Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides

Article abstract of DOI:10.1021/ac060509y

We report a new method called metal affinity capture that when coupled with tandem mass spectromery (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga^III or Fe^III and N_α,N_α-bis(carboxymethyl)lysme (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H) ⁺ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH < 5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the ⁶⁹Ga-⁷¹Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.

Full text of DOI:10.1021/ac060509y

Anal. Chem. 2006, 78, 6065-6073
Metal Affinity Capture Tandem Mass Spectrometry
for the Selective Detection of Phosphopeptides
Grady R. Blacken,† Michael H. Gelb,†,‡ and Frantisˇek Turecˇ ek*,†
Department of Chemistry and Department of Biochemistry, University of Washington, Seattle, Washington 98195
from phosphorylated Ser and Thr residues.4 Alternatively, a parent
ion scan can be employed to search for precursors of the common
product ions H2PO4 or PO3 in negative ionization mode.5,6
Infrared multiphoton dissociation of protonated phosphopeptides
was found to show selectivity to the phosphate groups and can
be potentially used in conjunction with LC separation of peptide
mixtures.7
We report a new method called metal affinity capture that
when coupled with tandem mass spectromery (MAC-
MSMS) allows for the selective detection and identification
of phosphopeptides in complex mixtures. Phosphopep-
tides are captured as ternary complexes with GaIII or FeIII
and Nr,Nr-bis(carboxymethyl)lysine (LysNTA) in solution
and electrosprayed as doubly or triply charged positive
ions. The gas-phase complexes uniformly dissociate to
produce a common (LysNTA + H)+ ion that is used as a
specific marker in precursor ion scans. The advantages
of MAC-MSMS over the current methods of phosphopep-
tide detection are as follows. (1) MAC-MSMS uses metal
complexes that self-assemble in solution at pH <5, which
is favorable for the production of positive ions by electro-
spray. (2) Phosphorylation at tyrosine, serine, and threo-
nine is detected by MAC-MSMS. (3) The phosphopeptide
peaks in the mass spectra are encoded with the 69Ga-
71Ga isotope pattern for selective recognition in mixtures.
Detection by MAC-MSMS of singly and multiply phospho-
rylated peptides in tryptic digests is demonstrated at low-
nanomolar protein concentrations.
-
-
Many phosphate-specific enrichment methods have been
developed to aid in the quantitation of relative phosphorylation
levels. Some reported phosphopeptide enrichment techniques are
variations of the isotope-coded affinity tags method8 and involve
base-induced phosphate elimination, followed by Michael addition
of a biotin or mercaptoethanol handle to the dephosphorylated
Ser or Thr residue. The chemically modified peptides can be
separated by a specific capture/elution on bound streptavidin or
by the use of sulfur-specific antibodies.9-12 Since phosphotyrosine
residues cannot undergo â-elimination of the phosphate moiety,
a modified approach, developed by Zhou et al.,10 uses ethyl
carbodiimide to catalyze the addition of cysteamine to the
phosphorylated residue allowing for purification over glass beads.
Disadvantages of these techniques stem from the excessive
sample handling steps resulting in lost sample, thus limiting
sensitivity.
Alternatively, immobilized metal ion affinity chromatography
(IMAC)13,14 can also be used to enrich for phosphopeptides without
the inherent need for antibody-based resins or sample derivati-
zation. With this method, the negatively charged phosphates are
captured by a metal cation, such as FeIII or GaIII, that is
immobilized on the surface of an agarose bead, which is deriva-
tized with a chelating agent such as nitrilotriacetic acid (NTA) or
Phosphorylation is one of the most important posttranslational
modifications of proteins1,2 that plays a role in several cellular
biochemical pathways, in particular in signaling.3 Thus, quantifying
phosphorylated proteins and detecting phosphorylation sites on
hydroxylated amino acids (Ser, Thr, Tyr) has been of considerable
interest in proteomic applications of mass spectrometry. Several
methods have been developed to selectively analyze the phos-
phoprotein content in complex biological samples. A common
feature of the current methods is that the protein in the sample
is first purified by electrophoretic or reversed-phase chromato-
graphic separation. The next step typically involves digestion with
a cleavage enzyme, such as trypsin, although some methods
include a phosphoprotein enrichment step prior to digestion.
Phosphopeptide analysis relies on separation by liquid chroma-
tography followed by tandem mass spectrometry (LC-MSn). This
involves a neutral loss scan which identifies peptides that on
collisional activation eliminate H3PO4 (98 Da) or HPO3 (80 Da)
(4) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180-
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* To whom correspondence should be addressed. E-mail: turecek@
chem.washington.edu.
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(11) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382.
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† Department of Chemistry.
‡ Department of Biochemistry.
