Collision-induced Dissociative Chemical Cross-linking Reagent for Protein Structure Characterization

Yiming ChenNov. 28, 2012

J. Mass. Spectrom 2010, 45, 178-189

Outline

• Introduction

• Chemistry of cross-linker

• Examples

• Conclusion

Outline

• Introduction

• Abstract

• Edman Degradation

• Instrumentation

• Chemistry of cross-linker

• Examples

Abstract

• Design an innovative cross-linker based on Edman degradation

• Form indicative mass shifted fragment ions and constant neutral losses

• Unambiguous identification of cross-linked peptide

Edman Degradation

N C S H2NO

HN Peptide

R

H+

HN

S

NH O

HN Peptide

R

H+

N NH

SNHO

R

HPeptide

N NH

SO

R

N NHO

S

R

heat

Peptide-NH2

Edman Degradation

• Phenylisothiocyanate is reacted with an uncharged terminal amino group

• Terminal amino acid is cleaved as a thiazolinone derivative

• Thiazolinone is treated with acid to form the more stable phenylthiohydantoin (PTH) that can be chracterized

Edman Degradation

• Sequence peptides with 60 or less amino acid residues

• Sensitivity: 10-100 pico-moles

• Has been automated

Instrumentation

• Linear MALDI-TOF

• nano-HPLC-MALDI-TOF/TOF-MS (offline)

• nano-HPLC/nano-ESI-LTQ-Orbitrap-MS

• Offline nano-ESI-LTQ-Orbitrap-MS

Outline

• Introduction

• Chemistry of cross-linker

• Synthesis

• Mechanism of Reaction

• Example

• Conclusion

SynthesisO

OHH2NSOCl2, MeOH O

OH2N

O

ONCS

CS2/CH2Cl2

HN

O

OHO

O

OO NOH

DCC

HN

O

OO

O N

O

O

+NH

O

OH

HN

O

O

O N

O

OH + H2NO

O

DCCHN

O

O

O N

O

NH O

O

Pd/H2O

N

O

NH O

OH2N +O

ONCSO

O HN

S

HN

O

N

O

NH O

O

LiOH/THF/H2OHO

O HN

S

HN

O

N

O

NH O

OHN O

OO

O

HN

S

HN

O

N

O

NH O

O N

O

O

NHS-BuTuGPG-NHS

Cross-Linker

Mechanism (1)

Inter-peptide cross-link

Mechanism (2)

Intra-peptide cross-link

Mechanism (3)

dead-end link

Example: substance P

• Sequence: Arg Pro Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (RPKPQQFFGLM-NH2)

• Associated with inflammatory processes and pain

• Molar Mass: 1347.63 g/mol

Example: substance P

Example: substance P

• Neutral loss of 184 m/z (doubly charged 843 m/z to 751 m/z) indicates cross-linking

• Two intrapeptidal cross-linking isomers can be distinguished by y10 fragment

• Dead-end link has characteristic neutral losses: 172 m/z or 202 m/z

Example: LHRH

• Sequence: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

• No intrapeptidal cross-link, only dead-end link

• Characteristic neutral losses: 172 m/z or 202 m/z

Example: lysozyme

• Lysozyme is part of the innate immune system, 14.3 kDa

• Up to 6 cross-linkers attached

• Digested by trypsin and resulting peptides are analyzed by nano-HPLC/MALDI-TOF/TOF-MS and nano-HPLC/nano-ESI-LTQ-Orbitrap-MS

Example: lysozyme

Example: lysozyme

Residue 6-14

Example: lysozyme

Residue 115-125

Example: lysozyme

• For residue peptide 115-125, m/z 836 is correspondents to intrapeptidal cross-linking

• Results suggests Thr-118 is also involved beside Lys-116

Outline

• Introduction

• Chemistry of cross-linker

• Examples

• Conclusion

Conclusion

• Easy to synthesize

• Clear characterization

• Investigate tertiary or quaternery structures

Thanks!