Amino acids tagging

Dieterich DC, Link AJ, Graumann J et al (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci USA 103 9482-9487... 

Our studies of non-canonical amino acids were motivated initially by an interest in making proteins with novel properties. Our interest broadened when our colleague Daniela Dieterich suggested that pulsed metabolic labeling of cellular proteins with non-canonical amino acids might provide a method for time-resolved analysis of protein synthesis in neurons. With Daniela and Erin Schuman, we developed this idea into the BONCAT (bio-orthogonal non-canonical amino acid tagging) method shown in Scheme 2 [33]. 

For the resolution of a secondary alcohol, forming an optically active alcohol enantiomer and the corresponding ester antipode, purification is usually carried out by silica gel chromatography because of the chemical nature of both final products, and adequate derivatization can be carried out, facilitating the isolation of the final products by filtration or distillahon [50]. Some elegant and more sophisticated acyl donors have been described for the KRs of secondary alcohols, which allow an easy purification of the final products and the removal of the acyl donor by simple extraction protocols. These include the use of cyclic anhydrides such as succinic anhydride [51,52] or vinyl esters with amino acid tags [53]. 

As shown in Figure 45.1, the bases appear in complementary pairs, A with T and G with C in this particular example, the sequence for one strand of DNA is A-T-C-G-T- while the other strand is -T-A-G-C-A-. The sequences of the bases attached to the sugar-phosphate backbone direct the production of proteins from amino acids. Along each strand, groups of three bases, called codons, correspond to individual amino acids. For example, in Figure 45.1, the triplet CGT, acting as a codon, would correspond to the amino acid serine. One codon, TAG, indicates where synthesis should begin in the DNA strand, and other codons, such as ATT, indicate where synthesis should stop. 

Sequence tagging The use of MS-MS to investigate the amino acid sequence of a peptide. 

The calculated value for the Hisg-tagged protein is 28,492 Da. N-terminal amino acid sequence analysis (Procise) was carried out on purified recombinant resilin. The following sequence (at 120 pmol yield) was obtained for the first 12 amino acid residues MHHHHHHPEPPV, as expected from DNA sequence analysis. 

Fig. 15 Amino acid sequences of artificial extracellular matrix (aECM) proteins. Each protein contains a TV tag, a histidine tag, a cleavage site, and elastin-like domains with lysine residues for crosslinking. The RGD cell-binding domain is found in aECM 1, whereas aECM 3 contains the CS5 cell-binding domain. aECM 2 and aECM 4 are the negative controls with scrambled binding domains for aECM 1 and aECM 3, respectively. Reprinted from [121] with permission from American Chemical Society, copyright 2004...

When the Arabidopsis Expressed Sequence Tag (EST) Database was searched with the tomato fmit Psubunit protein sequence two related cDNAs were identified (Figure 11). cDNA 2 is near full length and has been completely sequenced, cDNA 1 has also been sequenced but currently lacks approximately 100 amino acids of coding region. The two Arabidopsis cDNAs are 81% identical at the protein level and have lower identity to the protein encoded by tomato gene 1, 64 and 63% for cDNA 1 and cDNA 2, respectively. However, both cDNAs encode... 

AccQ-Tag ) system mentioned in the section on amines has also been used for amino acids.90... 

Separation of amino acids, peptides, and proteins Amino acids are interesting molecules by themselves from an analytical point of view for two reasons. They are inherently enantiomeric and are the building blocks of peptides and proteins. The separation of amino acids is usually done through a derivatization process due to the fact that the absorbance in the UV is low. The most frequently used derivatization is done by fluorescent tagging. Sensitivity can reach the subfemtomole level.136 139 Temperature control can be used to separate conformers.140 Two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 and four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 were separated at subzero temperatures by including glycerol as an antifreeze component of the buffer. 

Dinitrofluorobenzene (86, X = F) is, because of its reactivity, much used for tagging the NH2 group of terminal amino-acids in protein end group analysis. Once it has reacted with the NH2 it is very difficult to remove again and will thus withstand the subsequent hydrolysis of the protein to its constituent amino-acids. 

Gu, S Du, Y Chen, J., Liu, Z Bradbury, E.M., Hu, C.A., Chen, X. (2004). Large-scale quantitative proteomic study of PUMA-induced apoptosis using two-dimensional liquid chromatography—mass spectrometry coupled with amino acid-coded mass tagging. J. Proteome Res. 3, 1191 1200. 

Heterocyclic fluorophores based on the benzoxadiazole nucleous, namely 4-nitrobenz-2-oxa-l,3-diazole (NBD) 14 derivatives/analogs, have been widely used as derivatization reagents for analysis purposes. Examples include the amino- or thiol reactive 4-fluoro-7-nitrobenz-2-oxa-l,3-diazole (NBD-F) 15 and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-C1) 16 [45-50] and the thiol-reactive /V-((2-(iodoacetoxy)ethyl)-/V-methyl)amino-7-nitrobenz-2-oxa-1,3-dia-zole (IANBD ester) 17 [51] and 7-chlorobenz-2-oxa-l,3-diazole-4-sulfonate (SBD-C1) 18 [52], NBD-F and NBD-C1 derivatives can be excited at about 470 nm by using the relatively inexpensive and reliable argon ion lasers or newer diode pumped solid state (DPSS) lasers. NBD-F has been used as a labeling tag in various capillary electrophoresis (CE) experiments for amino acids [53-57] including the monitorization of in vivo dynamics of amino acids neurotransmitters [58]. 

We have employed a more systematic random mutagenesis approach by dividing the BDI into consecutive clusters of 10 amino acid residues (Boxes) that are individually replaced with a string of 10 alanine residues in the full-length Hisg-tagged allele to facilitate affinity purification of the mutant protein (Valasek et ah, 2004). Alternatively, clusters rich in... 

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