N,N-Dimethyl Leucines as Novel Isobaric Tandem Mass Tags for Quantitative Proteomics and Peptidomics

摘要

Herein, we describe the development and application ofna set of novel N,N-dimethyl leucine (DiLeu) 4-plex isobaricntandem mass (MS2) tagging reagents with highnquantitation efficacy and greatly reduced cost for neuropeptidenand protein analysis. DiLeu reagents servenas attractive alternatives for isobaric tags for relativenand absolute quantitation (iTRAQ) and tandem massntags (TMTs) due to their synthetic simplicity, labelingnefficiency, and improved fragmentation efficiency. DiLeunreagent resembles the general structure of a tandemnmass tag in that it contains an amine reactive groupn(triazine ester) targeting the N-terminus and ε-aminongroup of the lysine side chain of a peptide, a balancengroup, and a reporter group. A mass shift of 145.1 Danis observed for each incorporated label. Intense a1nreporter ions atm/z 115.1, 116.1, 117.1, and 118.1nare observed for all pooled samples upon MS2. Allnlabeling reagents are readily synthesized from commerciallynavailable chemicals with greatly reduced cost.nLabels 117 and 118 can be synthesized in one stepnand labels 115 and 116 can be synthesized in twonsteps. Both DiLeu and iTRAQ reagents show comparablenprotein sequence coverage (∼43%) and quantitationnaccuracy (<15%) for tryptically digested proteinnsamples. Furthermore, enhanced fragmentation ofnDiLeu labeling reagents offers greater confidence innprotein identification and neuropeptide sequencingnfrom complex neuroendocrine tissue extracts from anmarine model organism, Callinectes sapidus.