ORGANIC zyxwvutsrqpo MASS SPECTROMETRY, VOL. 24, zyxwvutsr 176-182 (1989) Sequence-specific Fragmentation of Benzyloxycarbonyl Dipeptides by Resonance-enhanced Multiphoton Ionization Kimberly R. Segar and Murray V. Johnston? Department of Chemistry and Biochemistry, Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado 80309-0215, USA The mechanism of fragment ion formation in resonance-enhanced multiphoton ionization (REMPI) of benzyl- oxycarbonyl (CBZ)-derivitized dipeptides is presented. At 266 nm, the entire multiphoton process can be thought of zy as a two-part scheme where ionization occurs by resonant two-photon ionization followed by photodissociation of the created ions. When the energy of two photons exceeds the molecular ionization energy by a significant amount, REMPI has the advantage of producing both parent ions and low appearance energy fragments in large amounts. For CBZ dipeptides, resonant two-photon ionization at 266 nm produces parent ions as well as A type sequence ions with high abundance. On the other hand, a three-photon process (resonant two-photon ionization followed by parent ion photodissociation) forms sequence-related ions which also involve complex fragmentations of the CBZ chromophore. These results are compared to mass spectra obtained by other ionization/dissociation methods and to REMPI mass spectra of related compounds. Factors related to molecular structure elucidation based upon REMPI mass spectra are discussed. Enhanced isomer distinction is demonstrated for CBZ-leu-ala-OCH, and CBZ-ile-ala-OCH, based upon REMPI fragmentation. INTRODUCTION Recent work in resonance-enhanced multiphoton ion- ization (REMPI) mass spectrometry has shown the potential this technique holds for obtaining mass spectra of important biomolecules such as metabolites of catecholamines,’ porphyrins,’ chlorophyll a,3 amino acids4 and pep tide^.^*^-^ In most cases, the parent ion alone can be produced by a judicious choice of the laser intensity and wavelength. Under these conditions, soft ionization is achieved at low laser intensity, while exten- sive fragmentation is induced at high laser intensity. In some cases, most notably involving amino acids and small peptides, true soft ionization is difficult to achieve and various fragment ions are observed along with the parent ion even at the lowest laser inten~ities,~*~ Fur- thermore, the relative abundances of the fragment ions can change markedly with laser ~ a v e l e n g t h . ~ , ~ It is clear from these studies that REMPI fragmentation depends strongly upon the laser wavelength and inten- sity chosen. In order to better understand these depen- dences, we have undertaken and present here a detailed investigation of the mechanism of ion formation in REMPI fragmentation of small peptides. Recently, we have illustrated several unique aspects of REMPI fragmentation through the use of simple aro- matic compounds. REMPI mass spectra tend to be more sparse than conventional electron impact (EI) ion- ization or collisionally induced dissociation ~ p e c t r a . ~ Furthermore, the presence or absence of certain frag- t Author to whom correspondence should be addreirsed. ment ions in the mass spectrum can be strongly depen- dent on laser wavelength. lo These characteristics are due to the narrow internal energy distributions of the parent ions produced by REMPI. When REMPI includes a resonant two-photon ionization step, parent ion internal energies are bounded by the difference between the energy of two photons and the ionization energy of the molecule. This boundary is typically 1.5 eV or less when radiation in the 25G-300 nm region is used to ionize small aromatic molecules. As the parent ion absorbs additional photons, this energy dis- tribution is maintained. Only those fragment ions that are efficiently produced within the range of parent ion internal energies available will be observed in the REMPI mass spectrum. Changing the laser wavelength will change the width and average values of these energy distributions and potentially the types of frag- ment ions produced. When molecular structure infor- mation is desired from the observation of specific parent ion fragmentation processes, the laser wavelength and intensity must be adjusted to allow these fragmentations to occur efficiently. For structure elucidation of small peptide molecules, one would like to abundantly produce both the parent ion and sequence ions in the mass spectrum. Although both types of ions have been observed with REMPI,6*7 it would be useful to understand what conditions are necessary to produce them. For example, is it possible to select a wavelength where only the parent ion is observed at low laser intensity while sequence ions are abundantly produced at high intensity? Or must a com- promise wavelength be found where the parent ion and sequence ions are both produced across a wide range of 003&49JX/89/03017&07 $05.00 zyxwvut 0 1989 by John Wiley & Sons, Ltd. zyxwvutsr Received zyx 9 zyx March 1988 Accepted (reaised) 10 August 1988