Antibody Sequencing by LC-MS/MS

Paul ShanBioinformatics 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

Interactive de novo-based sequence reconstruction

Experimental

• MaterialsI 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/MSOrbitrap with HCD

Database searching

Homolog protein sequence

GapLow confidence

Peptide De Novo Sequencing

• Local confidence score

• De novo sequence tag

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

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

(de novo) : LLLYDASTR(real) : LLIYDASTR |||||||+|(homolog) : LLIYDASNR

MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKRT

(de novo): EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR(real) : EPEDFALYYCQQRSNWPPSFTFGPGTRVDIKR ||||||+|||||||||||+ ||||||||||||(homolog) : EPEDFAVYYCQQRSNWPPW TFGPGTRVEIKR

de novo error

mutation

GapLow confidence

EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR

Contigs

LLLYDASTRVLTQSPASLSLSPER

...

Sequence Validation

• Coverage – Peptide map at 0.1 % FDR

• Confidence– De Novo sequence tag map

EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

VLTQSPASLSLSPERATLSCR

EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT

Report

CHAMPS - Antibody Sequencing Service

• Data analysis• Sample analysis

http://www.bioinfor.com/peaks/products/champs.html