Fluorescence-based peptide labeling and fractionation strategies for analysis of cysteine-containing peptides.
This study demonstrates that 1,5-I-AEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) can be used as a versatile fluorescence-based peptide quantification tool and provides readily interpretable tandem mass spectra for de novo peptide sequencing. Two AEDANS-cysteinyl-peptide fractionation strategies were evaluated. One AEDANS-cysteinyl-peptide fractionation strategy employs immobilized metal affinity chromatography (IMAC) to recover AEDANS-labeled peptides and reduce the complexity of peptide mixtures. In an alternate solid-phase approach, 1,5-I-AEDANS was coupled to an o-nitrobenzyl-based photocleavable resin to produce a resin that can label and isolate thiols and cysteine-containing peptides with a modified-AEDANS label (mAEDANS: 5-((4-amino-4-oxobutanoyl){2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). This fractionation protocol enriches cysteine-containing peptides more specifically than the IMAC strategy. Using micro-LC-ESI-MS with an on-line fluorescence detector and a Q-TOF mass spectrometer, we generated fluorescence-based elution profiles and corresponding positive ion mass spectra of AEDANS-labeled peptides. This study demonstrates that AEDANS-peptides produce positive ion ESI-MS mass spectra with detection limits comparable to those of the unlabeled peptide. Collision-induced dissociation (CID) of fluorescent AEDANS-peptides revealed readily interpretable product ion spectra with the label intact. Similar to the AEDANS-labeled peptide, an mAEDANS-labeled thiol is fluorescent and CID of a mAEDANS-labeled peptide also reveals an interpretable product ion spectrum with the label intact.
Clements
Adrienne
A
Department of Chemistry, University of Delaware, Newark, Delaware 19716, USA.
Johnston
Murray V
MV
Larsen
Barbara S
BS
McEwen
Charles N
CN
D000345:Affinity Labels / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D005453:Fluorescence; D015394:Molecular Structure; D009282:Naphthalenesulfonates / Q000737:chemistry; D010455:Peptides / Q000032:analysis*
D000345:Affinity Labels; D009282:Naphthalenesulfonates; D010455:Peptides; C006421:1,5-I-AEDANS; D003545:Cysteine