The product ion mass spectra obtained by CID of the b 9 ions derived by loss of neutral alanine from the MH ϩ ion of the peptides Tyr(Ala) 9 , (Ala) 4 Tyr(Ala) 5 , and (Ala) 8 TyrAla are essentially identical, indicative of full cyclization reaction to a common intermediate before fragmentation. This leads to abundant nondirect sequence ions in the product ion mass spectra of the b 9 ions. The product ion mass spectra of the b 8 ions from the first two peptides also are essentially identical. The fragmentation of the MH ϩ ions also leads to low intensity nondirect sequence ions in the product ion mass spectra. N-terminal acetylation blocks the cyclization and eliminates nondirect sequence fragment ions in the product ion mass spectra. (J Am Soc Mass Spectrom 2009, 20, 2248 -2253) © 2009 American Society for Mass Spectrometry T andem mass spectrometry plays an important role in the sequencing of peptides [1][2][3]. The most common approach has involved collision-induced dissociation (CID) of the protonated (or multiply-protonated) gas-phase peptide ions produced by soft ionization techniques. As a result of many studies, the fragmentation of protonated peptides is known, at least in a phenomenologic sense, as illustrated in Scheme 1 [4,5]. Under the frequently-used low-energy collision conditions, protonated peptides most often fragment at amide bonds. In the ideal case this leads to a series of b and/or y ions, which contain, respectively, the N-terminus and C-terminus residues. It is these series of b and y ions, which provide the sequence information, and any nondirect sequence ions [6] may lead to uncertainty in interpretation of the observed spectra.It has been clearly established [7,8] that the y ions are protonated amino acids (y 1 ) or protonated truncated peptides (y n ). Although it was originally proposed [4,5] that b ions had an acylium ion structure, extensive studies [9 -14], mainly of b 2 and b 3 ions, have presented strong evidence that, in many cases, cyclization has occurred at the C-terminus to form a protonated oxazolone. Recently, infrared multiple photon dissociation (IRMPD) experiments [15,16] have provided definitive evidence for a protonated oxazolone structure for the b 4 ion derived from Leu-enkephalin. More recently, several IRMPD studies [17][18][19] have shown that relatively simple b 2 ions also have a protonated oxazolone structure. On the other hand, when there is a strong nucleophile in the side chain, alternative cyclization reactions involving this nucleophile may occur [20 -24].Early studies [25,26] reported the observation of nondirect sequence ions in the fragmentation of doublyprotonated b ions containing lysyl or ornithyl residues, which was interpreted in terms of cyclization/reopening reactions before fragmentation. More recently, several groups [6,[27][28][29][30][31] have presented evidence that b 5 ions form fully cyclic structures; ab initio calculations [6,30] indicate that the pathway to these cyclic structures involves attack of the N-terminal amine function on t...