Structures of b and a product ions from the fragmentation of argentinated peptides

Article abstract of DOI:10.1021/ja9808245

Argentinated (silver-containing) oligopeptides fragment under low- energy collision conditions to yield abundant argentinated product ions. The structures of the [b₂ - H + Ag]⁺ and [a₂ - H + Ag]⁺ ions have been determined by means of tandem mass spectrometry and confirmed by comparison with synthesized derivatives of the candidate [b₂ - H + Ag]⁺ ion structure. The [b₂ - H + Ag]⁺ ion was found to be an N-argentinated oxazolone, which could subsequently form the [a₂ - H + Ag]⁺ ion and other product ions after collision activation. Tripeptides containing proline as their second residue were observed to form a relatively less abundant [b₂ - H + Ag]⁺ ion, which was postulated to be an argentinated ketene.

Full text of DOI:10.1021/ja9808245

7302
J. Am. Chem. Soc. 1998, 120, 7302-7309
Structures of b and a Product Ions from the Fragmentation of
Argentinated Peptides
Vicky W.-M. Lee,†,‡ Hongbo Li,†,§ Tai-Chu Lau,‡ and K. W. Michael Siu*,†,§,|
Contribution from the Institute for National Measurement Standards, National Research Council of
Canada, Montreal Road, Ottawa, Ontario, Canada K1A 0R6, Department of Biology and Chemistry,
City UniVersity of Hong Kong, Tat Chee AVenue, Kowloon, Hong Kong, and Ottawa-Carleton Chemistry
Institute, Carleton UniVersity, Colonel By DriVe, Ottawa, Ontario, Canada K1S 5B6
ReceiVed March 12, 1998
Abstract: Argentinated (silver-containing) oligopeptides fragment under low-energy collision conditions to
yield abundant argentinated product ions. The structures of the [b2 - H + Ag]+ and [a2 - H + Ag]+ ions
have been determined by means of tandem mass spectrometry and confirmed by comparison with synthesized
derivatives of the candidate [b2 - H + Ag]+ ion structure. The [b2 - H + Ag]+ ion was found to be an
N-argentinated oxazolone, which could subsequently form the [a2 - H + Ag]+ ion and other product ions
after collision activation. Tripeptides containing proline as their second residue were observed to form a
relatively less abundant [b2 - H + Ag]+ ion, which was postulated to be an argentinated ketene.
Introduction
containing equivalents of b + OH and a ions are dominant
product ions of alkali-metal-containing peptides.13,14
The bio-inorganic chemistry of the silver ion is rich and
fascinating. The Ag+ ion has long been used as a bactericide
in the form of eye drops for the newborns.1,2 Some of its
complexes display remarkable antimicrobial activities.3,4 The
metallothioneins, a class of small proteins believed to be
responsible for heavy-metal detoxification in mammals, exhibit
very high affinity for Ag+.5-7 Binding of the silver ion to other
proteins and peptides is also observed. Argentinated (silver-
containing) oligopeptides fragment upon low-energy collision-
induced dissociation (CID) to give abundant argentinated b, a,
y, and b + OH ions that are indicative of the peptide sequence.8,9
The protonated equivalents of b, a, and y ions are common
fragment ions of protonated peptides,10-12 while the alkali-metal-
Owing to its diversity, the fragmentation of argentinated
peptides, once understood, may potentially provide a higher
information content than that of protonated or alkali-metal-
containing peptides.
Much effort has been spent on understanding fragmentation
mechanisms and the resulting product ion structures of pro-
tonated peptides, which leads to a relative wealth of
information.10-12,15-42 Recent efforts from Wysocki’s,15-18
† National Research Council of Canada.
‡ City University of Hong Kong.
§ Carleton University.
|| Present address: Department of Chemistry, York University, 4700
Keele St., Toronto, Ontario, Canada M3J 1P3. phone: (416)650-8021.
