Sequence analysis, amino-terminal proteins

Any commercially available or homemade gel electrophoresis apparatus can be used for the elution-concentration system as long as the dimensions do not significantly vary. Electroblotting is carried out in a Bio-Rad Transblot cell or MiniTrans blot. For details on protein sequencing and HPLC peptide separation, reference is made to the article on amino-terminal protein sequence analysis by Heinz Nika and Ruedi Aebersold in this volume. The scalpels (No. 11) were from Paragon and the long needles (Cat. No. V2A 1415 LL-10) from Acufirm. The power supplies were from Pharmacia-LKB (EPS 500/400). 

Automated carboxy-terminal (C-terminal) protein sequence analysis enables the direct and unambiguous confirmation of the C-terminal sequence of native and expressed proteins, the detection and characterization of protein processing at the C-terminus, the identification of post-translational proteolytic cleavages, and partial sequence information on amino-terminally blocked protein samples. In order for C-terminal sequence analysis to be of immediate benefit, each of the 20 common amino acid residues must be detectable. Additionally, the scope of typically analyzable protein samples must span a usefully broad molecular weight range and degree of structural complexity. 

The sample application is compatible with diverse samples recovered in various buffers (phosphate, inorganic salts, HEPES) and solvents (HPLC fractions). Samples that have been subjected to amino-terminal sequence analysis using the HP G1005A N-terminal protein sequencing system and Zitex as a reaction support may be transferred to C-terminal sequencer columns and subjected to C-terminal sequence analysis with the HP G1009A C-terminal sequencing system. 

Protein chemists have sought a practical, automated carboxy-terminal (C-termi-nal) sequencing method for many years. In the biophannaceutical industry, C-ter-minal sequence information can be essential for the early characterization of recombinant proteins, as well as for routine batch analysis. Several automated chemical degradation schemes, analogous in principle to the Edman method for amino-terminal (N-terminal) sequencing, have been proposed (1,2). In 1992, we introduced a novel alkylation method for C-terminal protein sequencing (3,4). 

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... 

For routine determination of unknown N-terminal protein sequences, at least 50-100 pmole of the purified protein should be available. The only method for the accurate quantitation of low picomole amounts of protein is quantitative amino acid composition analysis. Twenty to fifty picomoles of protein should be set aside... 

Amino acid analysis itself does not directly give the number of residues of each amino acid in a polypeptide, but it does give amounts from which the percentages or ratios of the various amino acids can be obtained (Table 5.2). If the molecular weight and the exact amount of the protein analyzed are known (or the number of amino acid residues per molecule is known), the molar ratios of amino acids in the protein can be calculated. Amino acid analysis provides no information on the order or sequence of amino acid residues in the polypeptide chain. Because the polypeptide chain is unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-termuial end. 

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. 

The hybrid approach enhances the speed and efficiency of primary structure analysis and the range of proteins that can be sequenced. It also circumvents obstacles such as the presence of an amino-terminal blocking group or the lack of a key overlap peptide. Only a few segments of primary strucmre must be determined by Edman analysis. 

Gorman, J.J. (1984) Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis. Anal. Biochem. 160, 376. 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

Hardeman K., Samyn B., Van der Eycken J., and Van Beeumen J. (1998), An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids, Protein Sci. 7, 1593-1602. 

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