Mass spectrometry peptide sequencing using

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

As each resin bead contains only a single molecule the beads can be screened individually for bioactivity by either screening for activity of bound peptide in the biological assay or by cleaving the resultant peptide from the bead before undertaking the bioanalysis. The identity of any active compounds can then be determined by using mass spectrometry to sequence the active peptide. 

Figure 2. Peptide maps (A-C) and MALDI-TOF mass spectra (D-F) of PVDF-bound transferrin (53 pmol) digested with trypsin in the presence of 50 pi of 1% RTX-100/10% acetonitrile/100 mM Tris, pH 8.0 (A,D), 1% octylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (B,E), and 1% decylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (CJF) as described in Materials and Methods. Ninety percent of the digestion was analyzed by HPLC ( 29 pmol based on Table I) and 0.5% ( 150 fmol) was used for MALDI-TOF mass spectrometry. Peptides 1 and 2 in A-C were amino terminally sequenced (Table II) and analyzed by MALDI-TOF mass spectrometry (Figure 3).

As for all synthetic products to be tested in biological systems, a careful analytical characterization of peptide libraries is crucial in order to confirm their identity and establish their quality. Compared to individual peptides, however, the analysis of peptide libraries is complicated due to the fact that the peptides are either bound to a solid support or arranged in highly complex mixtures. This poses certain restrictions on which analytical methods can be used to characterize combinatorial libraries. For example, analytical methods that are based on the separation of product components, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), are only of limited use for the analysis of peptide libraries, in particular of those made up of complex nnixtures (>100 peptides per mixture). The analytical methods beneficially applicable to peptide libraries include amino acid analysis, mass spectrometry, and sequencing. 

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.]...

Mass spectrometry may be used to determine primary sequences. Partial sequential chemical or enzymic degradation of peptides or proteins can yield a mixture of peptides with progressively shorter sequences. For example, consider the following (hypothetical) polypeptide sequence that has been partially digested with a proteolytic enzyme (carboxypeptidase) to yield the following ladder" of fragments ... 

Mass spectrometry is often used for the sequence analysis of peptides from 2 to 20 amino acids in length. The procedure requires only microgram quantities of protein and is very sensitive cationic fragments are identified by their charge-to-mass ratio. In one procedure, peptides are treated with triethylamine and then with acetic anhydride. What will such a procedure do to amino groups Next, the modified peptide is incubated with a... 

Keough, T. Youngquist, R.S. Lacey, M.P. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry. Proc. Natl. Acad Sci. USA. 1999, 96, 7131-7136. 

E Peptide Sequencing Using Mass Spectrometry and Sequence Databases... 

Bradley, C. V., D. H. Williams, and M. R. Hanley Peptide Sequencing Using the Combination of Edman Degradation, Carboxypeptidase Digestion and Fast Atom Bombardment Mass Spectrometry. Biochem. Biophys. Res. Comm. 104, 1223 (1982). Williams, D. H., S. Santikarn, P. B. Oelrichs, F. de Angelis, J. K. Macleod, and R. J. Smith The Structure of a Toxic Octapeptide, Containing 4 D-amino acids, from the Larvae of a Sawfly. Lophyrotoma Interrupta. J. Chem. Soc. (London), Chem. Commun. 1982, 1394. 

Bier, M. E. Schwartz, J. C. Zhou, J. Syka, J. E. P Taylor, D. Land, A. James, M. Fies, B. Peptide sequencing using fast MS and MS/MS scan rates on a pLC/quadrupole ion trap. In Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 21-26,1995, p. 988. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Tandem mass spectrometry (MS/MS) produces precise structural or sequence information by selective and specific induced fragmentation on samples up to several thousand Daltons. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for sequencing by mass spectrometry. The smaller sequences can be used to deduce the sequence of the whole protein. 

---