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Antibody Sequencing by LC-MS/MS

Antibody Sequencing by LC-MS/MS. Paul Shan Bioinformatics Solutions Inc. Antibody. Protein sequence variation Sequencing by LC-MS/MS Full coverage High confidence Workflow of sequencing Multiple enzyme digestion High resolution MS/MS Data analysis

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Antibody Sequencing by LC-MS/MS

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  1. Antibody Sequencing by LC-MS/MS Paul Shan Bioinformatics Solutions Inc.

  2. Antibody • Protein sequence variation • Sequencing by LC-MS/MS • Full coverage • High confidence • Workflow of sequencing • Multiple enzyme digestion • High resolution MS/MS • Data analysis Interactive de novo-based sequence reconstruction

  3. Experimental • Materials I 5154 IgG1 Kappa and SAB4700580 IgG2 • Enzymes AspN, Chymotrypsin, GluC, LysC, Proteinase K, and Trypsin • Sample preparation Reduction / alkylation -> de-glycosylation ->  separation -> digestion • LC-MS/MS Orbitrap with HCD

  4. Database searching Low confidence Gap Homolog protein sequence

  5. Peptide De Novo Sequencing • Local confidence score • De novo sequence tag

  6. Assembly of De Novo Tags De Novo tags . . [200.12]EPEDFALYYCQQR . [200.12]EPEDFALYYCQQR CQQRSNWPPSFTF [204.09]PGTRVDLKR[200.12] [201.08]WPPSFTFGPGTR [204.09]PGTRVDLKR[200.12] CQQRSNWPPSFTF [201.08]WPPSFTFGPGTR Contig EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKRT

  7. Sequence Reconstruction Problem：de novo errors、database mutations (denovo) X: LSCAFK ||| (homolog) Z: SIAAFK de novo error (denovo) X: [LS]CAFK (real) Y: [SI]CAFK || +||| (homolog) Z: [SI]AAFK SPIDER mutation Solution：minimize de novo errors and mutations

  8. Low confidence Gap EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR LLLYDASTR de novo error VLTQSPASLSLSPER . . . (de novo): EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR (real) : EPEDFALYYCQQRSNWPPSFTFGPGTRVDIKR ||||||+|||||||||||+ |||||||||||| (homolog) : EPEDFAVYYCQQRSNWPPW TFGPGTRVEIKR Contigs mutation (de novo) : LLLYDASTR (real) : LLIYDASTR |||||||+| (homolog) : LLIYDASNR MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKRT

  9. Sequence Validation • Coverage • Peptide map at 0.1 % FDR • Confidence • De Novo sequence tag map EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VLTQSPASLSLSPERATLSCR

  10. Report

  11. CHAMPS- Antibody Sequencing Service • Data analysis • Sample analysis http://www.bioinfor.com/peaks/products/champs.html

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