Tyrosine deprotonation yields abundant and selective backbone cleavage in peptide anions upon negative electron transfer dissociation and ultraviolet photodissociation

Article abstract of DOI:10.1021/ja3032086

Tyrosine deprotonation in peptides yields preferential electron detachment upon NETD or UVPD, resulting in prominent N-Cα bond cleavage N-terminal to the tyrosine residue. UVPD of iodo-tyrosine-modified peptides was used to generate localized radicals on neutral tyrosine side chains by homolytic cleavage of the C-I bond. Subsequent collisional activation of the radical species yielded the same preferential cleavage of the adjacent N-terminal N-Cα bond. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages are regularly observed for peptides when using high-pH mobile phases.

Full text of DOI:10.1021/ja3032086

Communication
pubs.acs.org/JACS
Tyrosine Deprotonation Yields Abundant and Selective Backbone
Cleavage in Peptide Anions upon Negative Electron Transfer
Dissociation and Ultraviolet Photodissociation
Jared B. Shaw,† Aaron R. Ledvina,† Xing Zhang,‡ Ryan R. Julian,‡ and Jennifer S. Brodbelt*,†
†Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, United States
‡Department of Chemistry, University of California, Riverside, California 92521, United States
S
* Supporting Information
mode analyses16,17 provide complementary information to that
ABSTRACT: Tyrosine deprotonation in peptides yields
preferential electron detachment upon NETD or UVPD,
resulting in prominent N−Cα bond cleavage N-terminal
to the tyrosine residue. UVPD of iodo-tyrosine-modified
peptides was used to generate localized radicals on neutral
tyrosine side chains by homolytic cleavage of the C−I
bond. Subsequent collisional activation of the radical
species yielded the same preferential cleavage of the
adjacent N-terminal N−Cα bond. LC-MS/MS analysis of
a tryptic digest of BSA demonstrated that these cleavages
are regularly observed for peptides when using high-pH
mobile phases.
obtained in the positive ion mode, and the combined data sets
allow more comprehensive proteomics analyses by MS/MS.
However, CID of peptide anions is generally ineffective due to
extensive and uninformative neutral losses.18,19 Recently, a
number of dissociation methods including electron detachment
dissociation (EDD),20,21 negative electron transfer dissociation
(NETD),22,23 ultraviolet photodissociation (UVPD)24 at 193
nm, negative-ion ECD,25 and electron photodetachment
dissociation (EPD)26−28 have been shown to be viable
alternatives to CID for peptide anion characterization. Higher
pH conditions using basic mobile phases for LC-MS enhance
deprotonation of peptide sites not routinely deprotonated (e.g.,
tyrosine side chain with pKa ≈ 10). Deprotonation of
alternative sites may promote alternative fragmentation path-
ways as described herein.
ottom-up workflows for qualitative and quantitative
Bprotein identification utilizing liquid chromatography
tandem mass spectrometry (LC-MS/MS) have emerged as
one of the top technologies of choice for proteome analyses.1
The success of these strategies hinges upon the ability to
accurately predict fragmentation patterns of theoretical peptide
sequences generated in silico from protein sequence data-
bases.2,3 The benchmark for peptide cation dissociation is
collision-induced dissociation (CID);4,5 electron capture
dissociation (ECD)6,7 and electron transfer dissociation
(ETD)8,9 afford complementary information to CID and
enhanced identification of labile post-translational modifica-
tions (PTMs).10 The dissociation mechanisms and character-
istic product ion types of these methods have been investigated
in depth and are fairly well understood. A number of
preferential cleavage types have been reported for peptide
cations. For example, cleavages at protonated histidine and N-
terminal to proline residues are commonly observed upon CID
of protonated peptides, and in the absence of a mobile proton,
enhanced cleavage also occurs at the amide bond immediately
C-terminal to acidic residues.11,12 Disulfide bond cleavage in
multiply charged peptide and protein cations upon ECD and
ETD is readily observed and for lower charge states is more
favored than peptide/protein backbone cleavage.13−15
We report preferential cleavage N-terminal to deprotonated
tyrosine residues in peptide anions upon UVPD at 193 nm or
upon NETD. The dominant backbone cleavages of peptide
anions upon NETD21,22 typically result in a•- and x-type
product ions and upon UPVD23 a- and x-type with lower levels
of b, c, y, Y, and z ions. Conversely, NETD (Figure 1A), NETD
with simultaneous IR photoactivation (a process termed AI-
NETD) (Figure 1B), and UVPD (Figure 1C) of triply
deprotonated DRVYIHPFHLVIHN yield abundant c3 and
•
z11 ions which arise uniquely from cleavage N-terminal to the
tyrosine residue. This process is likewise notable upon NETD
and UVPD of the 3− charge states of peptides NEKYA-
QAYPNVS, Ac-DRVYIHPFHLVIHN, DRVYIHPFHLLVYS,
and KTMTESSFYSNMLA (not shown). The formation of
high-abundance c- and z-type product ions has not been
reported previously for NETD or UVPD at 193 nm; however,
the highly selective nature of the fragmentation suggests that a
favorable radical-directed cleavage process may occur upon
electron detachment from tyrosine-containing peptides.