Fax: (416)736-5936. E-mail: kwmsiu@yorku.ca.
(1) Petering, H. G. Pharmac. Ther. A 1976, 1, 127-130.
(2) Wigley, R. A.; Brooks, R. R. In Noble Metals and Biological Systems;
Brooks, R. R., Ed.; CRC Press: Boca Raton, FL, 1992; pp 277-297.
(3) Nomiya, K.; Kondoh, Y.; Nagano, H.; Oda, M. J. Chem. Soc., Chem.
Commun. 1995, 1679-1680.
(4) Nomiya, K.; Onoue, K. I.; Kondoh, Y.; Kasuga, N. C.; Nagano, H.;
Oda, M.; Sakuma, S. Polyhedron 1995, 14, 1359-1367.
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Chem. Soc. 1991, 113, 9354-9358.
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Anal. Chem. 1993, 65, 2859-2872.
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Soc. Chem. 1994, 116, 8368-8369.
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Spectrom. 1990, 1, 249-257.
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1991, 111, 173-189.
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S0002-7863(98)00824-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/08/1998

Structures of b and a Product Ions from the Fragmentation of Argentinated Peptides
1998 7303
J. Am. Chem. Soc., Vol. 120, No. 29,
Gaskell’s,19-27 Boyd’s,28-30 and other’s31-36 groups have es-
tablished the “mobile proton theory”, namely that the “external”
proton on a protonated peptidespresumably first attached to
the N-terminus or to the basic site on a side chainsis easily
transferred to amide nitrogen atoms on the peptide backbone
upon collision activation, thus producing a heterogeneous
population of precursor ion structures. (This is to be contrasted
with an alternative view in which the ionization process produces
a heterogeneous population of ionized structures whose pro-
tonation sites are fixed.) Protonation of an amide nitrogen
weakens the amide bond, which then fragments to yield either
the b or the y′′ (y + 2H) ion in a charge-directed cleavage.10-12,15
Subsequent elimination of CO from the b ion produces the a
ion.10-12 It is generally believed that the y′′ ion has the structure
of a protonated peptide or amino acid, the a ion an immonium
ion, and until recently the b ion an acylium ion.10-12
atoms. Some of these bonds are formed as a consequence of
self-solvation in the gas phase after solvent removal and do not
exist in solution prior to electrospray introduction. Low-energy
collision-induced dissociation of the [M + Ag]+ ion of
argentinated peptides yields a variety of silver-containing ions,
including [bn - H + Ag]+, [an - H + Ag]+, [bn + OH +
Ag]+, and [yn + H + Ag]+. The observation of fragmentation
along the peptide backbone parallels that in protonated peptides
and strongly suggests a heterogeneous population of argentinated
precursor ions. This heterogeneity could arise as a consequence
of collision activation or of ionization plus desolvation, i.e., from
silver ion migration to form a mixture of precursor structures
or from a distribution of fixed, silver-containing structures. The
fact that the silver ion is chelated to a number of sites would
appear, at least superficially, to decrease its likelihood of being
relatively mobile.
Recent studies of Harrison’s,37-39 Wesdemiotis’,40 and Hunt’s
groups41 showed that the b ion is not an acylium ion but a
protonated oxazolone, with the acylium ion structure being that
of the activated complex.37 Employing neutral fragment reion-
ization, Wesdemiotis and co-workers40,42 were able to demon-
strate that the C-terminal neutral fragment formed with proto-
nated oxazolone (the b ion) is a truncated peptide or amino acid;
however, the N-terminal neutral fragment formed with the y′′
ion is either an aziridinone or a diketopiperazine, but not an
oxazolone unless the peptide is N-acylated. These studies have
painted an interesting dichotomy in fragmentation depending
on which terminus the charge is located.
