Lysine Peroxycarbamates: Free Radical-Promoted Peptide Cleavage

Article abstract of DOI:10.1021/ja038615u

Strategies are reported that combine in one step a predictable chemical-based protein digestion with mass spectrometry. Lysine residue amino groups in peptides and proteins are modified by reaction with a peroxycarbonate derived from p-nitrophenol, and tert-butyl hydroperoxide. The peroxycarbonate reacts with lysine residues in peptides and proteins, and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID). Observed fragmentation of the peptides involves apparent free radical processes including Hofmann-Loeffler-type rearrangements that lead to peptide chain fragmentation. Strategies for directed cleavage of peptides by free radical promoted processes are feasible, and such strategies may well simplify schemes for protein analysis. Copyright

Full text of DOI:10.1021/ja038615u

Published on Web 12/31/2003
Lysine Peroxycarbamates: Free Radical-Promoted Peptide Cleavage
Douglas S. Masterson, Huiyong Yin, Almary Chacon, David L. Hachey, Jeremy L. Norris, and
Ned A. Porter*
Department of Chemistry, Vanderbilt UniVersity, NashVille, Tennessee 37235
Received September 19, 2003; E-mail: n.porter@vanderbilt.edu
The cornerstone methods used in protein identification are
enzymatic proteolysis and analysis of isolated small peptide
fragments by mass spectrometry.1,2 Chemical digestion of proteins
has also provided strategies that are helpful in assignment of peptide
sequences.3 Both chemical and enzymatic protein digestion, how-
ever, can be cumbersome and time-consuming, and attempts to
avoid this step by the use of powerful mass spectrometric “top-
down” approaches are being explored to provide protein primary
sequences.4,5
Strategies that combine in one step a predictable chemical-based
protein digestion with mass spectrometry might prove to be
convenient for rapid primary peptide sequence analysis. We report
here experiments in which lysine residue amino groups in peptides
and proteins are modified by reaction with the peroxycarbonate 1.
The resulting lysine peroxycarbamates6 undergo homolytic frag-
mentation under conditions of low-energy collision-induced dis-
Figure 1. Electrospray collision-induced dissociation ions formed from
lithium complex of 2 vs offset energy.
sociation (CID). One result of this process is predictable peptide
fragmentation in the mass spectrometer at or near the modified
followed by decarboxylation to form an aminyl radical, 3.6 shown
here as the lithium complex. Other proposed adducts formed by
subsequent fragmentation of 3 are shown below. An MS/MS
analysis of 3 confirmed that fragments 4-7 are daughter ions
derived from 3.
lysine residues of small peptides.
Reaction of p-nitrophenylchloroformate with tert-butyl hydro-
peroxide gave the peroxycarbonate 1.7 In basic water or water-
acetonitrile mixtures, 1 gave an almost immediate yellow color,
indicative of the formation of p-nitrophenoxide.8 In basic aqueous
acetonitrile solutions, RN-acetyl lysine methyl ester was converted
cleanly to 2 on reaction with 10 equiv of 1. The peroxycarbamate
2 (Ac-LPC-OMe)9 was stable to chromatography and was fully
characterized by spectroscopy and HRMS, see Supporting Informa-
tion. Analysis of 2 by electrospray showed a parent ion for the H,
Li, Na, K, and Ag peroxycarbamate adducts. Collision-induced
dissociation (CID) of the parent ion led to loss of -C (O)OOtBu
(m/z ) 117) for each of the adducts (Li, Na, K, and Ag gave similar
CID results, whereas the H+ adduct resulted only in loss of H+).
The Li adduct of 2, for example, gave a parent ion at m/z ) 325,
while CID on 2 at -15 eV gave a major ion at m/z ) 208, see
Figure 1. Increasing CID offset energies gave rise to smaller lithium
ion complexes at m/z ) 192, 150, and 137. CID fragmentation of
the lithium adduct of RN-acetyl lysine methyl ester, the precursor
to 2, does not give similar ions.
Several peptides were reacted with 1, and adducts or multiple
adducts were observed in every case by electrospray mass
spectrometry. Thus, Ac-Gly-Ser-Ala-Lys-Val-Ser-Phe (Ac-7; M +
1, m/z ) 853.4) gave a product with 1 at m/z ) 853.4 + 116,
presumably, Ac-7-LPC Ac-Gly-Ser-Ala-LPC-Val-Ser-Phe. Mass
spectral analysis of Ac-7-LPC with added LiCl gave monolithium
adducts as shown in Scheme 1, E ) Li. The formation of each of
the species, 9-13, can be understood to be based on generation of
an intermediate aminyl radical followed by remote hydrogen
abstraction and â-fragmentation of the intermediate carbon radical.
Of particular interest is the fact that adducts 9 and 11 result from
peptide bond fragmentation. The peptide 14, Tyr-Glu-Val-His-His-
Gln-Lys-Leu-Val-Phe-Phe gave a peroxycarbamate derivative that
also gave peptide cleavage under CID to give a species analogous
to 11, as shown in Scheme 1. The conversion of radical 8 to 11
has ample precedent in the well-known Hofmann-Lo¨ffler reac-
tion.10,11 The carbon radical formed from this sequence, as shown
in Scheme 1, gives 11 by a simple â fragmentation, another reaction
that is well-known in radical chemistry.
The chemistry associated with the fragmentations of the per-