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10.1021/ac060509y CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/04/2006
Analytical Chemistry, Vol. 78, No. 17, September 1, 2006 6065

iminodiacetic acid.13,15 After the flow-through portion is washed
away, the captured phosphopeptides can be eluted with a
phosphate-containing buffer, typically PBS. Since the immobilized
metal ion can also capture the negatively charged carboxylates
of both the peptide C-terminus and Asp and Glu side chains,
methyl esterification of these carboxylate groups is often employed
to ensure a more selective enrichment.16 An inherent limitation
of the IMAC method is nonspecific binding of hydrophobic
peptides to the gel beads. The interactions leading to this capture
are difficult to alleviate and are actually enhanced by methyl
esterification of carboxylic groups. Thus, ongoing efforts exist to
develop a more specific and comprehensive phosphopeptide
analysis. For example, TiO2 and ZrO2 columns have been recently
used for phosphopeptide enrichment.17 Many such efforts involve
combining two or more enrichment steps to ensure selective
isolation of phosphopeptides.7,18
In this study, we introduce a new approach to specific
phosphopeptide detection that relies on the metal coordination
properties of the phosphate group, but is independent of the amino
acid residue that is phosphorylated and does not require LC
separation of the peptide mixture. We call this method metal
affinity capture tandem mass spectrometry (MAC-MSMS). We
present a study of the complexation of phosphopeptides with NTA-
containing ligands via trivalent metal cations, FeIII and GaIII, in
solution with the goal of determining their utility as an analytical
tool in phosphopeptide analysis. The NTA-like lysine derivative,
NR,NR-dicarboxymethyllysine, was used as a metal chelating agent.
NTA binds strongly to four of the six FeIII or GaIII coordination
sites leaving two free sites for phosphopeptide binding. Upon
electrospray ionization (ESI)-MSn, these ternary complexes show
specific fragmentations that are promising for MAC-MSMS to be
developed into a useful tool in phosphopeptide analysis.
performed at collision gas setting of 5 psi and collision energy of
30-40 eV (for singly charged ions). The nebulizer gas and curtain
gas settings were 20 and 10 psi, respectively. A 50-V declustering
potential, 10-V collision cell entrance potential, and 15-V collision
cell exit potential were found to provide optimum phosphopeptide
detection. MSn spectra on the ion trap were obtained for confirma-
tion and initial characterization of phosphopeptide complexes.
MSMS data were acquired on a tandem quadrupole instrument
in Q1 scan, neutral loss scan, or parent ion scan mode.
Materials. NR,NR-Dicarboxymethyllysine was synthesized by
the method of Schmitt et al.19Briefly, Nꢀ-carboxybenzyllysine (H-
Lys(Z)-OH, Fluka), 4 mmol, was dissolved in 5 mL of 2 M NaOH,
and the solution was added dropwise with stirring at 0 °C to
bromoacetic acid (Fluka, 8 mmol) dissolved in 5 mL of 2 M NaOH.
The reaction was stirred for 2 h, at which point the cooling bath
was removed, and the reaction was stirred at room temperature
overnight. Following heating to 50 °C for 2 h, 20 mL of 1 M HCl
was added and the product crystallized after 4 h of cooling in ice.
The solid precipitate was redissolved in 7 mL of 1 M NaOH and
recrystallized from the same volume of 1 M HCl. The product
ꢀ
NR,NR-bis(carboxymethyl)-Nꢀ-(benzyloxycarbonyl)-
L
-lysine (N -
Z-NTA) was confirmed by melting point (170-175 °C)15 and ESI-
MS (MH+ m/z 397).
The Nꢀ-protected dicarboxymethyl lysine was dissolved in 10:1
CH3OH/H2O and hydrogenated over a Pd-on-charcoal catalyst at
room temperature and 1 atm of H2 for 2 h. Hydrogenation was
monitored by TLC in 4:1 CH3CN/H2O. The Pd catalyst was filtered
off, and the solvent was removed under vacuum. The nitrilotri-
acetic acid product (LysNTA) was purified by recrystallization
from 10:1 C2H5OH/H2O and characterized by electrospray mass
spectrometry. Collision-induced dissociation (CID) of (LysNTA
+ H)+ precursor ions at m/z 263 leads to common losses of NH3,
(CO + H2O), and (CO + 2H2O) giving rise to fragments at m/z
246, 217, and 199, respectively (Figure S1, Supporting Informa-
tion). Furthermore, the spectrum showed a major fragment ion
at m/z 130 that corresponds to an elimination of iminodiacetic
acid by a cleavage of the NsCR bond leaving (Lys + H - NH3)+,
which is also the major fragment observed in the CID spectrum
of protonated lysine.
