title: Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.
abstract: The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.
authors.affiliation: Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Borisov
authors.forename: Michael B
authors.initials: MB
authors.lastname: Goshe
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: V Sergey
authors.initials: VS
authors.lastname: Rakov
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D012259:Ribonuclease, Pancreatic / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D020543:Proteome; D012259:Ribonuclease, Pancreatic; D003545:Cysteine