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Anal. Chem.

2006, 78, 1743-1749

Selective Zirconium Dioxide-Based Enrichment of


Phosphorylated Peptides for Mass Spectrometric
Analysis
Hye Kyong Kweon and Kristina Hkansson*

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055

Due to the dynamic nature and low stoichiometry of


protein phosphorylation, enrichment of phosphorylated
peptides from proteolytic mixtures is often necessary prior
to their characterization by mass spectrometry. Several
phosphopeptide isolation strategies have been presented
in the literature, including immobilized metal ion affinity
chromatography. However, that technique suffers from
poor selectivity and reproducibility. Recently, titanium
dioxide-based columns have been successfully employed
for phosphopeptide enrichment by several research groups.
Here, we present, to our knowledge, the first demonstration of the utility of zirconium dioxide microtips for
phosphopeptide isolation prior to mass spectrometric
analysis. These microtips display similar overall performance as TiO2 microtips. However, more selective isolation of singly phosphorylated peptides was observed with
ZrO2 compared to TiO2 whereas TiO2 preferentially enriched multiply phosphorylated peptides. Thus, these two
chromatographic materials possess complementary properties. For r- and -casein, Glu-C digestion provided no
evident advantage compared to trypsin digestion when
combined with TiO2 or ZrO2 phosphopeptide enrichment.

Reversible phosphorylation of proteins catalyzed by kinases


and phosphatases is recognized as a primary regulatory mechanism in eukaryotic cells.1,2 This mechanism controls a wide variety
of cellular events, including signal transduction, gene expression,
metabolism, and cell growth, division, and differentiation. To
achieve detailed insights into phosphorylation-controlled cellular
regulation, it is important to identify phosphorylated proteins and
determine the precise sites of phosphorylation within those
proteins as well as the phosphorylation residency at a certain
metabolic stage. However, phosphorylation profiling still remains
a challenge due to its low and dynamic stoichiometry.
Mass spectrometry (MS) has been widely applied as a powerful
tool to characterize protein modifications, including phosphorylation, due to its high sensitivity and capability of rapid sequencing
by tandem mass spectrometric (MSn) techniques.3-8 However, the
* To whom correspondence should be addressed. E-mail: kicki@umich.edu.
Phone: (734) 615-0570. Fax: (734) 647-4865.
(1) Graves, J. D.; Krebs, E. G. Pharmacol. Ther. 1999, 82, 111-121.
(2) Hunter, T. Cell 2000, 100, 113-127.
(3) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602.
10.1021/ac0522355 CCC: $33.50
Published on Web 02/07/2006

2006 American Chemical Society

commonly low occurrence of phosphorylation is still an issue in


mass spectrometric analysis for localization of protein phosphorylation sites. Thus, prior isolation and enrichment of phosphopeptides from a proteolytic peptide mixture (resulting from an
enzymatic digest of a protein) is often required. This procedure
serves to eliminate interferences and to enhance the signal from
phosphopeptides, which often exhibit signal suppression in MS.
Commonly used enrichment strategies include immobilized metal
ion affinity chromatography (IMAC)9-11 incorporating Fe3+, Ga3+,
or other metal ions (including Zr4+; however, that approach is
markedly different from the use of zirconium oxide, which is
presented here), immunoprecipitation with phosphoproteinspecific antibodies,12,13 and the addition of an affinity tag to
phosphorylated amino acids through chemical reactions.14 Of
these methods, IMAC is the most widely used, both on-line and
off-line.11,15-17 However, nonspecific binding of nonphosphorylated
acidic peptides and the complexity of factors affecting phosphopeptide binding and release often result in low specificity and
sensitivity for target phosphopeptides. Recently, highly specific
phosphopeptide isolation has been demonstrated with titanium
dioxide columns in both off-line18 and on-line19,20 liquid chroma(4) Mann, M.; Ong, S.-E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A.
Trends Biotechnol. 2002, 20, 261-268.
(5) Schweppe, R. E.; Haydon, C. E.; Lewis, T. A.; Resing, K. A.; Ahn, N. G.
Acc. Chem. Res. 2003, 36, 453-461.
(6) Chalmers, M. J.; Kolch, W.; Emmett, M. R.; Marshall, A. G. J. Chromatogr.,
B 2004, 803, 111-120.
(7) Cantin, G. T.; Yates, J. R. J. Chromatogr., A 2004, 1053, 7-14.
(8) Meng, F.; Forbes, A. J.; Miller, L. M.; Kelleher, N. L. Mass Spectrom. Rev.
2005, 24, 57-77.
(9) Neville, D. C.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.;
Townsend, R. R. Protein Sci. 1997, 6, 2436-2445.
(10) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892.
(11) Nuhse, T. S.; Stensballe, A.; Jensen, O. N.; Peck, S. C. Mol. Cell. Proteomics
2003, 2, 1234-1243.
(12) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann, M.;
Lodish, H. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 179-184.
(13) Ficarro, S. B.; Chertihin, O.; Westbrook, V. A.; White, F.; Jayes, F.; Kalab,
P.; Marto, J. A.; Shabanowitz, J.; Herr, J. C.; Hunt, D. F.; Visconti, P. E. J.
Biol. Chem. 2003, 278, 11579-11589.
(14) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826-6836.
(15) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207-222.
(16) Haydon, C. E.; Eyers, P. A.; Aveline-Wolf, L. D.; Resing, K. A.; Maller, J. L.;
Ahn, N. G. Mol. Cell. Proteomics 2003, 2, 1055-1067.
(17) Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Methods 2005, 35, 256-264.
(18) Sano, A.; Nakamura, H. Anal. Sci. 2004, 20, 861-864.
(19) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Anal.
Chem. 2004, 76, 3935-3943.
(20) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen,
T. J. D. Mol. Cell. Proteomics 2005, 4, 873-886.