In this article, we report the results of our study on the
structures of the [bn - H + Ag]+ and [an - H + Ag]+ product
ions, and the mechanisms of their formation, from the [M +
Ag]+ precursor of oligopeptides. The 2-phenyl derivatives of
the candidate structure of the b ions, an oxazolone, were
synthesized,43 argentinated, and probed by means of tandem
mass spectrometry along with argentinated oligopeptides and
some of their N-acetyl derivatives. Energy-resolved CID was
performed on a representative tripeptide to yield the fragment
intensity versus collision energy relationship.
Experimental Section
On the basis of results of tandem mass spectrometry and
ZINDO calculations, Li et al.9 showed that, in argentinated
peptides, the silver ion is chelated to a number of sites, including
the amide nitrogen, the amide oxygen, and the methionine sulfur
Instrumental. Experiments were performed on a triple quadrupole
mass spectrometer equipped with electrospray introduction, PE SCIEX
API 300 (Concord, Ontario). Samples were typically 0.1 mM oligo-
peptides (Sigma, St. Louis, MO) in 50/50 methanol/water containing
0.2 mM silver nitrate (Aldrich, St. Louis, MO). These were continu-
ously infused with a syringe pump (Harvard Apparatus, Model 22,
South Natick, MA) at a typical flow rate of 2 µL/min into the
electrospray probe. The optimum probe position was established from
time to time but was typically with the tip about 2 cm from the interface
plate and with the spray off-axis from the orifice. Mass spectra were
acquired in the positive ion detection mode with unit mass resolution
at a step size of 0.1 m/z unit and at a dwell time of 10 ms/step.
Typically, 10 scans were summed to produce a mass spectrum. Tandem
mass spectrometry was performed with a nitrogen pressure of 2.5 mTorr
(23) Burlet, O.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1993, 4, 461-
469.
(24) Ballard, K. D.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1993, 4,
477-481.
(25) Cox, K. A.; Gaskell, S. J.; Morris, M.; Whiting, A. J. Am. Soc.
Mass Spectrom. 1996, 7, 522-531.
(26) Summerfield, S. G.; Bolgar, M. S.; Gaskell, S. J. J. Mass Spectrom.
1997, 32, 225-231.
(27) Summerfield, S. G.; Whiting, A.; Gaskell, S. J. Int. J. Mass
Spectrom. Ion Processes 1997, 162, 149-161.
(28) Alexander, A. J.; Boyd, R. K. Int. J. Mass Spectrom. Ion Processes
1989, 90, 211-240.
in q2 and at a collision energy at the center-of-mass frame (Ecm
)
typically of 1-2 eV. Apparent MS3 was achieved by raising the orifice
bias potential to induce fragmentation in the lens region, isolating the
product ion of interest with Q1, inducing further fragmentation in q2,
and mass-analyzing the second generation product ions with Q3.
Synthesis of 2-Phenyl Oxazolones. 2-Phenyl-5-oxazolone was
synthesized according to an established procedure.43 A mixture of N,N′-
dicyclohexylcarbodiimide and hippuric acid in 10 mL of CHCl3 was
stirred magnetically overnight to give a precipitate of dicyclohexylurea
while the filtrate gave 55% 2-phenyl-5-oxazolone as yellow plates.
Positive ion electrospray mass spectrometric analysis showed m/z 162
{MH}+. 1H NMR (400 MHz, CDCl3, 298 K, relative to Me4Si)
revealed δ 4.39 (s, 2H, NCH2CO), 7.45 (t, 2H, meta-H), 7.55 (t, 1H,
para-H), 7.95 (d, 2H, ortho-H). 13C NMR (400 MHz, CDCl3, 298 K):
δ 54.9 (s, 1C, CH2), 125.8 (s, 1C, aryl carbon with CNO), 127.0 (s,
2C, ortho-C), 128.7 (s, 2C, meta-C), 132.7 (s, 1C, para-H), 163.4 (s,
1C, NdCsO), 175.9 (S, 1C, carbonyl carbon). Synthesis of 2-phenyl-
4-methyl-5-oxazolone from N-benzoylalanine and N,N′-dicyclohexyl-
carbodiimide was similar.
