Matrix comparative sequence analysis

Retrieve the amino acid sequences of liver alcohol dehydrogenase from six organisms to perform phylogenetic analysis. Compare phylogenetic results from Fitch-Margoliash methods without (Fitch) versus with (Kisch) molecular clock using the Dayhoff PAM 001 matrix. 

Correct mass of purified proteins is determined using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). N-terminal sequencing and/ or LC-ES-MS can verify correct amino acid sequence. Folding of the recombinant protein should be compared to the natural counterpart by measuring CD-spectra. Furthermore, nuclear magnetic resonance (NMR) analysis can be performed to ensure the presence of tertiary structure, important for IgE binding activity (Neudecker et al. 2003). 

Mass analysis of peptide fragments frx>m a protein of "known sequence is the method of choice for speed and accuracy. Howevo, at least three major options are available for mass analysis, each varying in their merits. The three most widely used techniques are LSIMS, M/JLDI-TOF-MS, and ESI-MS. In LSIMS and MALDI, individual peptide fractions are analyzed by mixing with a matrix followed by ionization and mass analysis, while in ESI the samples can be separated on-line by LC, eliminating the need fcH individual p collecticMi. In this report we compare each of the techniques for the analysis of the heavy and light chains of the anti-CEA antibody CEA.11 H5 (1). 

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