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 Homolog protein sequence Gap Low confidence

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

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

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

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

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

10. Report

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