LysNTA metal ion complexes were formed with FeCl3 or GaCl3
(Aldrich). The formation constants for LysNTA-GaIII and LysNTA-
FeIII complexes of 1:1 ligand-metal ion stoichiometry are on the
order of 1013 and 1015 mol-1, respectively.20 Thus, a large excess
of metal is not required to drive the complex formation to
completion. A 2:1 ratio of metal to LysNTA was sufficient to
facilitate complete complex formation, as determined by the
absence of the free (LysNTA + H)+ ion (m/z 263) in the ESI-MS
spectrum of metal-LysNTA solutions (Figure S2, Supporting
Information).
EXPERIMENTAL SECTION
Methods. Electrospray ionization tandem mass spectra were
acquired on either an API III Perkin-Elmer Sciex triple quadrupole
or an Applied Biosystems API 4000 Sciex triple quadrupole. In
addition, a Bruker Esquire ion trap mass spectrometer was used
for the initial characterization of the complexes. All these instru-
ments were operated in positive ion mode. All peptides and peptide
complexes were sampled as solutions in 1:1 methanol/water
acidified with acetic acid to pH 3-5. Direct infusion via a syringe
pump was used at flow rates of 1-10 µL/min.
Optimum electrospray ionization was achieved at a capillary
voltage of 4000 V and a nitrogen drying gas flow rate of 5-6 mL/
min. Measurements on the Bruker Esquire ion trap instrument
were performed with a 30-V skimmer potential and a 70-V capillary
exit offset. Optimal trap drive settings were sample dependent
falling within the range of 55-65 of the instrument units. MSMS
measurements on the API 4000 triple quadrupole instrument were
The peptides used to characterize the ternary LysNTA-metal-
peptide complexes include NQLLpTPLR, QLLpTPLR, MpSGIFR,
MSGIFR, and pYWQAFR that were purchased from Genscript
Corp. (Piscataway, NJ). Peptides from the tryptic digestion of RS1-
casein were also used to characterize the formation of ternary
complexes with LysNTA and metal cations. Sequence grade
(15) Hochuli, E.; Dobeli, H.; Schacher, A. J. Chromatogr. 1987, 411, 177-184.
(16) Ficarro, S.; McCleland, M.; Stukenberg, P.; Burke, D.; Ross, M.; Shabanowitz,
J.; Hunt, D.; White, F. Nat. Biotechnol. 2002, 20, 301-305.
(17) (a) Pinkse, J. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R.
Anal. Chem. 2004, 76, 3935-3942. (b) Larsen, M. R.; Thingholm, T. E.;
Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D. Mol. Cell. Proteomics 2005,
4, 873-886. (c) Kweon, H. K.; Hakansson, K. Anal. Chem. 2006, 78, 1743-
1749.
(19) Schmitt, L.; Dietrich, C.; Tampe´, R. J. Am. Chem. Soc. 1994, 116, 8485-
8491.
(18) Tao, W.; Wollscheid, B.; O’Brien, R.; Eng, J.; Li, X.; Bodenmiller, B.; Watts,
J. D.; Hood, L.; Aebersold, R. Nat. Methods 2005, 8, 591-598.
(20) Martell, A. E.; Smith, R. M. Critical Stability Constants, Vol. 1: Amino Acids.
Plenum Press: New York, 1974; pp 116-118, 142-143.
6066 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

trypsin and bovine R-casein (>70% of the S1 variant) were
purchased from Aldrich. Tryptic digestion of R-casein was
performed at a 1:100 enzyme to substrate weight ratio in 20 mM
NH4HCO3 and incubated overnight at 37° C. Peptide aliquots were
purified by C-18 Ziptip (Millipore, Eugene, OR) in a process
involving a 10% HOAc binding and washing solution and 5 µL of
20% AcCN elution solution. The flow-through portions from this
purification were also examined as these were found to contain
significant amounts of tryptic phosphopeptides.
RESULTS AND DISCUSSION
Phosphoester Binding in Solution. Mass spectra of the
LysNTA-metal ion ternary complexes were initially acquired
using phosphorylated serine, threonine, and tyrosine. To quanti-
tatively characterize phosphoester binding to LysNTA-GaIII in
solution, formation constants were evaluated for the LysNTA-
GaIII-pSer ternary complex by monitoring the intensity of the
precursor ion at m/z 514 (the 69Ga isotope) as a function of pSer
concentration at constant LysNTA-GaIII concentrations. The
ternary formation constants are represented as â values,20 which
define the equilibrium constant for the formation of the LysNTA-
GaIII-pSer complex from free LysNTA, GaIII, and pSer (eq 1).
Figure 1. CID mass spectrum of the m/z 514 ion from ESI of
LysNTA-GaIII-pSer in 1% HOAc.
