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.

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides

“…The triply charged ion was more intense than the doubly charged one and in data-dependent acquisition mode was selected for MS/MS. The most abundant fragment ion is derived from the ICAT label (19). b, MS/MS spectrum of the doubly charged ion at m/z 947.4, which was labeled with N-ethyl-iodoacetamide.…”

“…Also, ICAT has an ether linker that is susceptible to fragmentations that produce abundant ICAT-specific fragment ions (Fig. 1a) (19). In contrast, the N-ethyl-iodoacetamide modification is stable upon collisional activation, and MS/MS spectra derived from lower charge state precursor ions typically provides more sequence-specific fragment ions (Fig.…”

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: The human mammary epithelial cell proteome

“…Nevertheless, several limitations associated with avidin affinity chromatography have been observed, including difficulty in removing nonspecifically bound peptides as well as reduced sample recovery due to irreversible binding of a subpopulation of the biotinylated peptides. The solid-phase approach for capturing cysteinyl peptides eliminates the need for avidin affinity chromatography, but contributes the new challenge of fragment ion production from the cysteine-bound tags during collision induced dissociation (CID) complicating the analysis [33]. Reversible labeling of cysteinyl peptides with 5, 5'-dithiobis (2-nitrobenzonic acid) changed the peptide hydrophobicity, which allows their specific isolation utilizing COFRADIC™; however, additional chromatographic steps are required in this method.…”

Stable isotopic labeling in proteomics

“…If this is not postanalytically corrected, erroneous peptide ratios will be determined. Furthermore, the original ICAT tag is very bulky and was reported to pose problems when interpreting MS/MS spectra of tagged peptides since many fragment ions resulted from the tag itself rather than from the actual peptide backbone [53]. More recent, MS/MS fragmentation hindrances were compensated by the introduction of solid-phase tags with cleavable linkers [54] that were further labeled with carbon-13 instead of deuterium by which coelution between light and heavy peptides .…”