(29) Alexander, A. J.; Thibault, P.; Boyd, R. K. Int. J. Mass Spectrom.
Ion Processes 1990, 98, 107-134.
(30) Tang, X.-J.; Thibault, P.; Boyd, R. K. Anal. Chem. 1993, 65, 2824-
2834.
(31) Yeh, R. W.; Grimley, J. M.; Bursey, M. M. Biol. Mass Spectrom.
1991, 20, 443-450.
(32) Kenny, P. T. M.; Nomoto, K.; Orlando, R. Rapid Commun. Mass
Spectrom. 1992, 6, 95-97.
(33) Schwartz, B. L.; McClain, R. D.; Erickson, B. W.; Bursey, M. M.
Rapid Commun. Mass Spectrom. 1993, 7, 339-342.
(34) Morgan, D. G.; Bursey, M. M. Biol. Mass Spectrom. 1993, 22, 502-
510.
(35) Fabris, D.; Kelly, M.; Murphy, C.; Wu, Z.; Fenselau, C. J. Am.
Soc. Mass Spectrom. 1993, 4, 652-661.
(36) Johnson, R. S.; Krylov, D.; Walsh, K. A. J. Mass Spectrom. 1995,
30, 386-387.
(37) Yalcin, T.; Khouw, C.; Csizmadia, I. G.; Peterson, M. R.; Harrison,
A. G. J. Am. Soc. Mass Spectrom. 1995, 6, 1165-1174.
(38) Yalcin, T.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. J.
Am. Soc. Mass Spectrom. 1996, 7, 233-242.
(39) Ambihapathy, K.; Yalcin, T.; Leung, H.-W.; Harrison, A. G. J. Mass
Spectrom. 1997, 32, 209-215.
Modeling. Molecular modeling was performed by means of AM1
and ZINDO, HyperChem (Hypercube, Inc., Guelph, Ontario). The
structures were found by means of an iterative process where the
geometry of the ion under consideration was optimized via a minimiza-
tion of the ion’s total energy.
(40) Nold, M. J.; Wesdemiotis, C.; Yalcin, T.; Harrison, A. G. Int. J.
Mass Spectrom. Ion Processes 1997, 164, 137-153.
(41) Arnott, D.; Kottmeier, D.; Yates, N.; Shabanowitz, J.; Hunt, D. F.
Proceedings of the 42nd ASMS Conference on Mass Spectrometry and Allied
Topics, Chicago, IL, 1994; p 470.
(42) Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Anal. Chem. 1993,
65, 1594-1601.
(43) Grahl-Nielsen, O.; Solheim, E. Anal. Chem. 1975, 47, 333-335.

7304 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Lee et al.
Figure 1. Product ion spectra at Ecm ) 2 eV: (a) [M + Ag]+, M )
2-phenyl-5-oxazolone; (b) [b2 - H + Ag]+ ion of [M + Ag]+, M )
N-benzoylglycine. See Scheme 1 for ion structures; X ) phenyl and R
) H.
