N-terminals blocking

Horner-Emmons reaction of N-terminal blocked aldehyde 1 with sulfonylphosphonates in the presence of sodium hydride gives the amino acid vinyl sulfone 2, which is deprotected with acid and converted into its chloride or tosylate salt 3 and coupled by the mixed anhydride method with an N-terminal protected peptide or amino acid to give the desired peptide vinyl sulfones 4 (Scheme 2). 4 5 N-Terminal protected aldehydes 1 are obtained from reduction of Boc amino acid V-methoxy-A-methylamides (Weinreb amides, see Section 15.1.1) by lithium aluminum hydride. 9 The V-methoxy-V-methylamide derivatives are prepared by reaction of Boc amino acids with N,O-dimethylhydroxylamine hydrochloride in... 

As an example where sequence information is available without the use of Edman or carboxypeptidase degradation, a study of calcineurin B may be cited. This work also allowed, most importantly, the determination of an N-terminal blocking group as myristic acid. 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

Identification of the Amino Terminal Peptide of N-terminally Blocked Proteins by Differential Deutero-Acetylation Using LC/MS Techniques... 

Figure 3. Contour plot of an LC/MS of a chymotryptic digest of modifled phosphorylase b, printed in black and white mode. Circles indicate the positions of peaks from a contour plot of the le-acetylated digest for comparison. The two significant shared peaks (indicated by arrows) are the N-terminal blocked peptide (at m/z=514 and 13 min elution time) and the leucine enkephalin internal standard (at m/z=SS6 and 21 min elution time).

These results show that the amino terminal peptide of N-terminally blocked proteins can be detected without analyzing every ion produced in an LC/MS experiment. 

Table I summarizes the sequencing results from alcohol dehydrogenase and the N-terminally blocked glycoprotein ovalbumin. The probable identity of these fragments is indicated. All fragments identified for both proteins by N-terminal sequencing corresponded to cleavage after cystine. The data from ovalbumin are particularly interesting. The structure of ovalbumin is well characterized (5) and contains only one disulfide bond between Cys 73 and Cys 120 yet sequence was obtained following Cys 11 and Cys 30. The bands for these fragments appeared more slowly than the odiers and were fainter in appearance.

Superoxide dismutase, an N-terminally blocked protein, was applied to a Zitex reaction membrane (approximately 1 nmol) in 10 xl of 1% aqueous trifluoroacetic acid. The first three cycles of C-terminal sequence analysis resulted in the identification of Lys (K) cycle-1, Ala (A) cycle-2, and lie (I) cycle-3 (Figure 3). The chemical background remained relatively stable as a thiohydantoin background increased, in part, attributed to internal cleavages as analogously observed for amino-terminal sequencing chemistry. 

As mentioned above, highly purified RNAP of S. acidocaldarius consists of 12 different components, B, A, A", D, E, F, G, H, I, K, L and N in near stoichiometric ratio which have been separated by SDS PAGE [91] (Fig. 5). Different N-terminal sequences have been obtained for all of these components, except for F and I which both appear N-terminally blocked [Lottspeich, F., unpublished results]. Internal peptides from F have been partially sequenced [Lottspeich, F. and Meitinger, C., unpublished]. Using... 

To overcome the drawbacks associated with the application of non-urethane N-terminal blocking groups, a new strategy had to be developed which enables a biocatalyst to cleave a urethane structure without the need for a direct attack on the urethane carbonyl group. The principle was successfully realized by the introduction of the p-acetoxybenzyloxycarbonyl (AcOZ) [30] and thep-phenyl-acetoxybenzyloxycarbonyl (PhAcOZ) [31] groups (Fig. 9). 

What percentage of the proteins you receive for sequence analysis are N-terminally blocked 24 25% (2-80) ... 

Studies of the rates of deamidation in a series of nonaqueous solvents have indicated a dependence of rate on the dielectric constant of the solvent. Although these experiments have generally involved solvents unsuitable for use in formulation, it is interesting to note a reported decrease of the rate of deamidation of N-terminal-blocked Boc-Asn-Gly-Gly in ethanol" (Boc = butoxycarbonyl). Similarly, the deamidation rate of the hexapeptide Val-Thr-Pro-Asn-Gly-Ala decreased with decrease in the dielectric constant of the solvent, an effect which was attributed to the destabilisation of the deprotonated peptide bond nitrogen anion which is involved in the formation of the cyclic imide intermediate. 

It should be obvious that the cleaner a preparation is, the better the resulting information will be. However, this does not mean that sequencing impure material is pointless. Even with the advent of micro techniques that enable sequencing from gel-purified material, co-migrating proteins are still encountered. In the worst scenario, one protein is N-terminally blocked (see the following). If the blocked protein is actually the one of interest, long-lasting confusion may follow easily. 

In the Merrifield method, the C-terminal amino acid is covalently attached to a solid support contained in a column. Each N-terminal blocked amino acid is added one at a time, along with other needed reagents, so the protein is synthesized from the C-terminal end to the N-terminal end. Notice that this is opposite to the way proteins are synthesized in nature (from the N-terminal end to the C-terminal end Section 27.13). Because it uses a solid support and is automated, Merrifield s method of protein synthesis is called automated solid-phase peptide synthesis. 

Figure 1 Charge sensitivities of the free energy of a solution of glycine dipeptide in methanol. The first three atoms/extended atoms (CMl, C, and O) builds up the acetyl N-terminal blocking group. The next five atoms/extended atoms (N, H, CA, C, and O) represent components of a glycine unit. The last three atoms/extended atoms at the C-terminal end (N, H, and CM2) terminate the peptide as an N-methyl amide.

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