From the m1 and m2 slopes thus obtained, and assuming that R1
) R2, â can be expressed by eq 5:
m1
â )
(5)
[LysNTA]0[GaIII]0(m1 - m2)
[LysNTA-GaIII-pSer]
â )
(1)
[LysNTA][GaIII][pSer]
The values of â were measured at several solution pH, as
summarized in Figure S3 (Supporting Information). The data
suggest that, in accordance with previous literature,14,22 the ternary
complex with pSer is most stable at a pH of <5, where log â e
8.9. The pH dependence can be explained by the phosphoserine
becoming less able to bind the metal center as the concentration
of OH- competitor ions increases. The acidic environment is
necessary to provide a bare, LysNTA-bound metal ion for
phosphate binding. A beneficial corollary of this result is that the
optimal pH for complex formation is also a desirable one for ESI-
MS detection in positive ion mode. Thus, detection of these
complexes by ESI-MS was performed in solutions of 1:1 methanol/
water acidified to pH 3-4 with acetic acid. The values of â e 109
mol-2 were further used to estimate the concentrations of LysNTA
(5-100 µM) and GaIII (10-200 µM) for the efficient formation of
phoshopeptide ternary complexes.
MSMS of Gas-Phase Complexes. Electrospraying the Lys-
NTA-GaIII-pSer complex produces mainly singly charged gas-
phase cations at m/z 514. The m/z value indicates that the ligands
are overall doubly deprotonated, but the deprotonation positions
(any of the four COOH groups or the phosphate) are unknown.
Therefore, we denote the ligands generically as LysNTA and pSer
without specifying their deprotonation in the complexes. The CID
spectrum of the m/z 514 ion is shown in Figure 1. The fragment
ions all retain the LysNTA-GaIII moiety and correspond to losses
of CH2dC(NH2)COOH from the pSer moiety (m/z 427), serine
(m/z 409), and phosphoserine (m/z 329). In contrast, CID of the
LysNTA-GaIII complexes with phosphopeptides lead to a different
fragmentation forming galliated phosphopeptide ions and a com-
mon (LysNTA + H)+ ion (m/z 263) as major products. Figure 2
illustrates the fragmentations of LysNTA-GaIII and LysNTA-FeIII
complexes with the synthetic phosphopeptide MpSGIFR. Com-
The equilibrium concentration of phosphoserine is equivalent to
the initial concentration of pSer minus the equilibrium concentra-
tion of the ternary complex. Furthermore, if excess LysNTA and
GaIII are used, then we can assume [LysNTA] = [LysNTA]0 and
[GaIII] = [GaIII]0 and the expression can be rewritten as (eq 2)
â )
[LysNTA-GaIII-pSer]
(2)
[GaIII]0[LysNTA]0{[pSer]0 - [LysNTA-GaIII-pSer]}
To determine the equilibrium concentration of the complex
precursor we assume that the ion intensity for the ternary
complex, Im/z 514, is proportional to its concentration scaled by a
response factor, Rm/z 514.
21 Substituting in eq 2 and rearranging it
gives an expression for Im/z 514 (eq 3) that shows that Im/z 514
depends linearly on [pSer]0.
â[GaIII]0[LysNTA]0[pSer]0
m/z 514(1 + â[LysNTA]0[GaIII]0)
Im/z 514
)
(3)
R
The slope, m (eq 4), still contains two unknowns, â and Rm/z
,
514
the latter of which is eliminated by constructing the linear
regression at two values of [LysNTA]0[GaIII]0.
â[GaIII]0[LysNTA]0
m/z 514(1 + â[LysNTA]0[GaIII]0)
m )
(4)
R
(21) (a) Gatlin, C. L.; Turecˇek, F. Anal. Chem. 1994, 66, 712-718. (b) Di Marco,
V. B.; Bombi, G. G. Mass Spectrom. Rev. 2006, 25, 347-379.
(22) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2992.
Analytical Chemistry, Vol. 78, No. 17, September 1, 2006 6067

Figure 2. CID mass spectrum of the LysNTA-GaIII (top) and LysNTA-FeIII complexes with MpSGIFR.
plexes of NQLLpTPLR and pYWQAFR gave quite analogous CID
spectra that are given as Supporting Information (Figures S4 and
S5, respectively). Major fragments result from cleavage at the
LysNTA-metal bond resulting in the (LysNTA + H)+ ion and a
metalated peptide ion whose charge is equal to the precursor ion
charge minus one.
nation, and the formation of the ternary complex can be forced
by increasing the concentration of LysNTA-GaIII.