Results and Discussion
Since the b ion of protonated peptides is found to be a
protonated oxazolone,37-41 and given the fact that b, a, and y
ions are abundant product ions observed in the CID of
protonated10-12 and argentinated peptides,8,9 a logical starting
candidate structure for the [b - H + Ag]+ ion is the argentinated
oxazolone. Figure 1a shows the product ion spectrum of the
[M + 107Ag]+ ion from electrospraying a solution containing
2-phenyl-5-oxazolone and Ag+ (all product ion spectra involving
Ag+ will be those of the 107Ag isotope; no subsequent isotope
identification will be made from now on). Figure 1b shows,
as a comparison, the product ions of the [b2 - H + Ag]+ ion
generated from fragmentation of [M + Ag]+ in the lens region,
where M is N-benzoylglycine. The [b2 - H + Ag]+ ion is
formed via cleavage of the carbon/hydroxyl bond in the
carboxylic group of glycine, but since the N-benzoyl group acts
as though it were an N-terminal amino acid residue, the b2 (as
opposed to b1) description is technically less confusing. The
close resemblance of the two spectra is self-evident and strongly
suggests that the [b2 - H + Ag]+ ion of argentinated N-benzoyl-
glycine is argentinated 2-phenyl-5-oxazolone. Similarly, a
comparison of the product ion spectrum of the [M + Ag]+ ion
of 2-phenyl-4-methyl-5-oxazolone and Ag+ (Figure 2a) and that
of the [b2 - H + Ag]+ from argentinated N-benzoylalanine
(Figure 2b) shows that the [b2 - H + Ag]+ ion of N-benzoyl-
alanine is argentinated 2-phenyl-4-methyl-5-oxazolone. As
Figure 2. Product ion spectra at Ecm ) 2 eV: (a) [M + Ag]+, M )
2-phenyl-4-methyl-5-oxazolone; (b) [b2 - H + Ag]+ ion of [M + Ag]+,
M ) N-benzoylalanine; (c) [b2 - H + Ag]+ ion of [M + Ag]+, M )
N-benzoylalanylalanine. See Scheme 1 for ion structures; X ) phenyl
and R ) CH3.
expected, the product ion spectrum of [b2 - H + Ag]+ of
argentinated N-benzoylalanylalanine (Figure 2c) is virtually
identical to that of argentinated N-benzoylalanine shown in
Figure 2b since in both cases the ion is argentinated 2-phenyl-
4-methyl-5-oxazolone. The softest basic site on oxazolone is
the nitrogen atom;44 furthermore, the silver(I) ion is one of the
softest Lewis acids known.44 This means the most likely
(44) Fleming, I. Frontier Orbitals and Organic Chemical Reactions; John
Wiley and Sons: London, 1976; pp 34-85.

Structures of b and a Product Ions from the Fragmentation of Argentinated Peptides
1998 7305
J. Am. Chem. Soc., Vol. 120, No. 29,
Scheme 1
argentination site on oxazolone is the nitrogen atom. Scheme
1 depicts proposed fragmentation reactions leading to the major
product ions for X ) Ph (phenyl): m/z 240/254 (II, R ) H/R
) CH3), m/z 210 (III), m/z 165/179 (V), and m/z 137/151 (VI);
the m/z 107 ion is Ag+. It is noteworthy that the major product
ions of argentinated oxazolones are very different from those
of protonated oxazolones. For protonated 2-phenyl-5-oxazolone
(m/z 162), the two major product ions are m/z 134 ([M - CO]+)
and m/z 105 ([PhCO]+) [ref 37 and our observations], while
for protonated 2-phenyl-4-methyl-5-oxazolone, the analogous
ions observed are m/z 148 ([M - CO]+) and m/z 105 ([PhCO]+)
(spectra not shown). Evidently, the central ion plays a critical
role in determining the fragmentation patterns.
Although attempts in synthesizing and isolating 2-methyl-5-
oxazolones were not successful and, therefore, a direct proof is
lacking, the similarity between the fragmentation patterns of
argentinated 2-phenyl-5-oxazolones and those of the [b2 - H
+ Ag]+ ions of argentinated N-acetyl peptides leaves little doubt
that these ions are argentinated 2-methyl-5-oxazolones. Figure
3 shows the product ion spectra of the [b2 - H + Ag]+ ions of
(a) argentinated N-acetylglycylglycine and (b) argentinated
N-acetylalanylglycine. The product ions seen and the fragmen-
tation reactions are depicted in Scheme 1 as well for X ) CH3:
m/z 178/192 (II, R ) H/R ) CH3), m/z 148 (III), m/z 137/151
(VI), and m/z 107 (Ag+).
