SHORT COMMUNICATION Individual b 2 ion fragmentation profiles combined with AspN digestion improve N-terminal peptide sequencing Dominic Winter & Wolf D. Lehmann Received: 17 November 2008 / Revised: 7 January 2009 / Accepted: 21 January 2009 / Published online: 12 February 2009 # Springer-Verlag 2009 Abstract The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b 2 ions, which are often the most abundant low- mass fragment ions. It was observed that b 2 ions are accompanied by a set of sequence-specific neutral losses of CO, H 2 O, NH 3 , and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b 2 ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b 2 ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b 2 ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b 2 ion annotation of AspN- generated peptides compared to the use of only a solitary b 2 ion m/z value. Recognition of the b 2 ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series. Keywords Peptide fragmentation . b 2 ions . Tandem mass spectrometry . Collision-induced dissociation Introduction The collision-induced dissociation (CID) spectra of pep- tides often show the b 2 ion as one of the most prominent fragment ions [1–3], whereas b 1 ions are only observed for the basic residues H, K, and R [4]. These b 1 ions are in general observed at low abundance only. Thus, the first peptide bond is a kind of blind spot for collision-induced dissociation of peptides. The low-mass region contains various classes of fragment ions, such as b ions, internal b ions [5], y ions, and their secondary fragmentation products caused by loss of NH 3 ,H 2 O, CO, and other small units, so that the recognition of the true b 2 ion may require additional information, such as the presence of the complementary y max-2 ion. The idea of this study was to test whether b 2 ions generate sequence-specific fragmenta- tion profiles, which might be useful for their individual recognition. As a simple subset to test this idea, we selected a set of synthetic peptides with all N-terminal dipeptide motifs which may occur in AspN-generated peptides since the specificity of AspN restricts the variability of the N-terminal motifs. AspN is a zinc-containing metalloendo- proteinase of the bacterium Pseudomonas fragi [6] and cleaves N-terminally to the acidic residues Asp, Glu, and cysteic acid [7, 8]. This cleavage characteristic restricts the variability of the N-terminal motifs to about 40 (20 starting with D, 20 with E) if the rare case of cysteic acid is not considered. Without restriction, as it is the case, e.g., for tryptic peptides, the number of possible N-terminal motifs is about ten times larger. On the other hand, the variability of the y 1 ion of AspN-generated peptides is higher compared to tryptic peptides (18 vs. 2), which is still a manageable number for manual control. In addition, all amino acid residues at the C-terminus give rise to abundant y 1 ions, which, according to our experience, are often even Anal Bioanal Chem (2009) 393:1587–1591 DOI 10.1007/s00216-009-2652-9 Electronic supplementary material The online version of this article (doi:10.1007/s00216-009-2652-9) contains supplementary material, which is available to authorized users. D. Winter : W. D. Lehmann (*) Molecular Structure Analysis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany e-mail: wolf.lehmann@dkfz.de