As mentioned above, the location of protons in the gas-phase
phosphopeptide complexes was not directly investigated in this
study, but a hypothesis can be made based on correlating the
parent and product ion m/z values. The LysNTA-GaIII-peptide
complexes, containing each of the three synthetic peptides,
acquire a net charge of +2 on electrospray. If we assume that
the ꢀ-NH2 group in LysNTA is protonated while the three NTA
COOH groups are deprotonated, then a net charge of +1 is added
by the LysNTA-GaIII component to the phosphopeptide. This
leaves the possibility that the phosphopeptide ligand is singly
deprotonated at the phosphate and doubly protonated along the
peptide backbone, probably at both the N-terminus and the basic
side chain of the C-terminal Arg. Thus, cleavage at the LysNTA-
metal bond to eliminate the (LysNTA + H)+ fragment at m/z 263
must be accompanied by the transfer of three protons from the
peptide moiety to the LysNTA carboxylates. Alternately, only two
protons would necessarily be transferred during fragmentation if
the precursor ion were instead deprotonated at only two LysNTA
COOH groups and deprotonated twice at the phosphate group of
the phosphopeptide. The fact that GaIII always remains coordinated
to the phosphopeptide following CID seems to favor structures
with doubly deprotonated phosphate groups, because those would
provide stronger binding to the metal ion.
Gallium(III) and iron(III) were both investigated as metal
centers in this study. The MSMS spectra of LysNTA-GaIII
complexes with each of the three synthetic phosphopeptides
provide significantly more b- and y-type gallium-free peptide
fragment ions than do the spectra of LysNTA-FeIII complexes
with the same peptides (Figure 2). The cause of variation in
fragmentation with metal ion was not studied in depth, but the
observation might be attributed to the relative stabilities of the
galliated and ferrated peptide fragment ions. The observed result
is consistent with the relative phosphopeptide-binding character-
istics of iron and gallium. Specifically, it has been noted that iron-
(III) binds to phosphopeptides more strongly than gallium(III).22
One advantage of gallium-containing fragments is that they can
be more readily identified than ferrated ones due to the well-
resolved isotope doublets stemming from the existence of two
naturally abundant gallium isotopes of 69 and 71 Da present in a
3:2 ratio. For example, in the CID spectrum of the singly charged,
LysNTA-GaIII-pSer ion, the galliated fragments can be identified
by the abundant ions at m/z 427, 409, and 329 (Figure 1). Similar
assignments can be made for galliated ions from the phospho-
peptides in the CID and precursor scan spectra (vide infra).
In another series of experiments, the molar ratio of LysNTA-
GaIII to the phosphopeptide was varied from 1000:1 to 30:1, while
the peptide concentration was kept at 300 nM. At the lowest
LysNTA-GaIII/phosphopeptide ratio, we observed ∼50% of the
peptide as the LysNTA-GaIII ternary complex and the other 50%
as protonated ions. Thus, phosphopeptide coordination to LysNTA-
GaIII to form ternary complexes efficiently competes with proto-
Scheme 1 illustrates the likely protonation sites for both the
precursor and the product ions in the dissociation of the ternary
complex (LysNTA-GaIII-MpSGIFR)2+. The product ion resulting
from the loss of (LysNTA + H)+ (m/z 263) from the Ga complex
corresponds to a galliated peptide with a charge that is one less
than the charge of the precursor ion. In the case of the synthetic
peptide complexes, this means that doubly charged precursors
fragment into a singly charged galliated peptide ion. In this case,
two mobile protons are transferred from the peptide to the
6068 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

Scheme 1
LysNTA carboxylates in a fashion described by Scheme 1. This
protonation scheme can also explain why the CID of the LysNTA-
GaIII-pSer complex (Figure 1) does not produce the (LysNTA +
H)+ fragment, because phosphoserine does not contain the
required two mobile protons.
confirmation of the peptide identity. As anticipated, the galliated
peptide product ion maintains a charge that is one less that the
complex precursor ion charge. Thus, the triply charged complex
produces a doubly charged galliated product ion.
Several other LysNTA-GaIII complexes with R-Cas phospho-
peptides were characterized by this ESI-MSMS approach. The
precursors at m/z 1173.7 (Figure S6) and at m/z 1119.5 (Figure
S7) correspond, respectively, to the triply charged complexes of
(LysNTA-GaIII)2-EKVNELSKDIGpSEpSTEDQAMEDIK (RS1-casein
residues 50-73) and (LysNTA)2-(GaIII)4-QMEAESIpSpSpSEEIVP-
NSVEQK (RS1-casein residues 74-94). Note that each phospho-
serine residue coordinates one GaIII ion. When selected for MSMS
in the tandem quadrupole instrument, the triply charged precursor
ion at m/z 1173.7 dissociates into a doubly charged galliated
peptide, m/z 1629.0, and the (LysNTA + H)+ ion (Figure S6).