N-derivatized) peptides are also argentinated oxazolones. Figure
4 shows the product ion spectrum of the [b2 - H + Ag]+ ion
of argentinated alanylalanylglycine, while Scheme 2 displays
the proposed product ion structures and fragmentation pathways.
Assignment of the major product ions: m/z 221, VIII, R1 ) R2
) CH3, [a2 - H + Ag]+; m/z 206, IX; m/z 178, X, an internal
ion; m/z 150, XI, an internal immonium ion; m/z 134, XII,
[HCNAg]+; and m/z 107, Ag+ was aided by H/D exchange by
repeating the experiment in D2O/CH3OD. The analogous ions
observed were m/z 251, d2-[b2 - H + Ag]+; m/z 223, d2-[a2 -
H + Ag]+; m/z 207, d1-(IX); m/z 179, d1-(X); m/z 151, d1-(XI);
m/z 135, d1-(XII), [DCNAg]+; and m/z 107, Ag+. The lineage
of product ions was developed using energy-resolved CID as
well as confirmatory precursor or product ion scans. Energy-
resolved CID yielded breakdown curves of the various product
ion intensities versus collision energy. Under a nitrogen
pressure of 2 mTorr in our q2, multiple collisions occur, thus a
product ion seen may be the result of sequential fragmentation
reactions. A suspected precursor/product relationship may then
be confirmed or rejected by raising OR to induce fragmentation
and formation of the precursor ion in the lens region, mass-
selecting the precursor ion with Q1, fragmenting the ion in q2,
and mass-analyzing the product ions with Q3. Figure 5 shows
the breakdown curves of the energy-resolved CID experiment
of the [b2 - H + Ag]+ ion of argentinated alanylalanylalanine.
Abundances of product ions of smaller m/z values generally
peak at higher collision energies. These observations emphasize
the need to select the appropriate collision energy for optimizing
the abundances of the ions of interest.
Moving one step further, the similarity between the frag-
mentation patterns of the [b2 - H + Ag]+ ions of argentinated
peptides, the [b2 - H + Ag]+ ions of argentinated N-acetyl
peptides, and argentinated 2-phenyl-5-oxazolones leads to the
conclusion that the [b2 - H + Ag]+ ions of argentinated (non-

7306 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Lee et al.
Figure 5. Energy-resolved CID: [b2 - H + Ag]+ ion of [M + Ag]+,
M ) alanylalanylalanine. See Scheme 2 for ion structures; R1 ) R2 )
CH3.
m/z 150, XI, R2 ) CH3; m/z 134, XII; and m/z 107, Ag+. The
observation of product ions with R2 ) CH3 (m/z 221, 178, and
150) is in accordance with a hypothesis in which they are formed
from product ions with R2 ) isobutyl after cleavage of the side
chain and loss of propene.45 Fragmentation of the side chain
to yield apparent methyl substitution was confirmed in the
product ion spectrum of the [b2 - H + Ag]+ ion of argentinated
leucylleucylleucine shown in Figure 6b. Most product ions are
identical to those in Figure 6a, many as a result of fragmentation
of the isobutyl side chain to yield methyl; m/z 333 is VII with
R1 ) R2 ) isobutyl, and m/z 305 is VIII again with R1 ) R2
) isobutyl.
Figure 3. Product ion spectra at Ecm ) 2 eV: (a) [b2 - H + Ag]+ ion
of [M + Ag]+, M ) N-acetylglycylglycine; (b) [b2 - H + Ag]+ ion of
[M + Ag]+, M ) N-acetylalanylglycine. See Scheme 1 for ion
structures; X ) CH3, R ) H for (a) and R ) CH3 for (b).
