MALDI sequencing

Edman Degradation and MALDI Sequencing Enables N- and C-Terminal Sequence Analysis of Peptides... 

Recently, fragmentations by post source decay (PSD) was introduced as a technique to obtain structural information in MALDI MS [6,7]. We describe here the combined use of automated Edman sequencing and MALDI sequencing for the determination of proteolytic peptide fragments in the low picomole range. 

Previously, we gave a short description of the principle of MALDI sequencing [11]. In the following we will briefly expand on the basics of the process as applied to peptide sequencing. 

Figure 4. MALDI sequencing of a chymotryptic peptide fragment.The overlapping mass ranges for metastable ions are due to steps in the reflector voltage.

Figure 6. The molecular ion peak (Figure 5) was subjected to MALDI sequencing.The mass range, in A, from 650 - 1000 Da displays theY serie LAHGK and the B seiieHFY in 5 the Y and the B serie YHLA from 1000 - 1350 Da are shown.

Heparin Analytical Tool. Recently, a heparin sequencing technology called property-encoded nomenclature/MALDI sequencing was developed (173). 

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

Experimentation showed that the protein was not glycosylated and that the sequence at the iV-amino acid terminus corresponded to that expected. The C-terminus sequence, however, did not correspond to that predicted and these data were interpreted in terms of the presence of a heterogeneous, truncated, protein. A study of the tryptic digest fragments from this protein with matrix-assisted laser desorption ionization (MALDI) with post-source decay enabled the authors to suggest the positions at which the parent protein had been truncated. 

The presence of three polypeptides in Table 5.8 tliat were not predicted from the relationship between the amino acid sequence and the enzyme used for digestion is worthy of note when interpretation of data of this sort is undertaken. The MALDI data showed six further unexpected polypeptides, none of which were detected in the LC-MS data ... 

The extraordinary complexity of human genes and their products has encouraged the development of extremely high-resolution analytical methods.75 Capillary electrophoresis is competitive with slab gel methods, with resolution up to the order of about 1,000 base pairs for sequencing, sizing, and detection of mutation. Reversed phase HPLC is useful for restriction digest mapping and MALDI-MS up to about 1000 base pairs. 

The major advantage of the tandem mass spectrometry approach compared to MALDI peptide fingerprinting, is that the sequence information obtained from the peptides is more specific for the identification of a protein than simply determining the mass of the peptides. This permits a search of expressed sequence tag nucleotide databases to discover new human genes based upon identification of the protein. This is a useful approach because, by definition, the genes identified actually express a protein. 

It should be pointed out that FAB, MALDI, and ESI can be used to provide ions for peptide mass maps or for microsequencing and that any kind of ion analyzer can support searches based only on molecular masses. Fragment or sequence ions are provided by instruments that can both select precursor ions and record their fragmentation. Such mass spectrometers include ion traps, Fourier transform ion cyclotron resonance, tandem quadrupole, tandem magnetic sector, several configurations of time-of-flight (TOF) analyzers, and hybrid systems such as quadrupole-TOF and ion trap-TOF analyzers. 

---