Monday, May 24, 2004 -- 10:15am -- Oral Presentation

Fragmenting and sequencing peptides by 157nm laser-induced photodissociation
James P. Reilly; Matthew S. Thompson; Weidong Cui; Indiana University, Bloomington, IN

Introduction
A tandem TOF instrument has been constructed to study the photodissociation of peptides using 157 nm light. Singly-charged peptides with C-terminal arginine yield unique fragment ion distributions primarily containing x-, v-, and w-type fragments. The charge is apparently not involved in the fragmentation process and it remains on the C-terminal fragment, greatly simplifying the interpretation of mass spectra. Peptides with basic N-terminal residues yield a- and d-type ion fragments. De novo sequencing software has been developed that takes advantage of the relatively simple mass spectra generated. When guanidination is used to increase the basicity of lysine residues, the method enables nearly complete sequence determination of tryptic peptides, including the differentiation of leucine and isoleucine

Methods
Experiments were performed on a homebuilt MALDI TOF/TOF instrument that uses a 157 nm F2 laser to fragment mass-selected precursor ions at the end of the first TOF stage. Fragment and undissociated precursor ions are then mass analyzed and detected in the second stage reflectron TOF. Studies have been performed using peptides that are commercially available or obtained from chromatographic separation of tryptic digests. Lysine-containing peptides are derivatized using O-methyl isourea using standard procedures. A de novo algorithm exploits the consecutive appearance of x-type fragment ions to derive sequence information. It then corroborates these assignments by considering adjacent v- and w-type ions.

Preliminary Results
We have performed 157nm photodissociation of dozens of singly charged peptides. In all cases for which a highly basic residue is located at the C-terminus, the resulting spectrum contains a remarkable and unprecedented series of x-, v-, and w-type ion fragments. The similarity of daughter ion peak intensities is consistent with dissociative chromophores being distributed uniformly throughout the peptide. The initial step in the ion fragmentation process appears to be cleavage of the peptide backbone bond between an a-carbon and a carbonyl carbon. This hypothesis is supported by the observation of a- and d-type ion fragments when an arginine on or near the N-terminus of the peptide ion sequesters the charge. Peptides that are deficient in highly basic residues yield fragmentation spectra that contain more conventional b- and y-type ion fragments, suggesting that mobile protons may play a role in their fragmentation. Guanidination of lysine-terminated tryptic peptides increases the basicity of this residue sufficiently that charge sequestering occurs and x-, v-, and w-type ion fragments are observed. The method consistently generates high-energy side chain cleavages that provide additional information about the identities of corresponding residues. For example, distinguishing isobaric leucine and isoleucine can be a challenge in many mass spectrometry experiments. However, these are readily differentiated by the 14 Da mass difference in their intense w ions. The propensities with which different amino acids form x, v or w fragments when excited with 157 nm light have also been investigated. This information is incorporated into our de novo sequencing algorithm in order to check sequence assignments that are initially derived by considering x-ion spacings. A number of examples of the application of this algorithm will be discussed.

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