The electron−hole recombination energy upon electron
transfer from peptide anions to fluoranthene radical cations has
been previously estimated to be 2.5−4.5 eV on the basis of the
difference in ionization energy (IE) of fluoranthene (IE = 7.9
eV) and the electron affinities (EA) of carboxylate (EA = 3.4
eV) and phosphate (EA = 5.4 eV) groups in phosphopep-
tides.23 The energy of a 193 nm photon is 6.4 eV. In a recent
Exploration of the negative ion mode for protein
identification affords an attractive alternative due to the fact
that a large portion of biologically significant PTMs, such as
phosphorylation, sulfonation, nitration, and glycosylation with
acidic glycans, as well as many naturally occurring peptides and
proteins, are acidic and thus readily form anions. Negative ion
Received: April 3, 2012
© XXXX American Chemical Society
A
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Journal of the American Chemical Society
Communication
yields results entirely consistent with the previously proposed
mechanism (Figure S2). The substantially increased abundan-
•
ces of the c3 and z11 ions upon NETD with simultaneous IR
photoactivation (Figure 1B) compared to NETD alone (Figure
1A) suggests that the additional vibrational energy provided by
IR photoactivation allows a greater portion of tyrosyl radical
ions to undergo the radical migration/N−Cα backbone
cleavage process.
CID of the charge-reduced precursor ([M − 3H]2−•
)
produced by NETD (Figure 2A) or by UVPD (Figure 2B) of
triply deprotonated DRVYIHPFHLVIHN yields the same
•
abundant c3 and z11 ions along with neutral losses of water,
carbon dioxide, and tyrosine side chain. This indicates that a
Figure 1. (A) NETD, (B) NETD with simultaneous IR photo-
activation, and (C) UVPD spectra of triply deprotonated
DRVYIHPFHLVIHN. * and ’ represent the precursor and loss of
water, respectively.
Figure 2. MS3 spectrum upon CID of the charge-reduced precursor
([M − 3H]2−•) produced by (A) NETD and (B) UVPD of triply
deprotonated DRVYIHPFHLVIHN . * and ’ represent the precursor
and loss of water, respectively.
top-down EDD study, Breuker et al. tabulated the EA of radical
functional groups in peptides and proteins.29 The EA of each
radical functional group is equivalent to the IE of the
corresponding deprotonated functional group, and all are
<3.5 eV. In contrast, ionization energies of neutral amino acids
are between 7 and 10 eV.30−32 Therefore, electron detachment,
a key step in the selective backbone cleavage showcased in this
report, likely occurs at sites of deprotonation.
portion of the [M − 3H]2−• ions formed upon electron
detachment from the triply deprotonated peptide precursors
contain stable radicals localized on the tyrosine side chain or c
and z ions held together through non-covalent interactions.
Subsequent collisional activation of these species provides
enough internal energy to surpass the activation barriers for
radical migration or non-covalent bond cleavage.
