ANIBAL, Stable Isotope-based Quantitative Proteomics by Aniline and Benzoic Acid Labeling of Amino and Carboxylic Groups

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 Creagent 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. Molecular & Cellular Proteomics 7:800 -812, 2008.

Quantitative Neuroproteomics of the Synapse

“…The Hystar program (Bruker) was used to define the HPLC and mass spectrometry methods that were used to develop the solvent gradient and MS tune method and integrate the instruments used (8). Other MS and LC systems can be used with appropriate modifications to these protocols (9–11).…”

2nd Congress of the International Society of Nutrigenetics/Nutrigenomics (ISNN)

“…These latter observations raise important questions regarding the potential pleiotropic effects of the 5-LO pathway on components of the metabolic syndrome. For example, recent studies in humans and mice have demonstrated that various pharmacological modifiers of the 5-LO pathway decrease inflammatory biomarkers and aortic lesion formation, respectively, [7][8][9], raising the possibility of developing 5-LO pathway inhibitors for the treatment of CVD. Our results suggest that it would be important for such therapeutic strategies to carefully consider potentially undesirable metabolic effects as well despite the pro-atherogenic role that 5-LO pathway genes appear to play in humans.…”

“…Our Nutrigenomics and Nutrigenetics research will be exemplified with own tool developments [5][6][7] and application areas like nutritional modulation of immunity, digestive health [8,9] diabetes and obesity as well as bioavailability and bioefficacy of health-beneficial ingredients [10,11].…”

Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq) for 5-Carboxylcytosine Detection in DNA

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