N-terminal sequence analysis

Hall et al. [62] identified in a separate study the same glycoprotein in H,K-ATPase vesicles isolated from porcine gastric mucosa. A stoichiometric ratio of 1.2 1.0 was found for the deglycosylated protein (35 kDa)/catalytic 94-kDa protein. Furthermore, compelling evidence that this glycoprotein is the H,K-ATPase p subunit was provided by N-terminal sequence analysis of three protease V8-obtained peptides of the 35-kDa core protein. These peptides showed 30% and 45% homology with the Na,K-ATPase pi and pi subunit, respectively. 

Characterization of Purified Proteins Assessing purity, 182, 555 determining size, molecular weight, and presence of subunits, 182, 566 amino acid analysis, 182, 587 limited N-terminal sequence analysis, 182, 602 peptide mapping, 182, 613 analysis for protein modifications and nonprotein cofactors, 182, 626 protein crystallization, 182, 646. 

During the period of time when the nature of the 19-nortestosterone acetate-dependent photoinactivation was under investigation, a new bacterial steroid isomerase was obtained from extracts of Pseudomonas putida (Biotype B) in nearly homogeneous form and some of its physical and enzymatic properties were characterized (64, 65, 66). The putida isomerase is similar in its molecular weight and quaternary structure to the testosteroni isomerase. Chemically, the most striking difference between the two isomerases is the presence of four residues of cysteine per polypeptide chain of the putida isomerase whereas no cysteine or cystine is present in the testosteroni isomerase. N-Terminal sequence analysis of the putida isomerase demonstrated substantial sequence homology between the two enzymes. 

For the majority of host-cell protein impurities, the most direct way of identification is through N-terminal sequence analysis. The automated... 

Matsudaira, P. (1990) Limited N-terminal Sequence Analysis, Methods Enzymd. 182, 602-613. 

During detailed characterization of a tryptic map of recombinant human tissue plasminogen activator (rtPA), a minor peak was resolved whose mass was not consistent with the expected set of tryptic peptides (L. Keyt and S.-L. Wu, unpublished observation). N-terminal sequence analysis of this fraction showed that it had a sequence containing residues 276-296 of rtPA with Lys-277 present as Hyl. We developed a modified amino acid analysis program capable of detecting hydroxylysine at levels down to 0.05 residues/mol to determine the distribution of Hyl in rtPA as well as in some other proteins derived from mammalian cells. 

Figure 2. N-terminal sequence analysis of rtPA 276-296 peptide with pxH-...

An early, reliable strategy for obtaining internal sequences involved electroblotting proteins to nitrocellulose membranes, in situ protease digestion, and subsequent isolation of peptides by reverse phase HPLC (7). This method has been employed in our laboratory for more than three years with a success rate of >95%. The two major limitations of this approach are 1) the blotting efficiency using nitrocellulose tends to be highly variable and frequently recovery at this step is low, and 2) the multiple step nature of this procedure further reduces overall recoveries. Even when recoveries are optimized at each step of the procedure, it is usually necessary to start with at least 5 to 10 times more protein than the amount required for direct N-terminal sequence analysis. 

The N-terminal sequence analysis of isoforms A and B showed the intial cycles as Pro-Ile-Gln and Val-Pro-Ile, respectively. These results are consistant with the molecular mass data described earlier (Table I), indicating that these isoforms are N-terminal truncated. 

Figure 3. N-terminal sequence analysis of norleucine-containing peptides digested from [15N]r-metHuLeptin. (a) Cycle 1-3 of peptide A9, Aps-Nle-Leu (b) Cycle 13-15 of peptide A5, Ser-Nle-Pro.

The techniques developed for the LF 3600 N-terminal protein sequencer (Beckman Instr.) provide a fast and efficient way to positively identify Ser (0-linked Sac), -Thr (0-linked Sac) and Asn (N-linked Sac) carbohydrate structures during N-terminal sequence analysis (Gooley et al., 1995). Liquid phase anhydrous trifluoroacetic acid (TEA) is used to extract glycosylated, polar amino acid derivatives from the reaction cartridge. These amino acids are then converted into PTH derivatives which can be... 

Gheorge, M. T. and Bergman, T., Deacetylation and internal cleavage of polypeptides for N-terminal sequence analysis, in Methods in Protein Structure Analysis, Atassi, M. Z. and Appella, E., Eds., Plenum Press, New York, 1994. 

Edman N-terminal sequence analysis can be performed to further confirm the sequence of the peptide. 

Pankuweit, S., 1. Portig, F. Lottspeich, and B. Maisch. 1997, Autoantibodies in sera of patients with myocarditis characterization of the corresponding proteins by isoelectric focusing and N-terminal sequence analysis. J.Mol.Cell Cardiol. 29 77-84. 

The primary structure was assessed by peptide mapping and N- and C-terminal sequencing. N-terminal sequence analysis showed that a single sequence was detected, MKAIFVLNAA, which corresponds exactly to the first 10 amino acids at the N terminus of P40 as predicted from the DNA sequence. Reverse phase HPLC analysis of a digestion of P40 with a lysine-specific endopeptidase, i.e. endoproteinase Lys-C, was used for identification and primary structure confirmation (Fig. 9). Endoproteinase Lys-C hydolyzes peptide bonds at the carboxylic side of lysine residues. The seventeen peaks resolved were characterized by mass spectrometry, allowing the confirmation of 99 % of the primary sequence. 

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