A comparison of Schemes 1 and 2 reveals differences in
fragmentation, between argentinated 2-substituted oxazolones
(I) and oxazolones of oligopeptides (VII). In Scheme 1, it is
proposed that the N-argentinated oxazolone (I) is interconverting
with the less energetically favorable O-argentinated isomer (IV),
which fragments to yield V and VI, both O-argentinated product
ions. This is to be contrasted with VII in Scheme 2 in which
Ag+ is chelated to both nitrogen atoms (ZINDO calculations
reveal that the Ag+ is not in plane with the ring structure and
is slightly closer to the N of the second residue (2.220 D) than
to that of the first (2.281 D)); hence, it is not available for
complexing with the carbonyl oxygen. However, loss of the
N-terminal fragment (HNdCHsR1) provides additional frag-
mentation routes to yield IX and X, and their fragmentation
products, XI and XII.
Figure 4. Product ion spectrum at Ecm ) 2 eV: [b2 - H + Ag]+ ion
of [M + Ag]+, M ) alanylalanylalanine. See Scheme 2 for ion
structures; R1 ) R2 ) CH3.
The protonated oxazolone structure of the b ion is believed
to form as a result of charge-induced cyclization of protonated
oligopeptide followed by elimination of a C-terminal amino acid
or peptide.26,37-41 Yalcin et al.37-39 and Nold et al.40 favored
protonation of the amide nitrogen of the amide bond to cleave
The identities of the product ions in Scheme 2 were confirmed
by the CID of the [b2 - H + Ag]+ ion of other argentinated
tripeptides. For example, Figure 6a shows the product ion
spectrum for argentinated alanylleucylglycine: m/z 291, VII,
R1 ) CH3, R2 ) isobutyl; m/z 263, VIII, R1 ) CH3, R2 )
isobutyl; m/z 248, IX, R2 ) isobutyl; m/z 221, VIII, R1 ) R2
) CH3; m/z 192, XI, R2 ) isobutyl; m/z 178, X, R2 ) CH3;
(45) McLafferty, F. W.; Turecˇek, F. Interpretation of Mass Spectra, 4th
ed.; University Science: Mill Valley, CA, 1993.

Structures of b and a Product Ions from the Fragmentation of Argentinated Peptides
1998 7307
J. Am. Chem. Soc., Vol. 120, No. 29,
Scheme 2
Scheme 3
whereas Summerfield et al.26 and Arnott et al.41 opted for
protonation of the amide oxygen. This second option is in line
with the known higher stability of protonation at the amide
oxygen than that at the amide nitrogen10-12,15-18 as well as
elegantly allowing the formation of both the b and the y′′ product
ions from the same precursor ion depending on details of the
ensuing fragmentation.26 It is, however, unlikely to be correct,
as it produces an O-protonated oxazolone, which presumably
will convert to the N-protonated isomer,46 but more importantly,
a neutral oxazolone fragment in the formation of the y ion,
which is not in accordance with recent observations. The neutral
fragment that accompanied the formation of the y′′ ion from
protonated oligopeptides was found to be either an aziridinone
or a diketopiperazine, and not an oxazolone.40,42 For argentin-
ated oligopeptides, the lack of O-argentinated product ions (vide
supra) and silver’s preference for N- over O-complexation44
make argentination of the amide oxygen arguably even less
likely. Scheme 3 shows the proposed mechanism for the
formation of the [b2 - H + Ag]+ ion from an argentinated
tripeptide whose Ag+ is originally chelated to N atoms (XIII).
Collision activation of XIII results in transfer of the proton at
the charge site to the amide nitrogen of the ensuing residue to
form XIV and subsequent cyclization to form argentinated
oxazolone (VIII) plus an amino acid. The transfer and loss of
an exchangeable proton is in accordance with the observation
of d2-[b2 - H + Ag]+ (vide supra). A variation of the
aforementioned fragmentation is depicted in Scheme 4, in which
chelation of Ag+ to the last two amide nitrogen atoms (XV)
and cyclization result in the loss of Ag+ and the formation of
(46) Results of AM1 calculations, in increasing ∆fH, are

7308 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Lee et al.
