Trends in the Design of New Isobaric Labeling Reagents for Quantitative Proteomics

Abstract

Modern mass spectrometry is one of the most frequently used methods of quantitative proteomics, enabling determination of the amount of peptides in a sample. Although mass spectrometry is not inherently a quantitative method due to differences in the ionization efficiency of various analytes, the application of isotope-coded labeling allows relative quantification of proteins and proteins. Over the past decade, a new method for derivatization of tryptic peptides using isobaric labels has been proposed. The labels consist of reporter and balanced groups. They have the same molecular weights and chemical properties, but differ in the distribution of stable heavy isotopes. These tags are designed in such a way that during high energy collision induced dissociation (CID) by tandem mass spectrometry, the isobaric tag is fragmented in the specific linker region, yielding reporter ions with different masses. The mass shifts among the reporter groups are compensated by the balancing groups so that the overall mass is the same for all forms of the reagent. Samples of peptides are labeled with the isobaric mass tags in parallel and combined for analysis. Quantification of individual peptides is achieved by comparing the intensity of reporter ions in the tandem mass (MS/MS) spectra. Isobaric markers have found a wide range of potential applications in proteomics. However, the currently available isobaric labeling reagents have some drawbacks, such as high cost of production, insufficient selectivity of the derivatization, and relatively limited enhancement of sensitivity of the analysis. Therefore, efforts have been devoted to the development of new isobaric markers with increased usability. The search for new isobaric markers is focused on developing a more selective method of introducing a tag into a peptide molecule, increasing the multiplexicity of markers, lowering the cost of synthesis, and increasing the sensitivity of measurement by using ionization tags containing quaternary ammonium salts. Here, the trends in the design of new isobaric labeling reagents for quantitative proteomics isobaric derivatization strategies in proteomics are reviewed, with a particular emphasis on isobaric ionization tags. The presented review focused on different types of isobaric reagents used in quantitative proteomics, their chemistry, and advantages offer by their application.

Keywords: ESI-MS; ionization enhancers; isobaric labeling reagents; quantitative proteomics; quaternary ammonium salts.

Figure 1

The D4/H4 forms of nicotinyl-N-hydroxysuccinimide (Nic-NHS) developed by James et al. [19] and N-terminally labeled peptide, which can be applied for quantitation. X-H or D atom.

Figure 2

Isotope Coded Affinity Tag proposed by Gygi et al. [21]. X depicts H or D atoms in the molecule.

Figure 3

Schematic presentation of iTRAQ structure (A) and peptide labeled by iTRAQ (B).

Figure 4

First (A) and second (B) generation of tandem mass tags developed by Thomson and co-workers [14].

Figure 5

The structure of commercially available TMT (A) and the peptide modified by tandem mass tag (B) [17].

Figure 6

N,N-dimethyl leucine (DiLeu) tag (A) developed by Xiang and co-workers [10] and DiLeu modified peptide (B).

Figure 7

Schematic presentation of Deuterium isobaric Amine Reactive Tag (DiART) [32] (A) and DiART modified peptide (B).

Figure 8

iTRAQ hydrazide (iTRAQH) proposed by Palmese et al. [12].

Figure 9

Schematic presentation of the glyco-TMT reagents containing hydrazide (A) or aminoxy carbonyl-reactive groups (B) [13].

Figure 10

The iodoTMT reagent for cystein-containing peptide labeling and quantification proposed by Pan and co-workers [46].

Figure 11

The structures of QAS-iTRAQ 2-plex (X = Br, I) [9].

Figure 12

Reaction of peptide with 2,4,6-trisubtituted pyrylium salt (R*-alkyl or aryl).

Figure 13

ESI-MS-MRM chromatogram obtained for H-Gly-Leu-OH derivatized with 2,4,6-triphenylpyrylium salt and registered for 1 attomole [66].

Figure 14

Scheme presenting the formation of a pyridinium-based duplex (the blue circle indicates the ¹³C labeling) [70].