Enzymatic Modification Of Targets For Proteomic Analysis

Another related approach uses intrinsic tissue transglutaminases activity to modify specific peptide sequences.239 A discussion of the usefulness of site-specific enzyme modification is included as it is considered complementary to the chemical approaches described above. Transglutaminases catalyze the formation of an isopeptide bond between a glutamine residue and a lysine residue (Figure 3.40). Blood Coagulation Factor Xlll/XIIIa (fibrin-stabilizing factor) is an example of a transglutaminases; factor XIIIa "stabilizes" fibrin clots by forming cross-links between specific regions in fibrin monomers.240 241 Tissue transglutaminases serve a variety of functions including adhesion and maturation of matrix proteins.242243 Transglutaminases have a high specificity for glutamine residues but are considerably more promiscuous with respect to the amine donor. For example, transglutaminases can catalyze the incorporation of dansylcadavarine into proteins (Figure 3.41).244 Esposito and colleagues245 have used two approaches to identify transglutaminase substrates by proteomic technology. 5-(Biotinamido) pentylamine (biotinylated cadavarine) (Figure 3.42) was used to identify glutamine sites while a biotinylated peptide (biotin-TVQQEL-OH) was used to identify lysine residues. A mixture of seminal vesicle protein IV, vasoactive intestinal peptide and P-casein was used as the test mixture. After transglutaminase treatment, the modified proteins were separated by reversed-phase HPLC. After hydrolysis with trypsin, the peptides were separated by HPLC and submitted to MS/MS analysis. In a subsequent study,246 this group used the same technical approach to identify transglutaminase substrates in human intestinal epithelial cells o o n n ch3

DAABD-Cl;[ 4-(dimethylaminoethylammosulfonyl)-7-chloro-2,1,3-benzoxad iazole]

TAABD-Cl; [7-chloro-2,1,3-benzoxadiazole-4-sulfonylaminoethyltrimethylam moniu

FIGURE 3.39 4-(Dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-CL) and 4-(trimethylammoniumethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiaz-ole (TAABD-Cl). Fluorogenic reagents for labeling cysteine peptides prior HPLC/MS analysis. See Matsuda, M. et al., Fluorogenic derivatization reagents suitable for isolaton and identification of cysteine-containing proteins utilizing high-performance liquid chro-matograpy-tandem mass spectrometry, Anal. Chem., 76, 728-735, 2004.

as part of a study on celiac disease. Transglutaminase is thought to be involved in the etiology of celiac disease.247 In the above study identifying transglutaminase substrates in human intestinal epithelial cells,247 more than 25 proteins were identified. The reader is directed to several recent publications248,249 for information on technical approaches to the study of transglutaminase substrates.

Nucleic acid-based microarrays (DNA microarrays) have been useful in measuring gene expression via transcription. While DNA microarray technology is distinct from genome sequence analysis, technical applications depend on the knowledge of linear gene structure.250 DNA microarrays measure gene function by measuring RNA


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Why Gluten Free

Why Gluten Free

What Is The Gluten Free Diet And What You Need To Know Before You Try It. You may have heard the term gluten free, and you may even have a general idea as to what it means to eat a gluten free diet. Most people believe this type of diet is a curse for those who simply cannot tolerate the protein known as gluten, as they will never be able to eat any food that contains wheat, rye, barley, malts, or triticale.

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