Ph.D Thesis

Ph.D StudentRabkin Emilia
SubjectCharge Derivatization of Peptides for Mass Spectrometry
DepartmentDepartment of Chemistry
Supervisor DR. Chagit Denekamp
Full Thesis textFull thesis text - English Version


The purpose of this work is to develop new and better methodologies for the determination of amino acids and peptides with mass spectrometry. The mass difference between consecutive ions within a series allows determining the identity of the consecutive amino acids and thus to deduce the peptide sequence. Frequently however, the collision induced dissociation (CID) spectra of protonated peptides show two or more incomplete series of ions. Hence, in the current research a novel charge derivatization procedure was developed, in which fixed positive charge is placed at the N-terminus of the analyzed peptide by a rapid chemical process. The derivatized peptide is then analyzed under CID conditions resulting in complete series of bi ions accompanies by the ai satellites. We find that the presence of the cationic moiety increases the sensitivity and facilitates the formation of informative fragmentation products.

However, fragmentation of the analyzed peptide at its backbone, as in the case of CID of closed-shell ions, does not allow the distinction between structural isomers or similar molecular weight amino acid residues. Hence, fragmentation of the side chains is required in addition to primary backbone cleavages. We present here a novel radical derivatization procedure, in which a triphenylcorrole(CuIII) complex moiety (tpc<Cu>) is placed at the N-terminus of the analyzed peptide by a rapid chemical process. Upon negative electrospray ionization, the derivative produces an anion radical peptide adduct, that facilates side chain fragmentations of the amino acids, under collision induced dissociation conditions.

Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry has been extensively used for the analysis of macromolecules, biomaterials and polymers owing to its soft ionization properties. Nevertheless, MALDI analysis of low molecular mass compounds is problematic because of their high volatility and reduced tendency to be ionized. Another drawback is the interference of low molecular mass compounds with the matrix used in the MALDI. In order to overcome these problems it is desirable to develop methods that either increase the molecular weight of the sample or allow ionization without matrix. The present work demonstrates the use of a new charge derivatization approach for small amines, as well as amino acids and peptides, in combination with MALDI-TOF mass spectrometry. The derivatization reaction is performed directly on a MALDI plate, thus allowing straightforward mass analysis by MALDI-TOF MS.