Anal. Chem. 1883, 65, 2824-2834
2824
Fragmentation Reactions of Multiply-Protonated Peptides and Implications for Sequencing by Tandem Mass Spectrometry with Low-Energy Collision-Induced Dissociation+ Xue-Jun Tang? Pierre Thibault, and Robert K. Boyd' Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 321
The low-energy collision-induced dissociation reactions of a series of multiply-protonated peptides have been investigated by tandem mass spectrometry. It is known that doubly-protonated tryptic peptides undergo facile fragmentation yielding redundant sequence information. The present work has shown that this fortunate circumstance seems likely to be the exception rather than the rule. The presence of additional basic residues, at positions other than the C-terminus, complicates the spectra. The most important such complication discovered in the present work involves wholesale transfer of one or two residues from thee-terminal end of a doubly-charged b fragment to the side chain of a lysine residue located near the N-terminus, resulting in mass shifts of the products of subsequent second-stage fragmentations. Other examples of the participation of the flexible lysine side chain a r e suggested but could not be confirmed to the same extent. The role of Coulombic repulsion in facilitating fragmentation has been explored via investigations of triply- and quadruply-protonated basic peptides bearing one charge for every three o r four amino acid residues. Such species yielded almost no sequence information under low-energy collision conditions, due to the localization of the ionizing protons on highly basic sites rather than on the peptide backbone. It is proposed that collisionally activated mobilization of protons from the basic sites, where they a r e originally located upon formation, to the backbone is a necessary condition for structurally useful fragmentation to occur. It was not possible, on the basis of the present work, to deduce mechanistic generalizations and predictive schemes which would permit structural interpretations of such fragment spectra for unknown peptides. INTRODUCTION The introduction of fast-atom bombardment mass spectrometry in 1981, by Barber et al.,l marked the beginning of a remarkable transformation in the applicability of mass spectrometry to biological problems. More recently, techniques based upon ion formation from charged microdroplets NRCC No. 38878. Present address: Dept. of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461. (1) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J.Chem. SOC.,Chem. Commun. 1981, 324-327. t
8
0003-2700/93/0365-2824$04.00/0
at atmospheric pressure, developed from early experiments of Thomson et al.? of Aleksandrov et al.2 and of Fenn et al.? have extended the range of analytes accessible to mass spectrometry to much larger and even more labile molecules. The electrospray technique3p4relies upon electrical instability of the charged liquid effluent to achieve the nebulization required, so that its efficient operation is restricted to liquid flow rates up to 5-10 I.tL/mineven when additional precautions are taken5 to optimize this process. The introduction of ion spray (pneumatically assisted electrospray) by Henion et al.6 has permitted extension of the phenomenon to flow rates of up to 200 pL/min, compatible with standard HPLC columns operated under gradient conditions. Electrospray techniques have found application7-16 in accurate measurements of molecular weights of peptides and proteins by exploiting the phenomenon of multiple protonation, which yields a coherent series of progressively more highly protonated species (M + nH)"+. An excellent review has been published by Smith et al.16 The extent of this protonation (maximum value for n) appears16 to be related to the number of basic amino acid residues (usually arginine, lysine, and histidine) which are present in the protein and which are accessible to the ambient acidic (protonating) species. Such highly redundant information, arising from the range of values for n observed in practice, permits accurate and precise measurements of protein molecular mass (