Previous work has shown that 266 nm UVPD of iodo-
tyrosine-containing peptide and protein cations results in
homolytic cleavage of the C−I bond, thus yielding a localized
radical on the tyrosine residue.33,34 Subsequent CID of these
peptide/protein radical cations led to dominant backbone
fragmentation in the form of a ions C-terminal to the tyrosine
residue.33,34 This process is illustrated below:
The likely sites of deprotonation in DRVYIHPFHLVIHN
include the side chains of aspartic acid and tyrosine, and the C-
terminus, which are all expected to be deprotonated in the
triply charged species. Delocalization of the negative charges
over backbone amides may also occur as a means of charge
solvation.21 The IEs of deprotonated aspartic acid and the C-
terminus have been estimated to be 3.34 eV, and the IE of
deprotonated tyrosine as 2.17 eV.29 Due to the lower IE and
greater absorption cross section of deprotonated tyrosine,
electron detachment is most favored at the deprotonated
phenol of tyrosine over the carboxylates of aspartic acid or the
C-terminus. In the doubly deprotonated species a majority of
the ions are expected to be deprotonated at the carboxylates of
aspartic acid and the C-terminus, not the tyrosine side chain. In
fact, NETD and UVPD of doubly deprotonated DRVIHPFHL-
hν
CID
[M + nH + I]n+ ⇒ [M + nH]n+• =⇒ fragments
(1)
In addition to selective a ion formation adjacent to the
modified tyrosine residue, abundant secondary cleavages
producing complementary a and y ions were observed N-
terminal to proline residues in the vicinity of the modified
tyrosine residue. These experiments were recently extended to
peptide anions,35 thus providing a convenient method for
creating radicals on tyrosine side chains in peptide anions in the
present study. The resulting ions allow determination of
whether the selective formation of c and z ions N-terminal to
tyrosine upon NETD and 193 nm UVPD proceeds through a
tyrosyl radical.
•
VIHN (Figure S1A,B) yielded very low abundance c3 and z11
product ions. This outcome is expected if it is tyrosine
deprotonation that specifically promotes the preferential
cleavage.
Breuker et al. proposed a mechanism for the formation of c
and z ions N-terminal to tyrosine residues upon EDD (see
Scheme S2 in ref 29), and it is reasonable to consider a similar
pathway during NETD and 193 nm UVPD. In fact, deuterium
labeling of the β carbon atom of tyrosine in our experiments
B
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Journal of the American Chemical Society
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UVPD at 193 nm (Figure 3A) of doubly deprotonated
DRVYIIHPFHLVIHN (where YI represents iodo-tyrosine)
yields predominantly an intact charge reduction product ([M
− 2H + I]2−•) via electron photodetachment and a wide array
of low abundance a, x, c, and z ions as expected. Homolytic
dissociation (RDD). This is consistent with the analogous
results reported previously showing that y ion formation N-
terminal to proline proceeds through a radical mechanism.33,35
The fact that the tyrosine residue is not deprotonated in the 2−
charge state of the iodo-tyrosine-containing peptide and is
deprotonated in the 3− charge state of the non-iodinated
peptide likely accounts for the observation of the abundant y7
ion. UVPD of triply deprotonated DRVYIIHPFHLVIHN
(Figure 3C) shows that the presence of iodine on the tyrosine
side chain does not impede the selective cleavage N-terminal to
the deprotonated tyrosine residue. Dominant c3 and [z11 + I]•
ions are produced. The absence of y7 ions upon UVPD of the
3− charge state of the iodinated peptide supports our
hypothesis that the phenol oxygen needs to be protonated
for y ion formation. Collectively, these results suggest that the
formation of tyrosyl radicals, whether promoted by NETD or
UVPD of peptides containing a deprotonated tyrosine residue,
is a key step that precedes the selective cleavage N-terminal to
the tyrosine residue.