Although two LysNTA-GaIII are apparently coordinated to the
two peptide phosphorylation sites, only one (LysNTA + H)+ is
lost through CID. It is possible that the proximity of the two
LysNTA-GaIII bound phosphorylation sites allows for the vacated
GaIII to bind the remaining LysNTA forming a stabilized, bridged
structure, preventing loss of this second LysNTA in CID. The
stability of such a GaIII-LysNTA-GaIII bridge might also be
responsible for the observed composition of the complex ion at
m/z 1119.5. The peptide in this complex contains four phospho-
rylated serine residues and a free threonine residue. Since the
four pSer residues fall within a five-residue segment of each other,
the four bound GaIII ions are likely in proximity, allowing binding
to only the observed two LysNTA ligands, which create two
bridged structures disallowing further LysNTA binding. Indeed,
each of the LysNTA-GaIII peptide complexes detected contains
no more than two gallium-bound LysNTA moieties, possibly due
to the proximity of these phosphorylation sites in the casein motif.
The role of the multiple glutamic acid residues near the phos-
phorylated segment in preventing further LysNTA binding in the
The mobile proton assumption received support from our study
of phosphopeptide methyl esters in which the carboxylic protons
were replaced by methyl groups.16 ESI of methyl esters of
NQLLpTPLR, QLLpTPLR, MpSGIFR, and pYWQAFR showed
formation of ternary complexes with GaIII-LysNTA, although with
efficiencies that were 5-10% of those of the corresponding
phosphopeptides with free carboxylic groups. However, upon
collisional activation, the complexes did not produce the diagnostic
(LysNTA + H)+ ion at m/z 263. This indicates that the peptide
carboxyl proton plays a role in the dissociation. It appears plausible
that the phosphopeptide carboxylate participates in displacing the
LysNTA ligand and is coordinated to the metal cation in the GaIII-
contaning fragment ions.
As a further characterization of phosphopeptide complexes
with LysNTA-GaIII, an RS1-casein tryptic digest was analyzed by
ESI-MSMS. A 10 µM solution of this tryptic digest, without added
LysNTA or GaCl3, in 1:1 CH3OH/H2O in 1% HOAc, was analyzed
on a Bruker Esquire ion trap ESI-MS. The doubly charged, singly
phosphorylated peptide, YKVPQLEIVPNpSAEER (m/z 976.7) was
selected for MSMS characterization (Figure 3, top). CID of this
ion results in loss of H3PO4 giving the major product ion at m/z
927.8. Several fragments of the b and y ion series also appear in
the spectra, confirming the identity of the peptide. In contrast,
the MSMS spectrum of the triply charged, LysNTA-GaIII complex
with the phosphorylated R-Cas tryptic peptide at m/z 761 (Figure
3, bottom) shows major product ions that include a doubly charged
galliated peptide fragment, m/z 1010, and a singly charged
(LysNTA + H)+ ion at m/z 263. A few b and y ions are also
produced by CID of this ternary complex, allowing for further
Analytical Chemistry, Vol. 78, No. 17, September 1, 2006 6069

Figure 3. CID mass spectrum of the LysNTA-GaIII ternary complex with R-casein tryptic phosphopeptide YKVPQLEIVPNpSAEER.
complex is unclear. It cannot be ruled out that the three-
dimensional structures of these peptides also allow the Glu side-
chain carboxylates to occupy GaIII coordination sites and further
prevent LysNTA binding. Thus, according to these results, the
bridging of LysNTA species to multiple GaIII creates a stabilized
structure that prevents both the loss of multiple LysNTA in CID
and the binding of more than two LysNTA to the GaIII bound,
multiply phosphorylated casein motif.
Parent Ion Scanning of Phosphopeptide Complexes. In
light of these data, the common product ion, (LysNTA + H)+ at
m/z 263 was targeted as a possible phosphopeptide marker from
the CID of phosphopeptide complexes with LysNTA-GaIII. The
prescribed method involves parent ion scanning on a triple
quadrupole mass spectrometer, whereby precursor ions are
selected in a scanning mode by Q1 for CID fragmentation in Q2.
Subsequently Q3 selects only fragment ions at or near m/z 263
for detection, thus identifying only precursors that are associated
with phosphopeptide complexes. Figure 4 (bottom) illustrates the
data collected from the parent ion scan analysis of phosphopeptide
GaIII complexes. A mixture of four synthetic peptides, NQLLpT-
PLR, pYWQAFR, MpSGIFR, and MSGIFR, each at 300 nM, was
combined with excess LysNTA-GaIII as described above. The
three precursors appearing at m/z 559.7, 639.7, and 681.2 and their
71Ga satellites correspond to doubly charged ternary complexes
of LysNTA-GaIII with each synthetic phosphopeptide. The precur-
sor at m/z 672.8 and its 71Ga satellite correspond to the (LysNTA-
GaIII-NQLLpTPLR)2+ complex that eliminated NH3 on CID.