Analytical Chemistry, Vol. 78, No. 6, March 15, 2006 1743

tography/MS and matrix-assisted laser desorption/ionizationMS.20 This approach is based on specific chemisorption of
phosphate groups on the surface of titanium oxide and was shown
to result in less nonspecific binding than IMAC. In addition,
column preparation is more straightforward, eliminating the metal
ion chelating and washing steps required for preparation of IMAC
columns. An alternative strategy involving magnetic Fe3O4/TiO2
core/shell nanoparticles as affinity probes for phosphopeptides
has also been recently demonstrated.21 Finally, utilization of a
metal hydroxide, Al(OH)3, for phosphopeptide and phosphoprotein
binding also proved effective and more selective than commercial
phosphoprotein enrichment kits.22 Here, we present enrichment
of phosphorylated peptides on the surface of zirconium dioxide
loaded in a microtip. ZrO2 has drawn attention as chromatographic
column material due to its physical (e.g., toward extremes of pH)
and thermal stability and its unique surface chemistry.23-25
However, to our knowledge, its use for phosphopeptide isolation
has not previously been reported in the literature. We investigated
the performance of these microtips for trypsin and Glu-C proteolytic digests of R- and -casein and compared ZrO2 phosphopeptide binding specificity and recovery to TiO2-based enrichment.
Mass spectrometric phosphopeptide analysis was performed with
electrospray ionization (ESI) in both negative and positive ion
modes with a Fourier transform ion cyclotron resonance (FT-ICR)
instrument.
EXPERIMENTAL SECTION
Reagents and Sample Preparation. R-Casein and -casein
from bovine milk (Sigma, St. Louis, MO) were prepared in 25
mM ammonium bicarbonate (Fisher Scientific, Fair Lawn, NJ)
buffer to a concentration of 25 M. Trypsin (Promega, Madison,
WI) digestion was performed for 15 h at 37 C at an enzyme/
substrate ratio of 1:50. Glu-C (Roche, Penzberg, Germany)
digestion also proceeded for 15 h but at 25 C at an enzyme/
substrate ratio of 1:100. Microtips filled with ZrO2 and TiO2 (25
or 50 g) were either provided as a gift or purchased from Glygen
(Columbia, MD) and used without further modification. For
optimization of the phosphopeptide enrichment conditions, binding
solutions of different pH were prepared by adding various
concentrations (see below) of formic acid (Acros Organics, Fair
Lawn, NJ) or ammonium bicarbonate to HPLC grade water
(Fisher). Washing solutions consisted of pure water (pH 6.5), 250
mM ammonium acetate (pH 6.9), 100 mM ammonium bicarbonate
(pH 8.0), or 75 mM ammonium hydroxide (pH 10.5). Finally, 0.5%
piperidine (pH 11.5) or 0.25 or 0.5% ammonium hydroxide (pH
10.8 and 11, respectively) were used for eluting bound phosphopeptides. Peptide mixtures from the enzymatic digests were
fractionated into 1-100-pmol aliquots, dried down in a vacuum
concentrator (Eppendorf, Hamberg, Germany), reconstituted in
binding solution, and loaded onto microtips that had been
equilibrated with the same binding solution. Unbound peptides
(21) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912-5919.
(22) Wolschin, F.; Wienkoop, S.; Weckwerth, W. Proteomics 2005, 5, 43894397.
(23) Nawrocki, J.; Rigney, J.; McCormick, A.; Carr, P. W. J. Chromatogr., A 1993,
657, 229-282.
(24) Nawrocki, J.; Dunlap, C.; McCormick, A.; Carr, P. W. J. Chromatogr., A
2004, 1028, 1-30.
(25) Hoth, D. C.; Rivera, J. G.; Colon, L. A. J. Chromatogr., A 2005, 1079, 392396.

