Sequencing of peptide amino acids

Table 2.2 Molecular masses and amino acid sequences of peptides isolated from the extract of acrospires of Arena sativa oat...

Because of their known specificity, these enzymes, especially trypsin and chymotrypsin, have been widely utilized in helping to determine the amino acid sequences of peptides (see Section 13.7). Hydrolysis using these... 

Messenger RNA Translation Predict the amino acid sequences of peptides formed by ribosomes in response to the following mRNA sequences, assuming that the reading frame begins with the first three bases in each sequence. 

The comparison of the amino acid sequence of the above-mentioned bitter peptides shows a large proportion of hydrophobic amino acids in each peptide. And the amino acid sequence of peptides also plays an important role in the intensity of the bitter taste. For example, the bitterness of Phe-Pro is more intense than that of Pro-Phe, and the bitterness of Gly-Phe-Pro is more intense than that of Phe-Pro-Gly (23). C-terminal groups of all bitter peptides in pepsin hydrolysates of the above-mentioned soy protein were characterized by the location of the Leu residue (14-17). The research on the relationship between the structure and bitter taste intensity of Arg-Gly-Pro-Pro-Phe-Ile-Val (BP-Ia) showed that Pro and Arg located on center and the N-terminal site, respectively, played an important role in the increment of bitter taste intensity besides the hydro-phobic amino acids located on C-terminal site (24-26). This may indicate that the peptide molecular structure formed by the arrangement of Arg, Pro and hydrophobic amino acid residues contributes to the bitter taste intensity of the peptide. 

Mass spectrometry is a powerful qualitative and quantitative analytical tool that is used to assess the molecular mass and primary amino acid sequence of peptides and proteins. Technical advancements in mass spectrometry have resulted in the development of matrix-assisted laser desorption/ion-ization (MALDI) and electrospray ionization techniques that allow sequencing and mass determination of picomole quantities of proteins with masses greater than 100kDa (see Chapter 7). A time-of flight mass spectrometer is used to detect the small quantities of ions that are produced by MALDI. In this type of spectrometer, ions are accelerated in an electrical field and allowed to drift to a detector. The mass of the ion is calculated from the time it takes to reach the detector. To measure the masses of proteins in a mixture or to produce a peptide map of a proteolytic digest, from 0.5 to 2.0 p.L of sample is dried on the tip of tlie sample probe, which is then introduced into tire spectrometer for analysis. With this technique, proteins located on the surfaces of cells are selectively ionized and analyzed. 

The amino acid sequence determination of peptides through de novo peptide sequencing procedure is one of the most familiar applications of mass spectrometry. A precise knowledge of the amino acid sequence of peptides is required in many situations - to understand their biological functions, to characterize components of the metabolic cycle of precursor proteins, to map changes in the metabolic profile of a peptide family caused by... 

The amino acid sequences of peptide fragments obtained from a normal protein and from the same protein synthesized by a defective gene were compared. They were found to differ in only one peptide fragment. The primary sequences of the fragments are shown here. 

Edman degradation was originally developed for determination of the primary structure (i.e., amino acid sequence) of peptides and proteins. Sequence analysis is not regularly performed for quality control in routine peptide synthesis but is more often employed for problem solving. As described earlier in this chapter, efficient characterization of synthetic peptides can be readily obtained by a combination of RP-EDPLC and mass spectrometry. Amino acid analysis is also valuable if MS is not available. If an incorrect mass or a discrepancy in the amino acid composition is found, one obvious alternative is to resynthesize the peptide. But, in order to deduce the cause of a failed synthesis, additional analyses must be performed. Both Edman degradation and tandem MS can be used to obtain sequence information... 

Edman degradation a method for determining the amino acid sequence of peptides and proteins (5.4) electronegativity a measure of the tendency of an atom to attract electrons to it in a chemical bond (2.1) electron transport to oxygen a series of oxidation-reduction reactions by which the electrons derived from oxidation of nutrients are passed to oxygen (19.1) electrophile an electron-poor substance that tends to react with centers of negative charge or polarization (7.5)... 

Describe the sequential Edman degradation method and the automated determination of the amino acid sequences of peptides. 

Table 2 Amino acid sequences of peptides containing optimal consensus sequences phosphorylated by different protein kinases and apparent binding constants (JCapp, M ) of 35 and 36 to the peptides as determined by fluorescence change...

Very often, no exact database match can be found even with high quality MS/MS mass spectra. It depends directly on the completeness and accuracy of the database searched, i.e. whether the genome is complete or incomplete, and on the quality of the transcripted EST sequences. These problems raise e question whether it is a novel protein, a known protein with a post-translational modification or if the failure to produce a database match due to inter-species variation, database sequence errors, or unexpected proteolytic cleavages. To address this problem, de novo interpretation of MS/MS data is an alternative where the amino acid sequence of peptides is derived by interpreting the mass differences between the generated MS/MS fragment ions sequence. 

In the final section of the chapter we discuss procedures to obtain the amino acid sequence of peptides, which is a vital component of protein identification. The fragmentation rules of peptides and guidelines for interpretation of peptide-mass spectrum are also discussed. By following these systematic steps, de novo sequencing of peptides and hence of a protein can be achieved. 

K. Biemann and S. A. Martin, Mass spectrometric determination of the amino acid sequence of peptides and proteins. Mass Spectrom. Rev. 6, 1-75 (1987). 

Once the identity of phosphopeptides in a digestion mixture is revealed, it is imperative to recognize which amino acid residue (Ser, Thr, or Tyr) is phospho-rylated. This task is accomplished by determining the amino acid sequence of peptide fragments in the protein digest after their fractionation into individual components by RP-HPLC, either off- or online with tandem mass spectrometry [34], The tandem MS methods described below have proved to be practical for this purpose. 

Tandem mass spectrometry (MS/MS) is very useful for the amino acid sequencing of peptides, and has been used widely in both protein biochemistry and pro-teomics to identify proteins, to deduce the sequence of a peptide, and to detect and locate post-translational modifications. Until around a decade ago, the concept of amino acid sequencing by MS-technologjes was synonymous with ESI-MS/MS, but today MALDI-MS/MS techniques are implemented in high-performance instruments such that the quality of MALDI tandem mass spectra is comparable with that of ESI-MS/MS spectra. Currently, MALDI tandem mass spectrometers exist in a number of geometries, including TOF-TOF, Q-TOF, ion trap and orbitrap analyzers that each provide unique analytical features for the sequencing of peptides and proteins by MS/MS (details of the instrumentation for different types of MS/MS are provided in Chapter 2). 

Traditionally, proteins were initially characterized by de-novo sequencing using automated Edman degradation and amino acid composition analysis. Today, however, these techniques tend to be replaced by MS, which not only provides more flexibility and sensitivity but is also amenable to the analysis of protein and peptide mixtures. Tandem mass spectrometry (MS/MS) is used for amino acid sequencing of peptides. MALDI-MS/MS is very powerful for peptide characterization and identiflcation via sequencing and sequence database searching. 

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