PMID- 16905328
OWN - NLM
STAT- MEDLINE
DCOM- 20061221
LR  - 20181113
IS  - 1044-0305 (Print)
IS  - 1044-0305 (Linking)
VI  - 17
IP  - 11
DP  - 2006 Nov
TI  - Reversed-phase liquid chromatography in-line with negative ionization 
      electrospray mass spectrometry for the characterization of the disulfide-linkages 
      of an immunoglobulin gamma antibody.
PG  - 1590-8
AB  - In this report, we present a new approach for the determination of the disulfide 
      bond connectivity in proteins using negative ionization mass spectrometry of 
      nonreduced enzymatic digests. The mass spectrometric analysis in negative ion 
      mode was optimized to allow in-line analysis coupled directly to the HPLC system 
      used for the separation of the peptides resulting from enzymatic digestion. We 
      determined the disulfide structure of a human immunoglobulin gamma 2 (IgG2) 
      antibody containing 18 unique cysteine residues linked via 11 unique disulfide 
      bonds. The efficiency of the gas-phase dissociation of disulfide-linked peptides 
      using negative electrospray ionization was evaluated for an ion trap mass 
      spectrometer and an orthogonal acceleration time-of-flight mass spectrometer. 
      Both mass spectrometry techniques provided efficient in-source fragmentation for 
      the identification of the disulfide-linked peptides of the antibody. Both 
      instruments were limited in the number of disulfide bonds that could be 
      dissociated. Seven of the 11 unique disulfide linkages have been determined, 
      including the linkage of the light chain to the heavy chain. Only the disulfide 
      connectivity of the hinge peptide H6H7H8H9 (C6C7VEC8PPC9PAPPVAGPSVFLFPPKPK) could 
      not be determined (numbering the cysteine residues sequentially from the 
      N-terminus and labeling the heavy chain cysteines "H" and the light chain 
      cysteines "L"). However, we identified the dimer of peptide 
      C6C7VEC8PPC9PAPPVAGPSVFLFPPKPK linked via four disulfide bonds based on the 
      unique molecular weight of this dipeptide. The established linkages were H1 to 
      H2, H10 to H11, H12 to H13, L1 to L2, L3 to L4, and L5 to H3H4. The intrachain 
      linkages of the light chain (L1 to L2, L3 to L4), and heavy chain (H10 to H11, 
      H12 to H13) domains were identical to the linkages found in IgG1 antibodies.
FAU - Chelius, Dirk
AU  - Chelius D
AD  - Amgen Inc., Thousand Oaks, California 91320, USA. dchelius@amgen.com
FAU - Huff Wimer, Mary E
AU  - Huff Wimer ME
FAU - Bondarenko, Pavel V
AU  - Bondarenko PV
LA  - eng
PT  - Journal Article
DEP - 20060814
PL  - United States
TA  - J Am Soc Mass Spectrom
JT  - Journal of the American Society for Mass Spectrometry
JID - 9010412
RN  - 0 (Disulfides)
RN  - 0 (Immunoglobulin gamma-Chains)
RN  - 0 (Recombinant Proteins)
SB  - IM
MH  - *Chromatography, High Pressure Liquid
MH  - Disulfides/*chemistry
MH  - Humans
MH  - Immunoglobulin gamma-Chains/*chemistry
MH  - Recombinant Proteins/chemistry
MH  - Spectrometry, Mass, Electrospray Ionization/*methods
EDAT- 2006/08/15 09:00
MHDA- 2006/12/22 09:00
CRDT- 2006/08/15 09:00
PHST- 2006/05/25 00:00 [received]
PHST- 2006/07/06 00:00 [revised]
PHST- 2006/07/07 00:00 [accepted]
PHST- 2006/08/15 09:00 [pubmed]
PHST- 2006/12/22 09:00 [medline]
PHST- 2006/08/15 09:00 [entrez]
AID - S1044-0305(06)00635-0 [pii]
AID - 10.1016/j.jasms.2006.07.008 [doi]
PST - ppublish
SO  - J Am Soc Mass Spectrom. 2006 Nov;17(11):1590-8. doi: 10.1016/j.jasms.2006.07.008. 
      Epub 2006 Aug 14.