Characterization of Diisocyanate Haptenation of Serum Albumin by Multiplexed Tandem Mass Spectrometry

Details

• Personal Author:
  Green B ; Hettick J ; Nayak A ; Siegel P ; Wisnewski A
• Description:
  The link between diisocyanate exposure and occupational disease is well established, however, few targets of diisocyanate in vivo have been described. Furthermore, the chemistry of the diisocyanate-protein modification remains unknown, as does the mechanism of allergic disease. The studies presented here have applied modern analytical proteomics methodologies (in particular, multiplexed tandem mass spectrometry) to characterize the conjugation chemistry of industrially relevant diisocyanates on model proteins. The site(s) of diisocyanate conjugation upon the model protein(s) are characterized by enzymatic digestion of the conjugated protein, followed by fragmentation of the resultant enzymatic peptides by collision-induced dissociation (CID) in a quadrupole time-offlight (qTOF) mass spectrometer. CID produces sequence-informative fragmentation and unambiguous determination of the conjugation site is possible on the basis of mass shifts of resultant fragment ions. In these experiments, CID is performed in a multiplexed manner (e.g. alternating low and high collision energy scans) to allow high mass accuracy scans of parent and daughter fragment ions that may be temporally correlated in silico post run. Such an experiment allows greatly improved sequence coverage over more traditional "data-directed" experiments that require the instrument to make real-time decisions about which m/z ratios to select for fragmentation and often fail to target low abundance peptides that would otherwise be of interest to the analyst. Characterization of the haptenation of the model protein, human serum albumin (HSA), by 2,4- and 2,6-toluene diisocyanate (TDI) and 4,4-methylene diphenyl diisocyanate (MDI) resulted in the observation of a variety of reactive chemistries, including direct conjugation, hydrolysis, inter- and intra-molecular cross linking, and polymerization of the diisocyanate upon a reactive amino acid site. Conjugation proceeds in a dose-dependent manner, with as few as ten reactive sites at 1:1 (HSA:TDI) ratio and as many as 37 reactive sites at a 1:40 (HSA:TDI) ratio. Conjugation at lysine residues is the most prevalent, although conjugation of two glutamine residues, as well as the N-terminal aspartic acid residue is observed. Preferred conjugation sites include the N-terminal amine, Lys199, and so-called dilysine (KK) motifs, all of which are expected to have lower pKa values than that of the free lysine amino acid. MDI was observed to react with a subset of 20 of the 37 putative TDI conjugation sites. This is in good agreement with conjugation data for p-tolyl isocyanate, suggesting it is the increased reactivity of TDI due to the second isocyanate moiety on the ring, rather than the size or relative hydrophobicity of MDI that is responsible for the difference in reactivity with albumin. Conjugates produced from vapor exposures are hypothesized to more closely resemble those formed in vivo. Characterization of these conjugates demonstrates that domains II and IIIB of HSA are modified less by vapor phase TDI exposures, suggesting that the orientation of serum albumin at the air/liquid interface may play an important role in how HSA is haptenated by diisocyanates. In aggregate, these experiments increase our understanding of the reactive chemistry of diisocyanates, and suggest possible regions of the serum albumin molecule that may be of interest as biomarker candidates. [Description provided by NIOSH]
• Subjects:
  Biomarkers Chemistry Hypersensitivity Isocyanates Occupational Health Safety
• Keywords:
  4,4'methylenebisphenyl Isocyanate Allergic Reactions Amino Acids Biological Effects Chemical Reactions Diisocyanates MDI
• Publisher:
  Work Wellness and Disability Prevention Institute (WWDPI)
• Document Type:
  Text
• Genre:
  Abstract or Summary
• Place as Subject:
  Connecticut ; Maryland ; OSHA Region 1 ; OSHA Region 3 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  70
• NIOSHTIC Number:
  nn:20054300
• Citation:
  Isocyanates & Health: Past, Present and Future, April 3-4, 2013, Potomac, Maryland. Vancouver, BC, Canada: Work Wellness and Disability Prevention Institute (WWDPI), 2013 Apr; :70
• Contact Point Address:
  Dr. Justin Hettick, Research Scientist Team Leader, National Institute for Occupational Safety and Health, Health Effects Laboratory Division. 1095 Willowdale Road, Morgantown, WV 26505
• Email:
  jhettick@cdc.gov
• CAS Registry Number:
  Diphenylmethane diisocyanate (CAS RN 101-68-8) ; Toluene 2,4-diisocyanate (CAS RN 584-84-9) ; Toluene diisocyanate (CAS RN 26471-62-5)
• Federal Fiscal Year:
  2013
• Peer Reviewed:
  False
• Source Full Name:
  Isocyanates & Health: Past, Present and Future, April 3-4, 2013, Potomac, Maryland
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:3e13d5d83bf201fbfb4b169b07b252703815480ea99e3e404db8d780655b66b8b0b6a1882fe3a2deebcf676638371aa26899a66d2a829654c29e809d18cfb68b
• Download URL:
  https://stacks.cdc.gov/view/cdc/216892/cdc_216892_DS1.pdf
• File Type:
  [PDF - 705.30 KB ]