Modified amino acids, identification

Identification of the N-terminal Amino Acid in Polypeptides (TLC of Modified Amino Acids)... 

A comprehensive analysis of protein phosphorylation includes the identification of the phosphoprotein or phosphopeptide, the localization of the modified amino acid, and if possible, the quantification of phosphorylation. 

Absolute identification of the modified amino acids may require more than one enzyme digest to produce different peptides. Some kinds of modifications that are easily identified by MS include phosphorylation of threonine or serine sulfation or phosphorylation of tyrosine deamidation of asparagine or glutamine O-or N-linked glycosylation oxidation of methionine or cysteine and N-terminal modification by formylation or prenylation. Combining enzymatic maps (tryptic mapping) with MS/MS may identify single amino acid variants of the protein that cannot otherwise be seen. 

In Steps 3 and 4, the protein is cleaved into smaller fragments, and the amino acid sequence is determined. Automated instruments can perform a stepwise modification starting from the N-terminal end, followed by cleavage of each amino acid in the sequence and the subsequent identification of each modified amino acid as it is removed. This process is called the Edman degradation. 

Posttranslational modification (PTM) with functional groups is a universal mechanism for diversifying the activities of proteins. PTMs can affect many properties of proteins, such as localization, activity status, interaction networks, solubility, folding, turnover, or stabUity. It is therefore of vital importance to accurately determine the identities of modified proteins, the modified amino acid residues, and the covalently attached group. This chapter describes the process of PTM identification using the adenylylation (i.e., the covalent transfer of an adenosine monophosphate (AMP)) of rat sarcoma related in brain (Rab) proteins hy Legionella pneumophila enzymes as an example. It also deals with the development of PTM-specific antibodies from synthetic peptides. This account underlines the importance of chemical biology in the elucidation of PTMs. 

N-terminal sequencing) peptide identification, moderate cost, widely used, can identify modified amino acids required, will only identify proteins/peptides, large sample size, requires purified proteins, sample consumed... 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

The vast majority of research focused on selenium in biology (primarily in the fields of molecular biology, cell biology, and biochemistry) over the past 20 years has centered on identification and characterization of specific selenoproteins, or proteins that contain selenium in the form of selenocysteine. In addition, studies to determine the unique machinery necessary for incorporation of a nonstandard amino acid (L-selenocysteine) during translation also have been central to our understanding of how cells can utilize this metalloid. This process has been studied in bacterial models (primarily Escherichia colt) and more recently in mammals in vitro cell culture and animal models). In this work, we will review the biosynthesis of selenoproteins in bacterial systems, and only briefly review what is currently known about parallel pathways in mammals, since a comprehensive review in this area has been recently published. Moreover, we summarize the global picture of the nonspecific and specific use of selenium from a broader perspective, one that includes lesser known pathways for selenium utilization into modified nucleosides in tRNA and a labile selenium cofactor. We also review recent research on newly identified mammalian selenoproteins and discuss their role in mammalian cell biology. 

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