Transcript 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
Interactive de novo-based sequence reconstruction
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
Database searching
Low confidence
Homolog protein sequence
Gap
Peptide De Novo Sequencing
• Local confidence score
• De novo sequence tag
Assembly of De Novo Tags
De Novo tags
[200.12]EPEDFALYYCQQR
[204.09]PGTRVDLKR[200.12]
CQQRSNWPPSFTF
[201.08]WPPSFTFGPGTR
...
[200.12]EPEDFALYYCQQR
CQQRSNWPPSFTF
[201.08]WPPSFTFGPGTR
[204.09]PGTRVDLKR[200.12]
Contig
EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR
MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKRT
Sequence Reconstruction
Problem：de novo errors、database mutations
(denovo)
X:
(homolog) Z:
LSCAFK
|||
SIAAFK
de novo error
(denovo)
(real)
SPIDER
X:
Y:
(homolog) Z:
[LS]CAFK
[SI]CAFK
|| +|||
[SI]AAFK
mutation
Solution：minimize de novo errors and mutations
Low confidence
Gap
MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKRT
EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR
LLLYDASTR
VLTQSPASLSLSPER
..
.
Contigs
de novo error
(de novo):
(real)
:
EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR
EPEDFALYYCQQRSNWPPSFTFGPGTRVDIKR
||||||+|||||||||||+ ||||||||||||
(homolog) : EPEDFAVYYCQQRSNWPPW TFGPGTRVEIKR
mutation
(de novo)
(real)
:
:
(homolog)
:
LLLYDASTR
LLIYDASTR
|||||||+|
LLIYDASNR
Sequence Validation
• Coverage
– Peptide map at 0.1 % FDR
EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
• Confidence
– De Novo sequence tag map
EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT
VLTQSPASLSLSPERATLSCR
Report
CHAMPS - Antibody Sequencing Service
• Data analysis
• Sample analysis
http://www.bioinfor.com/peaks/products/champs.html