oxycarbamate (Li-2, m/z ) 325) shown in Figure 1 is consistent
with an initial free radical dissociation of the weak -O-O- bond
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-
Gly-Lys-Lys-Arg (Ac-15, (M+3)3+, m/z ) 712.7) gave products
upon reaction with peroxycarbonate 1 in which one, two, or three
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C O M M U N I C A T I O N S
Figure 3. MALDI mass spectrum intensity vs m/z of (a) Ac-15 and (b)
peroxycarbamate-modified Ac-15 consisting of a mixture of 1, 2, and 3
LPC modifications.
the parent peptide. Each of these ions corresponds to fragmentation
of the peptide at one of the three lysine residues in the peptide,
with formation of species analogous the enamide 11 with E ) H,
as shown in Scheme 1. The ion observed at m/z ) 1423, for
example, corresponds to 11 with E ) H and R1 ) Ac-Ser-Tyr-
Ser-Met-Glu-His-Phe-Arg-Trp-Gly, while for m/z ) 1804, R1 )
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-
and for m/z ) 1932, R1 ) Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-
Trp-Gly-Lys-Pro-Val-Gly-Lys. Sequencing experiments on the ion
with m/z ) 1423 are consistent with the proposed structure. Loss
of CdCH(CH2)2-NH2 from this ion is observed as well as peptide
fragmentation expected from the sequence.
Figure 2. Electrospray mass spectrum (M+3)3+ of peroxycarbamate-
modified Ac-15. Inset, expansion of (Ac-15+2LPC+3H)3+
.
Scheme 1. Mechanism for Formation of Typical Products Formed
in Dissociation of LPCs Derived from Peptides
We note that proteins having molecular weights of 10000 or
greater gave fragmentation patterns by electrospray CID or MALDI
that were complex and not readily interpreted. The preliminary
results communicated here make it clear, however, that strategies
for directed fragmentation of peptides by free radical-promoted
processes are feasible12 and suggest that such strategies might
simplify schemes for protein analysis.
Acknowledgment. We thank Drs. D. Wright and R. Caprioli
and Ms. L. Manier for assistance.
Supporting Information Available: Preparation of percarbonate
1 and full characterization of the peroxycarbamate 2, MS/MS data for
Ac-7, and MS/MS data for intermediate 3 (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
free lysine side chain amino groups were converted to peroxycar-
bamates. Figure 2 shows the electrospray mass spectrum for the
LPC modified Ac-15. The analogous peptide 15, having a free
terminal amine, NH2-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-
Lys-Pro-Val-Gly-Lys-Lys-Arg, gave products upon reaction with
1 that had up to four amino groups modified. CID of these modified
peptides showed, in every case, loss of n -C(O)OOtBu, where n
is the number of peroxycarbamates in the peptide species analyzed.
References
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Thus, dissociation of (Ac-15+LPC+3H)3+ gave (Ac-15+3H)3+
,
presumably the radical species analogous to 8, formed by loss of
one peroxycarbamate group while (Ac-15+2LPC+3H)3+ lost two
-C(O)OOtBu, and so forth.
(7) Usual precautions regarding peroxides were followed for compounds 1
and 2.
(8) The half-life of 1 in pH 8.2 phosphate buffer (50% MeOH) is ap-
proximately 112 s. Details of the kinetics will be published in due course.
(9) For convenience, we adopt here the shorthand LPC for lysine peroxy-
carbamate. The compound 2 is therefore Ac-LPC-methyl ester. The
Matrix assisted laser desorption ionization (MALDI) of the
percarbamate-modified peptides also gave backbone fragments
formed by loss of the -C(O)OOtBu groups. The MALDI experi-
ment for Ac-15 shown in Figure 3 is typical. The spectrum
displayed in Figure 3b is for the same mixture of LPC-modified
Ac-15 whose electrospray mass spectrum is shown in Figure 2.
By MALDI, no LPC adducts were observed, but a complex set of
ions near m/z ) 2135, that of the parent peptide Ac-15, was
observed. The LPC-modified peptide clearly did not survive the
laser desorption process intact. Fragmentation of the weak peroxide
bond presumably leads to aminyl radicals as outlined in Scheme
1. In addition, ions at m/z ) 1423, 1804, and 1932 were prominent
in the LPC-modified compound, while they were not observed for
peptides use the following shorthand (peptide+nLPC+mE)m+
.
(10) (a) Majetich, G.; Wheless, K. Tetrahedron 1995, 51, 7095-7129. For a
discussion of the chemistry of protein aminyl radicals, see: (b) Hawkins,
C, L.; Davies, M. J. Biochim. Biophys. Acta. 2001, 1504, 196-219.
(11) See also: (a) Griep-Raming, J.; Meyer, S.; Bruhn, T.; Metzger, J. O.
Angew. Chem., Int. Ed. 2002, 41, 2738-2742. (b) Julian, R. R.; May, J.
A.; Stoltz, B. M.; Beauchamp, J. L. Angew. Chem., Int. Ed. 2003, 42,
1012-1015. (c) Yin, H.; Hachey, D. L.; Porter, N. A. J. Am. Soc. Mass
Spectrom. 2001, 12, 449-455.
(12) Free radicals have been suggested in the electron capture chemistry of
peptides and proteins. See, for example: (a) Leymarie, N.; Costello, C.
E.; O’Connor, P. B. J. Am. Chem. Soc. 2003; 125, 8949-8958. (b) Syrstad,
E. A.; Stephens, D. D.; Turecek, F. J. Phys. Chem. A 2003, 107, 115. (c)
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