage of glycans on glycopeptides. Suitable for different MS instruments. (e.g MALDI & ESI source) Capture feature mass of QIT, LTQ original data Spectrum feature of low CID, high CID, ETD and HCD N- CID Slide 6 Validation of diagnostic ions QIT MS Mode Peptide Sequence: Asp fragmentation Cys modification Miss cleavage Mass Path/Chain Slide 7 Moderate level of cleavage on peptide and glycan. Spectrum quality usually unsatisfied for the need of high-throughput identification. Background High Energy CID Low Energy CID Comprehensive cleavage on glycan, very little cleavage on peptide. Specifically designed algorithm is needed for interpretation. Slide 8 Development of GRIP Glycopeptide Reveal & Interpretation Platform Automatic filtration of peptides without glycosylation Automatic interpretation of glycosylation in real sample Adjustable parameters (e.g glycan markers / databases) Compatibility of common proteomic software (e.g TPP) De novo + Database Search Suitable for Standard Glycoprotein Exhaustive search all possible glycan and get consensus result + Slide 9 GRIP Raw Spectra Intensity Filtration Deconvolution Precursor Filtration Network Construction Glyco-related Mass Capture Network Module Relational Tree Glycopeptide Composition Database Prediction Result Filtration Module Slide 10 GRIP DirecTag DirecTag (2008. JPR) Slide 11 Relational networks were constructed from peaks of glycopeptide fragments. Our Software GRIP Improved Code Redundant edges are cut down to Reduce the Complexity of Network Single Mass Prediction Software FindMod GlycoMod GlycoPep ID GlycoSuiteDB Slide 12 Test in HRP Slide 13 Peaklist MassDatabase MassMass ErrorDatabase Entry 4054.81434054.948833VVHAVEVALATFNAESNGSYLQLVEISR_[Hex]3[HexNAc]2[Fuc]1 4274.05764274.02318VVHAVEVALATFNAESNGSYLQLVEISR_[Hex]4[HexNAc]3 4970.39354971.2773178VVHAVEVALATFNAESNGSYLQLVEISR_[Hex]4[HexNAc]5[NeuNAc]1 4987.43154988.2926173VVHAVEVALATFNAESNGSYLQLVEISR_[Hex]5[HexNAc]5[Fuc]1 5003.44425004.2875169VVHAVEVALATFNAESNGSYLQLVEISR_[Hex]6 [HexNAc]5 GlycoPep ID 2007 AC Slide 14 Glycopeptide Composition Database Peptide Database Glycan Database Slide 15 MN HRP: 37 17 N 5 4 2 2 2 6*5*3*3*3 = 810 13770 Slide 16 Next Step: Serum GRIP can extract enough information for N-Glycopeptide identification. Slide 17 Glycopeptide Database Peptide Database Glycan Database 1.Glycopeptide1 2.Glycopeptide2 3.Glycopeptide3 4.Glycopeptide4 5. GRIP PNGase 2DLC-MSMS 1.De-glycopeptide1 2.De-glycopeptide2 3.De-glycopeptide3 4.De-glycopeptide4 5. TPP SEQUEST The Retrosynthetic State-Transition Library 2009, Proteomics 365 Glycan Compositions CID HCD 1. 2. Slide 18 A. B. CID/HCD Pair C. CID Slide 19 All MS/MS spectra Shuffle spectra (based on the original) Database searching by GRIP Database searching by GRIP (same parameters) Target result Decoy result 2% FPR threshold Threshold (log) TargetDecoy-1Decoy-2Decoy-3FPR 0.750000N/A 0.50000N/A 0.250000N/A 030000.00% -0.25100000.00% -0.5310123.23% -0.75820322.03% 1500431.56% -1.251944863.09% -1.522171093.92% -1.7523381294.15% -22511217115.31% -9944216517417038.39% Test Result of Standard ASF Slide 20 44 Test Result of Standard ASF Slide 21 GRIP Score 0.1 (2.8%FPR) SEQUEST/ PeptideProphet p 0.99 All paired HCD spectra are manually interpreted for Y1 ions. 14,014(65.7%) spectra validated by HCD No overlapping on spectrum level Human serum 1)Typsinization 2)Enrichment 3)LC-CID/HCD-MS/MS (LTQ-Orbitrap, 2DLC x 2 ) 62,878 CID-MS/MS spectra 62,878 CID-MS/MS spectra 21,314 peptide spectra 21,314 peptide spectra 1,174 Glyco- peptide spectra 1,174 Glyco- peptide spectra Threshold (log) TargetDecoy1Decoy2Decoy3FPR 0.75440000.00% 0.52050000.00% 0.253620000.00% 06280200.11% -0.258024440.50% -0.5944610120.99% -0.7510601918201.79% 11743225432.84% -1.2512975246634.14% -1.5142595871136.90% -1.7515821511431779.92% -2183523524826613.61% -99419921042021204248.96% B B A A C C CID/HCD Pair Validation Slide 22 CID/HCD Pair Validation DEGLYCOPEPTIDE Slide 23 Threshold (log) Spectra Number DeltaCN >0.5 Y1 Score > 0%TrueFalseFPR 0.7544423685.7%3600% 0.520519215178.7%15100% 0.2536232925276.6%25110.4% 062853841577.1%41410.24% -0.2580268051575.7%50871.36% -0.594480760074.4%590101.67% -0.75106089564572.1%632132.02% 117498268369.6%667162.34% CID/HCD Pair Validation Slide 24 No overlapping on spectrum level Human serum 1)Typsinization 2)Enrichment 3)LC-CID-MS/MS (LTQ-Orbitrap,2DLC x 3) 251,886 CID- MS/MS spectra 251,886 CID- MS/MS spectra GRIP Score 0.178 (1.8% FPR) SEQUEST/ PeptideProphet p 0.99 53,567 peptide spectra 53,567 peptide spectra 4,341 Glyco- peptide spectra 4,341 Glyco- peptide spectra Threshold (log) TargetDecoy1Decoy2Decoy3FPR 0.75690000.00% 0.55010000.00% 0.2510740110.06% 017744630.24% -0.2526111115100.46% -0.534893633310.96% -0.7543417277821.77% 51791461381472.77% -1.2559342872452884.61% -1.566024734264586.85% -1.75726976170872610.07% -2794911771131112814.41% -991794611712113321193464.97% B B A A C C Large Scale Identification in Human Serum Slide 25 Large Scale Identification in Human Serum Slide 26 Slide 27 Slide 28 Slide 29 CID/HCD Pair Validation Slide 30 Slide 31 Large Scale Identification in Human Serum Slide 32 Large Scale Identification in Human Serum Slide 33 de novo Slide 34 GRIP Glycopeptide Composition Database Glycopeptide Fragment Database DEGLYCOPEPTIDE Glycopeptide Structure Database Fragmentation New Method ? Slide 35 9 1 9 9 2 43 30 5 572 63 N 4 4 Simulation using Glycoworkbench Slide 36 Slide 37 N 1 0 T F F Slide 38 N- Slide 39 N Slide 40 Slide 41 20mM01.01178.01178.2 Peptide-2D-110511.tgp Node_Tolerance = 1 Da Precursor_Tolerance = 10 ppm sp|P01859|IGHG2_HUMAN R.EEQFN#STFR.V 172-180 N_GLYCAN 1157.5227 Slide 42 Slide 43 Slide 44 StructureMatrix 228423 6 7 8 19 9 10 11 20 12 13 14 21 15 16 17 22 4 6 7 8 9 10 11 19 20 5 12 13 14 15 16 17 21 22 Slide 45 N- 2284 N- Slide 46 Acknowledgement CNHUPO 973 863