A tandem time-of-flight mass spectrometer was built for photodissociation (PD) of singly protonated peptides and small proteins generated by matrix-assisted laser desorption/ionization. PD was performed in a second source after deceleration of precursor ions. The delayed extraction/post-acceleration scheme was used for the product ions. For the PD at 193 nm of small singly protonated peptides, the present instrument showed much better sensitivity and resolution for product ions than the previous one (Moon JH, Yoon SH, Kim MS, Bull. Korean Chem. Soc. 2005; 26: 763) even though the overall spectral patterns obtained with the two instruments were similar. The present instrument was inferior in precursor ion selection and background noise level. PD was achieved for precursor ions as large as the singly protonated ubiquitin (m/z 8560.63), indicating that the photoexcitation is capable of supplying a sufficient amount of internal energy to dissociate large singly protonated proteins. As the precursor ion m/z increased, however, product ion signals deteriorated rather rapidly. As in the PD of small peptide ions with m/z around 1000, the types of the product ions generated from singly protonated peptides with m/z in the range 2000–4000 were mostly determined by the positions of arginine residues. Namely, an and dn ions dominated when an arginine residue(s) was near the N-terminus while vn, wn, xn and yn dominated when the same residue(s) was near the C-terminus. In addition, dn, vn and wn ions were generated according to the correlation rules previously observed in the collisionally activated dissociation. Isoleucine and leucine isomers could be easily distinguished based on the wn and dn ions. Copyright © 2007 John Wiley & Sons, Ltd.