N,N-dimethyl leucines as novel isobaric tandem mass tags for quantitative proteomics and peptidomics

Abstract

Herein, we describe the development and application of a set of novel N,N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS(2)) tagging reagents with high quantitation efficacy and greatly reduced cost for neuropeptide and protein analysis. DiLeu reagents serve as attractive alternatives for isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency, and improved fragmentation efficiency. DiLeu reagent resembles the general structure of a tandem mass tag in that it contains an amine reactive group (triazine ester) targeting the N-terminus and epsilon-amino group of the lysine side chain of a peptide, a balance group, and a reporter group. A mass shift of 145.1 Da is observed for each incorporated label. Intense a(1) reporter ions at m/z 115.1, 116.1, 117.1, and 118.1 are observed for all pooled samples upon MS(2). All labeling reagents are readily synthesized from commercially available chemicals with greatly reduced cost. Labels 117 and 118 can be synthesized in one step and labels 115 and 116 can be synthesized in two steps. Both DiLeu and iTRAQ reagents show comparable protein sequence coverage (approximately 43%) and quantitation accuracy (<15%) for tryptically digested protein samples. Furthermore, enhanced fragmentation of DiLeu labeling reagents offers greater confidence in protein identification and neuropeptide sequencing from complex neuroendocrine tissue extracts from a marine model organism, Callinectes sapidus.

Figure 1

MALDI-FTICR mass spectrum of neuropeptide allatostatin (AST)-I (APSGAQRLYGFGL-NH₂) (A) before and (B) after DiLeu labeling, and CaM (PLRRTLSVAA-NH₂) (C) before and (B) after DiLeu labeling, 145.1 Da mass shift is produced for each incorporated label.

Figure 2

ESI QTOF de novo sequenced MS² spectrum BSA tryptic peptide (YICDNQDTISSK) labeled by (A) iTRAQ 4-plex, (B) DiLeu 4-plex. General structures of un-fragmented reporter group (URG) of iTRAQ (inset 1 in A) and DiLeu (inset 1 in B) are shown. Peptides were isobarically labeled by iTRAQ (inset 2 in A) and DiLeu (inset 2 in B). (C) Low reporter ion (RI) intensities, presence of URG and incomplete fragments of b and y ions are shown for iTRAQ 4-plex labeled BSA tryptic peptide (LKPDPNTLCDEFK). (D) Intense RI, absence of URG and complete set of b and y ions are shown for the same peptide labeled by 4-plex DiLeu labeling. (E) MALDI TOF/TOF MS² spectrum of AST-I labeled by DiLeu 4-plex showing quantitation capability in inset.

Figure 3

ESI QTOF tandem MS fragmentation (A) before and (B) after DiLeu labeling of B-type allatostatin neuropeptide (GNWNKFQGSW-NH₂)

Figure 4

Quantitation of BSA tryptic peptide (YICDNQDTISSK) using A) iTRAQ, B) DiLeu. Quantitative dynamic range of the same peptide is illustrated in (C). Means and standard deviations of BSA are shown in insets.

Figure 5

ESI QTOF MS² spectra of neuropeptides from the pericardial organs tissue extract of blue crab C. sapidus. (A) DiLeu 4-plex labeled B-type allatostatin neuropeptide (GNWNKFQGSW-NH₂), and (B) orcokinin neuropeptide (NFDEIDRSGFGFA). Relative abundance changes for neuropeptide expression levels among different animals are highlighted in inset tables.

Figure 6

DiLeu 4-plex labeled AST-I peptides show negligible retention time difference (29.65 min) of extracted ion chromatograms.

Scheme 1

Formation of (A) dimethyl amino aicd a₁ ion and (B) iTRAQ reporter ion.

Scheme 2

(A) Synthesis of isobaric labels and (B) activation using 4-(4, 6-Dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)/ N-Methylmorpholine (NMM)

Scheme 3

General structure of dimethyl leucine isobaric tags is shown in (A). Formation of new peptide bond at N-terminus or ε-amino group of the lysine side-chain and isotope combination of isobaric tags are illustrated in (B). Quantitation of 4-plex isobarically labeled peptide is illustrated in (C).