TY - JOUR
T1 - To tag or not to tag
T2 - A comparative evaluation of immunoaffinity-labeling and tandem mass spectrometry for the identification and localization of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation
AU - Guo, Jia
AU - Prokai, Laszlo
N1 - Funding Information:
The presented research was supported by the grant AG025384 from the National Institutes of Health (Bethesda, MD, USA) and by the Robert A. Welch Foundation (endowment number BK-0031 ).
PY - 2011/10/19
Y1 - 2011/10/19
N2 - Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N'-aminooxymethylcarbonyl-hydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also happen. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.
AB - Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N'-aminooxymethylcarbonyl-hydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also happen. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.
KW - Collision-induced dissociation
KW - Immunoaffinity-labeling
KW - Protein carbonylation
KW - Redox proteomics
KW - Tandem mass spectrometry
UR - http://www.scopus.com/inward/record.url?scp=80054873045&partnerID=8YFLogxK
U2 - 10.1016/j.jprot.2011.07.013
DO - 10.1016/j.jprot.2011.07.013
M3 - Article
C2 - 21835276
AN - SCOPUS:80054873045
SN - 1874-3919
VL - 74
SP - 2360
EP - 2369
JO - Journal of Proteomics
JF - Journal of Proteomics
IS - 11
ER -