To gauge the frequency with which the preferential cleavage
N-terminal to tyrosine might occur in a bottom-up proteomics
experiment, we performed LC-MS/NETD and LC-MS/UVPD
of a bovine serum albumin (BSA) tryptic digest using 0.05%
ammonium hydroxide mobile phases (pH ∼10.5). LC-MS/MS
spectra were interpreted using the MassMatrix database search
engine36,37 which was adapted to search the appropriate ion
series for each dissociation method. Tyrosine-containing
peptide matches in which the charge state exceeded the
number of acidic functionalities (i.e., E-, D-, and C-terminus)
were manually interpreted to confirm preferential cleavage N-
terminal to deprotonated tyrosine residues. The peptide
sequence, charge state, and the percentage of total sequence
ion peak area for Y-selective c and z ions for those peptides that
produced preferential cleavage upon NETD are shown in Table
1. Figure S4 shows the NETD spectrum of the 4− charge state
Figure 3. (A) 193 nm UVPD of doubly deprotonated
DRVYIIHPFHLVIHN (YI represents iodo-tyrosine) and (B) sub-
sequent CID of [M − 2H]2−•. (C) 193 nm UVPD of [M − 3H + I]3−.
*, ’, and I represent the precursor, loss of water, and iodine,
respectively.
Table 1. BSA Tryptic Peptides Exhibiting Preferential
Cleavage N-Terminal to Tyrosine upon NETD
cleavage of the C−I bond (i.e., [M − 2H]2−•) also occurs at
∼10% relative abundance. Very low abundance c3 and [z11 + I]•
ions are observed and are expected on the basis of the results
from the non-iodinated peptide (i.e., in the 2− charge state YI is
not expected to be deprotonated, and thus Y-directed cleavage
is not favored). Based on previously observed results with
iodinated peptides, collisional activation of [M − 2H]2−• is
expected to yield selective a ion formation C-terminal to the
iodinated tyrosine residue. Instead, CID of [M − 2H]2−•
a
peptide sequence
charge state
HPYFYAPELLYYANK
RHPYFYAPELLYYANK
GLVLIAFSQYLQQCPFDEHVK
RHPEYAVSVLLR
3−, 4− (39, 56%)
3−, 4− (69, 88%)
4− (20%)
3− (63%)
3− (23%)
4− (53%)
LGEYGFQNALIVR
LGEYGFQNALIVRYTR
a
The percentage of sequence ion peak area due to the c and z ions
from Y-selective cleavage is given in parentheses for each charge state.
•
(Figure 3B) yields dominant c3 and z11 ions corresponding
to cleavage N-terminal to the tyrosine residue. In fact, the a4
and x10 ions which arise from cleavage C-terminal to the
tyrosine residue are observed at only 1% relative abundance. In
addition, y7 ions are observed at 90% relative abundance which
corresponds to cleavage C-terminal to proline. The UVPD/
CID experiments of other iodo-tyrosine peptides similarly
reproduced the same tyrosine-selective cleavages that occur
from non-iodinated peptides containing a deprotonated
tyrosine. However, the abundant y7 ion formation C-terminal
to proline is not observed upon NETD or UVPD of the triply
deprotonated non-iodinated peptide or upon CID of [M −
3H]2−• ion produced by NETD or UVPD of triply charged
DRVYIHPFHLVIHN. CID of the doubly deprotonated even
electron species (Figure S3) also fails to yield y7 ions, a results
which indicates the y7 ions are produced by radical directed
of RHPYFYAPELLYYANK from BSA. For peptides with
multiple tyrosine residues, c and z ion formation was observed
N-terminal to all tyrosine residues. Of the 16 tyrosine-
containing peptides identified by each method, 6 yielded
preferential cleavage N-terminal to tyrosine residues. However,
3 of the 16 peptides contained N-terminal tyrosine residues;
thus c and z ion formation N-terminal to the tyrosine residue is
not possible. The 7 remaining tyrosine-containing peptides did
not produce charge states consistent with tyrosine deprotona-
tion, explaining why preferential cleavage was not observed
upon NETD.
In summary, tyrosine deprotonation during negative ion
mode analyses yields selective and enhanced c and z ion
formation N-terminal to the tyrosine residue upon NETD and
C
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Journal of the American Chemical Society
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and additional spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
jbrodbelt@cm.utexas.edu
Funding
Funding from NIH (R21GM099028) and the Robert A. Welch
Foundation (F1155) is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
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