Detection of the pYWQAFR peptide is especially promising since
pY species cannot be detected by neutral loss scans. Figure 4
(top) shows a neutral loss scan for the 98 Da difference corre-
sponding to the elimination of orthophosphoric acid (H3PO4) from
singly protonated phosphopeptide precursor ions. While peaks
are detected for MpSGIFR (m/z 790.3) and NQLLpTPLR (m/z
1034.4), the peak for pYWQAFR that would be expected at m/z
950 is absent. Apparently, H3PO4 is not easily eliminated from the
phenyl phosphate group of tyrosine. The [LysNTA-GaIII-
pYWQAFR]2+ precursor ion is detected with a somewhat lower
intensity than those for the phosphoserine and phosphothreonine
peptides, perhaps reflecting the decreased ionization efficiency
of the more hydrophobic pYWQAFR peptide.
The nonphosphopeptide, MSGIFR, was added to the mixture
above to test the selectivity of the parent scanning method. As
6070 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

Figure 4. Top spectrum: neutral loss (98 Da difference) scan of a mixture of singly protonated peptides. Bottom spectrum: precursor scan
for the m/z 263 common fragment ion from NTA-GaIII complexes of the same peptide mixture.
shown in Figure 4 (bottom), the doubly charged complex with
this nonphosphorylated peptide (theoretical m/z 519.7) is not
detected while its phosphorylated analogue, MpSGIFR is detected
as the 69Ga-71Ga doublet at m/z 559.7-560.7. The selectivity of
LysNTA-GaIII binding to phosphopeptides and hydrophilic pep-
tides that contain several acidic Asp or Glu residues was examined
with an R-casein tryptic digest, as presented below.
Detection of phosphopeptides from an R-casein (R-Cas) tryptic
digest by parent scanning of phosphopeptide complexes was
compared to detection of these phosphopeptides by neutral loss
scanning on a triple quadrupole mass spectrometer. For direct
comparison of these detection techniques, the R-Cas tryptic
phosphopeptide, YKVPQLEIVPNpSAEER, residues 119-134, was
targeted for analysis by both neutral loss scanning of the peptide
digest and parent ion scanning of the peptide complexes with
LysNTA-GaIII. The neutral loss scanning method was optimized
for losses of orthophosphoric acid, H3PO4, from the triply (32.6
Da), doubly (49 Da), and singly (98 Da) charged precursors. Since
the doubly charged precursor at m/z 976.7 of the targeted
phosphopeptide is detected with the greatest intensity relative to
the other charge states, the neutral loss of 49 Da was targeted
for detection of this phosphopeptide at digest concentrations of
400 (Figure 5a) and 40 nM (Figure S8).
For comparison, solutions consisting of 100 µM NTA, 200 µM
GaCl3, and either 400 (Figure 5b) or 40 nM (Figure S9) R-Cas
tryptic digest were analyzed by parent ion scanning for the
common product ion at m/z 263. Both the neutral loss scan and
parent ion scanning methods were able to detect the targeted
phosphopeptide, YKVPQLEIVPNpSAEER, at these digest con-
centrations. However, the neutral loss scan was unable to detect
several multiply phosphorylated peptides that the parent ion scan
identifies. This is a common drawback of neutral loss scanning,
since multiply phosphorylated peptides maintain a larger number
of negatively charged components and their detection by ESI-
MS in positive ion mode is suppressed. Furthermore, these data
illustrate an inherent advantage of the parent ion scan over the
neutral loss scan, that is the ability to detect multiple charge states
of the same peptide in a single experiment. For example, both
the triply charged, m/z 761, and quadruply charged, m/z 571,
precursors of the targeted phosphopeptide complex, LysNTA-
GaIII-YKVPQLEIVPNpSAEER, are detected by parent ion scan-
ning. Thus, the precursor ion scan is able to detect a greater
number of phosphorylated species than scanning for any single
neutral loss, 32.5, 49, or 98 Da. While the advantages of precursor
ion scanning over neutral loss scanning in detecting multiply
phosphorylated species have not been investigated, two hypoth-
eses might be offered. First, since complexation of the negatively
charged phosphate moiety to the LysNTA-GaIII seems to result
in a net gain of one positive charge to the peptide, ionization of
these complex precursors in positive ion mode might be enhanced
relative to the free phosphopeptides. This enhancement is similar
to that achieved with copper ternary complexes for the detection
of amino acids and other coordinating analytes.23,24 Second, since
the product ion m/z does not depend on the precursor charge
state, we can identify phosphopeptides in several charge states
with a single scanning acquisition. This is not possible with neutral
loss scanning since elimination of phosphoric acid will result in
different apparent neutral losses depending on the precursor ion
charge. Thus, precursor ion scan of LysNTA-GaIII complexes has
the definite advantage of being a universal, charge-independent
detection method for phosphopeptides in positive ion mode.
