Abstract
Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states.
Highlights
Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry
Several strategies have been developed based on stable, non-radioactive isotopes like 2H, 13C, 15N, and 18O and can be classified in three stable isotope labeling experiment categories: (i) metabolic stable isotope labeling like stable isotope labeling by amino acids in cell culture (SILAC) using growth medium with stable isotopelabeled amino acids [6] or via isotope-labeled nutrients like [15N]ammonium salt [7, 8]; (ii) isotope tagging by chemical reaction such as ICAT, isotope-coded protein labeling (ICPL), or isobaric tagging for relative and absolute quantification [5, 9, 10]; and (iii) enzyme-catalyzed reactions like
Both reactions are based on carbodiimide chemistry to activate carboxylic groups for further reaction with primary amino groups
Summary
2D, two-dimensional; ANIBAL, aniline benzoic acid labeling; ANI, aniline; BA, benzoic acid; ICPL, isotopecoded protein labeling; iTRAQ, isobaric tagging for relative and absolute quantification; SILAC, stable isotope labeling by amino acids in cell culture; NHSS, N-hydroxysulfosuccinimide sodium salt; DMF, dimethylformamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl); IPI, International Protein Index; RAM, restricted access material. We describe a new isotope-coded chemical reaction optimized for electrospray ionization called aniline benzoic acid labeling (ANIBAL) that relies on a twin/dual chemistry approach tagging carboxylic and amino groups at the protein level. Both reactions are based on carbodiimide chemistry to activate carboxylic groups for further reaction with primary amino groups. The ANIBAL technique was first applied to protein standards for optimization, to a milk fraction containing six major abundant proteins for dynamic range assessment, and to a bacterial whole cell lysate (in a 1:1 ratio) to confirm the applicability to a complex sample
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