Abstract:
Investigation of negative ion mass spectrometry (MS) is important to proteomics due to its superior ability to analyze acidic peptides and provide complementary information to positive ion mode. This dissertation focuses on the deprotonation and dissociation of amino acids and peptides, which provides fundamental knowledge and assists in development of negative ion MS. Gas-phase acidities (GA) of acidic peptides were determined experimentally and compared to computational values. In electrospray ionization (ESI), either one major structure or multiple structures with very similar GAs were formed. Peptides with acidic residues at the C-terminus are more acidic than those at the N-terminus. Replacing glutamic acid residues (E) with aspartic acid (D) can increase the acidities when E is at the C-terminus but has no effect if E is at the N-terminus.
Bond dissociation energies of deprotonated amino acids, dipeptides, and their amides were investigated by combining MS and computations. The loss of H2O occurs in collision-induced dissociation (CID) from all amides except glycine amide. Loss of the C-terminus is only seen in CID of deprotonated dipeptides. In addition, an intense y1 ion forms from dipeptides and their corresponding amides.
In the comparison among radical-based dissociation techniques, negative ion in-source decay (nISD) has the best performance for peptides. nISD gives the highest sequence coverage and consistently generated singly charged c- and z-ions. Besides, nISD produced the least neutral loss products and is the fastest technique. The disadvantage of nISD is its inability to select precursor ions.
For the investigation of nISD with oxidizing matrices on acidic peptides, 4-nitro-1-naphthylamine (4N1NA) was used as a MALDI matrix for the first time and was found to be the most useful matrix. The overall sequence coverage obtained with 4N1NA is higher than with three other matrices. The formation of c- and a-ions indicates that 4N1NA has both reducing and oxidizing characteristics.