Figure 5b shows peaks in addition to those of the phospho-
peptide complexes. In particular, the acidic tryptic peptide
HQGLPQEVLNENLLR, which has three COOH groups, forms a
triply charged ternary complex with GaIII-LysNTA (ion E) that
fragments to produce the (LysNTA + H)+ ion at m/z 263. In
contrast, peaks of GaIII-LysNTA complexes for other nonphos-
phorylated acidic tryptic fragments, e.g., FFVPFPEVFGK,
EDVPSER, VPQLEIVPNS AEER, YLGYLEQLLR, and EPMIGVN-
QELAYFYPELFR, are not present at above-background relative
intensities. This indicates that while complexation to GaIII-
(23) Gatlin, C. L.; Turecˇek, F.; Vaisar, T. J. Am. Chem. Soc. 1995, 117, 3637-
3638.
(24) Gatlin, C. L.; Turecˇek, F. J. Mass Spectrom. 2000, 35, 172-177.
Analytical Chemistry, Vol. 78, No. 17, September 1, 2006 6071

Figure 5. Top spectrum: (a) neutral scan (49 Da difference) of a phosphopeptide mixture from trypsinolysis of R-casein at 400 nM. Bottom
spectrum: (b) precursor scan or the m/z 263 common fragment ion from LysNTA-GaIII complexes of the same peptide mixture.
LysNTA is not completely selective for phosphopeptides, only a
few peptides containing acidic residues (D and E) are picked up
by the precursor ion scan.
product ion that could be used as a marker in phosphopeptide
analysis. Complexes of GaIII gave a greater amount of spectral
information without a significant loss in signal relative to FeIII
complexes. A triple quadrupole, parent ion scanning method was
developed to detect several phosphopeptides through their
LysNTA-GaIII-phosphopeptide complexes in peptide mixtures.
Detection of phosphopeptides from an RS1-Cas digest was per-
formed by both neutral loss scanning for the common loss of
phosphoric acid and by parent ion scanning for the common
(LysNTA + H)+ product ion from peptide complexes with
LysNTA-GaIII. The parent scanning method provides the ability
to detect phosphopeptides in several charge states in a single
scanning data acquisition. Furthermore, complexation to LysNTA-
GaIII enhanced detection of multiply phosphorylated peptides by
ESI-MSMS in positive ion mode. The advantages of this parent
scan method will undoubtedly prove to be sample dependent. Of
course, there are inherent advantages to precursor scanning in
positive ion mode as opposed to precursor scanning in negative
ion mode or neutral loss scanning. Precursor scanning in positive
ion mode allows for fragmentation data, and thus sequence
information, to be acquired. In contrast, scanning in negative ion
One feature that distinguishes the potentially interfering
HQGLPQEVLNENLLR peptide from the phosphopeptides is the
much diminished intensity of its higher charge states. In particular,
whereas the A, B, and C phosphopeptides give abundant precur-
sors for the +3 and +4 states (Figure 5b), the E peptide gives
only a weak peak for the +4 precursor at m/z 523. Despite the
less than 100% selectivity for phosphopeptides, the presence of a
small number of acidic tryptic peptides in the precursor scan does
not impair the multiplex detection of phosphopeptides, especially
if the latter is based on more than one charge state. In most
proteomic applications, acidic peptides can be identified from the
known protein sequence and distinguished from tryptic peptides
carrying phosphorylated S, T, and Y residues.
CONCLUSIONS
Ternary complexes of LysNTA-GaIII/FeIII with phosphopeptide
were formed in aqueous solution and, when subjected to ESI-
MSMS analysis, were found to yield a common (LysNTA + H)+
6072 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

-
mode (for loss of PO3 or H2PO3-) requires a second run at
ACKNOWLEDGMENT
Funding of this research by NIH/NDDK (Grant DK67859) is
gratefully acknowledged. We also thank Dr. Martin Sadilek for
technical assistance with mass spectrometric measurements.
reversed polarity to acquire such information. Similarly, fragmen-
tation data cannot be obtained during a neutral loss scan. Instead,
the neutral loss scan must trigger a product ion scan in a data-
dependent manner. Furthermore, precursor scanning allows for
the simultaneous detection of several charge states of the same
peptide whereas a separate scan is required for each charge state
to detect peptides in neutral loss scanning. Thus, only parent ion
scanning allows detection of multiple charge states of the same
component. For these reasons, parent ion scanning in positive
ion mode offers definite advantages over parent ion scanning in
negative ion mode or neutral loss scanning, when applied to
peptide analysis.
SUPPORTING INFORMATION AVAILABLE
Figures S1-S9 of ESI and CID mass spectra of peptide
complexes. This material is available free of charge via the Internet
at http://pubs.acs.org.
Received for review March 20, 2006. Accepted July 6,
2006.
AC060509Y
Analytical Chemistry, Vol. 78, No. 17, September 1, 2006 6073