Determination of peptide sequence

Jones, B. N., Paabo, S., and Stein, S. (1981). Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phthaldialdehyde precolumn labeling procedure. J. Liq. Chro-matogr. 4, 565-586. 

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... 

Since the sensitivity of MS methods is similar to that of Edman chemistry, both methods will continue to be used. Because MS methods cannot determine the N- or C-terminal sequences in intact proteins, there will be a continued need for the Edman sequencer. However, MALDI- and ESI-MS methods can give accurate molecular masses for intact proteins, which when compared to their predicted sequences and databases (Eng et al., 1994), can verify a given structure, giving confidence to the N- and C-terminal sequence predicted from the peptide maps. MS-MS provides the fast and sensitive approach to sequence determination of peptides/proteins. 

Derivatization of amino acids with phenylisothiocyanate (XXXXII) to give phenylthiohydantoins (XXXXIIl) is utilized in the Edman method for sequence determination of peptides and proteins. The first step, performed under mildly basic conditions, may be regarded in principle as an acylation ... 

The sequence determination of peptides is crucial to understand their biological functions, to profile metabolic changes in a particular peptide family that can occur as a result of pathological stress or therapeutical treatment, and to assess the purity of newly synthesized peptides. As discussed above, sequencing of peptide from a protein digest is a major step in identification of proteins. 

Klarskov, K., Breddam, K., and Roepstorff, P. (1989) C-terminal sequence determination of peptides degraded with carboxypeptidases of different specificity and analyzed by Cf plasma desorption mass spectrometry. Anal. Biochem. 180, 28-37. 

Sequence Determination of the Brain Peptide Leucine Enkephalin A group of peptides that influence nerve transmission in certain parts of the brain has been isolated from normal brain tissue. These peptides are known as opioids, because they bind to specific receptors that also bind opiate drugs, such as morphine and naloxone. Opioids thus mimic some of the properties of opiates. Some researchers consider these peptides to be the brain s own pain killers. Using the information below, determine the amino acid sequence of the opioid leucine enkephalin. Explain how your structure is consistent with each piece of information. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

Fourier transform mass spectrometry (FTMS) is a rapidly growing technique of increasing analytical importance. Foremost among its many attributes are its high mass resolution and wide mass range capabilities, as well as its ability to store ions. This relatively new technique has been employed in a wide variety of applications, ranging from the exact mass measurement of stable nuclides to the determination of peptide sequences. The future holds considerable promise both for the expanded use of FTMS in a diverse range of chemical problems, as well as advances in the capabilities of the technique itself. 

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. 

Takaki, A., Yoshitake, S., Ishiguro, M., and Funatsu, M. (1972). Anticoagulant peptide obtained from fibrinogen degradation products by plasmin. II. Sequence determination of the peptide. Proc. Japan Acad., 48, 525-538. 

For determination of peptide sequences as well as protein modifications, MS/MS has become the definitive approach. However, for database searches MS/MS spectra have to be perfect. This is not always given because there are differences in tendencies of peptide bonds to fragment and certain amino acids show unique fragmentation characteristics. 

Partial sequence determination of a peptide gave the following ... 

Figure 1.14 displays an example of FABMS and MS/MS applied to the detection of peptides in a mixture and the sequence determination of one of them. 

Joo SH, Xiao Q, Ling Y, Gopishetty B, Pei D. High-throughput sequence determination of cyclic peptide library members by partial edman degradation/mass spectrometry. J. Am. Chem. Soc. 

N-terminal sequence determination of the intact protein and isolated peptides was performed by automated Edman degradation using a Hewlett Packard G1005A protein sequencer (7,8). Automated C-terminal sequence analysis was done in collaboration with Perkin Elmer-Applied Biosystems Division (9-12). 

The sequence of amino acids in the polypeptide chain (i.e., the primary structure of a polypeptide or protein) can be established by selective chemical and enzymatic cleavage of the protein followed by separation, amino acid analysis, and sequence determination of all peptide fragments. The entire amino acid sequence is established by overlapping identical regions of the individual fragments. The following problem illustrates the procedure. 

Figure 15.14. Determination of peptide sequence using nanoelectrospray ionization, and a very high-resolution mass analyzer (Q-TOF). In the first quadrupole, a doubly charged peptide ion of m/z — 625.41 was selected and later fragmented. The m/z CID spectrum yields the FGDYGSIDYGR sequence, shown at the top.23 [Reprinted, with permission, from E. Gustafsson, K. Thoren, T. Larsson, P. Davidsson, K. Karlsson, and C. L. Nilsson, Identification of Proteins from Escherichia coli Using Two-Dimensional Semi-Preparative Electrophoresis and Mass Spectrometry. Rapid Communications in Mass Spectrometry 15, 2001, 428-432. Copyright 2001 John Wiley Sons, Ltd.]...

A solid-state NMR technique for the determination of peptide backbone conformations at specific sites in unoriented samples under MAS has been described and demonstrated on labeled samples of tripeptide AlaClyGly and 17-residue peptide. Experiments and simulations show that both c ) and xj/ backbone dihedral angles can be extracted from a single data set. This technique, called DQCSA spectroscopy, may be especially useful when analyzing the backbone conformation of a polypeptide at a particular doubly labeled site in the presence of additional labeled carbons along the sequence. 

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