1744

Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

were removed with washing solution, and bound peptides were


eluted with the high-pH elution solutions. Eluted peptide samples
were dried down and reconstituted in 2-propanol/acetonitrile/
water (1:1:2) with 0.25% piperidine for negative ion mode and in
acetonitrile/water (1:1) with 0.1% formic acid for positive ion mode
ESI-FT-ICR MS analysis.
Mass Spectrometry and Data Analysis. Mass measurements
were performed with a 7-T hybrid quadrupole (Q)-FT-ICR mass
spectrometer (Bruker Daltonics, Billerica, MA) equipped with an
Apollo electrospray ion source. The ESI flow rate was 50 L/h,
and nebulization was assisted by N2 gas. Peptide ions were
externally accumulated for 0.5 s in a hexapole prior to being
transferred to the ICR cell and captured by gated trapping. The
external accumulation and transfer events were looped twice prior
to excitation and detection. Spectra were acquired with XMASS
(version 6.1, Bruker Daltonics) from m/z 250 to 2500 with 512k
data points and summed over four or eight scans. Data analysis
was performed with the MIDAS analysis software.26 Data were
Hanning apodized and zero filled once prior to fast Fourier
transformation and magnitude calculation. Lists of expected
proteolytic peptide masses were generated with the Peptide Mass
tool on the Expasy web site (www.us.expasy.org) using accession
numbers P02662 and P02663 for the two forms (S1 and S2) of
R-casein and P02666 for -casein. Another tool on the Expasy web
site, Compute pI, was used to compute pI values of certain
peptides. ZrO2 and TiO2 phosphopeptide binding selectivity was
determined by dividing the sum of the abundances of all phosphopeptide isotopomers with the sum of all observed isotopomers
in the same spectrum before and after phosphopeptide enrichment. Only peaks with a signal-to-noise (S/N) ratio above three
were included in the analysis. The noise level of a spectrum was
determined from the root-mean-square signal in a region devoid
of peptide peaks. The Peptide Mass tool automatically considers
phosphorylation when that modification is present in the database.
However, we identified additional phosphopeptides by allowing
different numbers of phosphate groups. Those assignments were
confirmed by collision-activated dissociation in the external
hexapole with argon as collision gas. Neutral loss of 98 Da
(corresponding to phosphoric acid) was used as a marker ion.
RESULTS AND DISCUSSION
Optimization of Zirconium Dioxide Phosphopeptide Enrichment Conditions. Zirconium oxide is known to have amphoteric properties; that is, it can react either as a Lewis acid or
base depending on the pH of the reaction solution. This property
is a result of unsatisfied valencies of both oxygen and zirconium
atoms in the surface layer.23 In acidic solution, ZrO2 behaves as a
Lewis acid with positively charged zirconium atoms, thereby
displaying anion-exchange properties.27 For example, high binding
affinity for polyoxy anions, including phosphate, borate, carboxylate, and sulfate, has been demonstrated.28 The binding constant
of phosphate ions is markedly higher than for other Lewis
bases,29,30 suggesting that high binding selectivity of phosphory(26) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun.
Mass Spectrom. 1996, 10, 1839-1844.
(27) Amplett, C. B. Inorganic Ion Exchangers; Elsevier: Amsterdam, The
Netherlands, 1964.
(28) Kraus, K. A.; Philips, H. O.; Carlson, T. A.; Johnson, J. S. Proceedings of
the Second International Conference on Peaceful Uses of Atomic Energy,
Geneva, Switzerland, 1958; 3.

Figure 1. Negative mode ESI FT-ICR mass spectra (8 scans) from


100 pmol of a tryptic digest of R-casein obtained prior to ZrO2
phosphopeptide enrichment (a) and following enrichment at various
binding pH values (b-e). Phosphopeptides are labeled with numbers
that are identified in Table 1. Nonphosphorylated peptides observed
following enrichment are labeled with their corresponding amino acid
residue numbers and R-casein isoform. *, noise.

