antibody sequencing by lc-ms/ms paul shan bioinformatics solutions inc
TRANSCRIPT
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Antibody Sequencing by LC-MS/MS
Paul ShanBioinformatics Solutions Inc.
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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
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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
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Database searching
Homolog protein sequence
GapLow confidence
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Peptide De Novo Sequencing
• Local confidence score
• De novo sequence tag
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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
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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
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(de novo) : LLLYDASTR(real) : LLIYDASTR |||||||+|(homolog) : LLIYDASNR
MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKRT
(de novo): EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR(real) : EPEDFALYYCQQRSNWPPSFTFGPGTRVDIKR ||||||+|||||||||||+ ||||||||||||(homolog) : EPEDFAVYYCQQRSNWPPW TFGPGTRVEIKR
de novo error
mutation
GapLow confidence
EPEDFALYYCQQRSNWPPSFTFGPTGRVDLKR
Contigs
LLLYDASTR
VLTQSPASLSLSPER
...
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Sequence Validation
• Coverage – Peptide map at 0.1 % FDR
• Confidence– De Novo sequence tag map
EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
VLTQSPASLSLSPERATLSCR
EIVLTQSPASLSLSPGERATLSCRASQSVSSYLAWYQHKPGQAPRLLIYDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFALYYCQQRSNWPPSFTFGPGTRVDIKRT
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Report
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CHAMPS - Antibody Sequencing Service
• Data analysis• Sample analysis
http://www.bioinfor.com/peaks/products/champs.html