Scheme 4
Scheme 5
protonated oxazolone, b2+ (XVI), a minor product ion observed
in the CID of the [M + Ag]+ ion of argentinated tripeptides.
The proposed mechanism for the formation of the [b2 - H
+ Ag]+ ion depicted in Scheme 3 requires that the amide
hydrogen of the second residue be transferred to the third residue
of the tripeptide, which means that it cannot be operative in
any tripeptide that contains proline as the second residue.
Indeed, the [b2 - H + Ag]+ product ions of alanylprolylglycine
(APG) and glycylprolylalanine (GPA) were found to be
comparatively less abundant than the [b2 - H + Ag]+ product
ions of non-proline-containing tripeptides under identical ex-
perimental conditions (Table 1). Scheme 5 depicts a proposed
mechanism for the formation of the [b2 - H + Ag]+ ions of
tripeptides that contain proline as the second residue. Similar
to XIII, XVII has its Ag+ bound to N atoms of the first and
second residue. However, the hydrogen that is transferred to
the neutral fragment is a -CH- hydrogen on the R-carbon of
the proline residue since it lacks an -NH- hydrogen. This
mechanism is identical to the one proposed by Teesch et al.14
for alkali-metal-containing oligopeptides. H/D exchange showed
that the [b2 - H + Ag]+ ion formed is d2, in accordance with
transfer of a -C-H instead of an -N-H hydrogen to the
neutral fragment. The [b2 - H + Ag]+ ion involving proline,
XVIII, is postulated here to have an argentinated ketene
structure, the acylium equivalent of metalated peptide fragments;
the acylium ion was hypothesized to be the structure of the
activated complex of the b ion prior to association into the a
ion and CO.37,38 The low abundances of the [b2 - H + Ag]+
product ions of APG and GPA, relative to those of the [b2 - H
+ Ag]+ ions of non-proline-containing tripeptides (Table 1),
are in accordance with the hypothesis that the acylium, relative
to the oxazolone structure, is of higher energy content, and hence
the former [b2 - H + Ag]+ ions have comparatively low
abundances.
Figure 6. Product ion spectra at Ecm ) 2 eV. (a) [b2 - H + Ag]+ ion
of [M + Ag]+, M ) alanylleucylglycine. See Scheme 2 for ion
structures, R1 ) CH3 and R2 ) isobutyl. VIII′, X′ and XI′ have their
R2 ) CH3 after elimination of propene. (b) [b2 - H + Ag]+ ion of [M
+ Ag]+, M ) leucylleucylleucine. VII′′ and VIII′′ have their R1 ) R2
) isobutyl. See (a) for the identification of the other ions.
Conclusions
The [b2 - H + Ag]+ product ion of argentinated oligopep-
tides is found to be an N-argentinated oxazolone. This product
ion can further fragment to yield the [a2 - H + Ag]+ and other

Structures of b and a Product Ions from the Fragmentation of Argentinated Peptides
1998 7309
J. Am. Chem. Soc., Vol. 120, No. 29,
Table 1. Percent of [b2 - H + Ag]+ abundance of the base peak
proton displacement and migration, cyclization, and elimination
of the C-terminal fragment as a neutral amino acid or peptide.
Tripeptides having the second residue as proline form their
relatively less abundant [b2 - H + Ag]+ ion via transfer of the
-CH- hydrogen R to the cleaved amide bond. The [b2 - H
+ Ag]+ ion of these peptides is hypothesized to be an
argentinated ketene.
[M + Ag]+ (Ecm ) 1.2 eV)
peptide
% abundance
AGG
ALG
APG
GGA
GAA
GLA
GPA
29
37
9
32
94
73
4
Acknowledgment. Financial support to V.W.M.L. and
T.C.L. from the RGC of Hong Kong are gratefully acknowl-
edged. H.L. thanks Carleton University for a studentship.
product ions. Reaction schemes leading to these ions have been
proposed. The [b2 - H + Ag]+ ion is postulated to form via
JA9808245