lated peptides over nonphosphorylated acidic peptides should be


achievable with ZrO2 upon careful selection of the pH. Figure 1a
shows a negative mode ESI FT-ICR mass spectrum of a 100 pmol
nonenriched tryptic digest of R-casein. Identified phosphopeptides
are labeled with numbers from 1 to 12 and included in Table 1.
Only the most abundant phosphopeptides are labeled in the
spectrum. Spectra obtained following ZrO2 enrichment of phosphopeptides from the same digest at different binding solution
pH values are shown in Figure 1b-e (100 pmol/spectrum). All
these enrichment experiments involved water washing and elution
in 0.5% piperidine. These data show that it is critical to use lowpH solutions to achieve high phosphopeptide binding selectivity.
At pH 2 (Figure 1b), only one nonphosphorylated peptide (doubly
deprotonated EPMIGVNQELAYFYPELFR, corresponding to amino
acid residues 148-166 in the S1 form of R-casein) is detected with
S/N > 3. This peptide contains three acidic glutamate residues
and has a pI of 4.25. The data obtained at pH 3 (Figure 1c) do not
differ drastically from those obtained at pH 2. However, already
at this slightly higher pH, an increased abundance of nonphos(29) Blackwell, J. A.; Carr, P. W. J. Chromatogr., A 1991, 549, 43-57.
(30) Blackwell, J. A.; Carr, P. W. J. Chromatogr., A 1991, 549, 59-75.

phorylated peptides is seen with the peptide mentioned above


being detected both in its doubly and triply deprotonated forms.
In addition, a singly deprotonated peptide with one glutamate
residue (VNELSK, corresponding to amino acid residues 52-57
in the S1 form of R-casein with a pI of 5.97) is observed. However,
phosphopeptides dominate the mass spectrum. Binding in water
(Figure 1d) still provides phosphopeptide enrichment compared
to the spectrum obtained without the use of ZrO2 tips (Figure
1a). However, the selectivity is further compromised. At higher
pH (Figure 1e) for which the Lewis acid property of ZrO2
diminishes, phosphopeptides are no longer the dominant species
and a very limited amount of peptides binds to the tip.
In chromatographic applications of zirconia, both the pH and
the ionic strength of the mobile phase can influence retention
properties due to a mixed-mode ligand- and ion-exchange analyte
binding mechanism.23 We did not control the ionic strength in
our pH-dependence experiments; however, the carboxylate ions
originating from the formic acid added to lower the pH would be
expected to decrease phosphopeptide binding due to competition
for ZrO2 acidic sites. Thus, because we observed increased rather
than decreased binding at lower pH, we believe the pH is far more
important than ionic strength under our experimental conditions.
To achieve maximum recovery of bound phosphopeptides, we
also optimized the washing and elution conditions and found that
pure water was the best washing solution. We also found that a
pH as high as 10.8-11.5 was needed for optimum elution
performance. The highest phosphopeptide recovery was achieved
with 0.5% piperidine (pH 11.5) and that elution condition was used
in all experiments presented below.
ZrO2 and TiO2 Phosphopeptide Enrichment from Tryptic
Digests of Phosphoproteins. To establish how ZrO2 phosphopeptide enrichment compares to TiO2 enrichment, we applied the
protocol established above to both ZrO2 and TiO2 microtips (50
g). Panels a-c in Figure 2 show this comparison for a tryptic
digest of R-casein. In our hands, either technique is more selective
than IMAC, similar to the results obtained previously by Larsen
et al.20 However, we found that the relative abundance of singly
versus multiply phosphorylated peptides varies between ZrO2 and
TiO2 with singly phosphorylated peptides being enriched to a
higher extent with ZrO2: For the R-casein tryptic digest, the most
abundant signal following ZrO2 enrichment (Figure 2b) originates
from the 2- charge state of the singly phosphorylated peptide
YKVPQLEIVPNSAEER, corresponding to amino acid residues
119-134 in the S1 form of R-casein (peptide 7 in Table 1) followed
by the 2- charge state of the doubly phosphorylated peptide
DIGSESTEDQAMEDIK, corresponding to amino acid residues
58-73 in the S1 form of R-casein (peptide 6 in Table 1). Abundant
signal (31% relative abundance) is also observed from the 3charge state of the singly phosphorylated peptide 7 as well as
minor signal from its quadruply deprotonated form. In addition,
a shorter version of peptide 7, corresponding to no missed trypsin
cleavage sites (amino acid residues 121-134 in the S1 form of
R-casein, peptide 4 in Table 1), is detected following ZrO2
enrichment. By contrast, the doubly phosphorylated peptide 6 is
the most abundant species following TiO2 enrichment (Figure 2c),
and the abundance for both the 2- and 3- charge states of the
singly phosphorylated peptide 7 is lower in that spectrum (53 and
12%, respectively) compared to the spectrum obtained following
Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

1745

Table 1. List of Phosphopeptides Found in Negative Mode FT-ICR Mass Spectra from Proteolytic Digests of r- and
-Casein without (w) Phosphopeptide Enrichment and Following ZrO2 and TiO2 Enrichment

no.

peptide identity

enzyme

phosphorylation

monoisotopic
mass

method

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

R-casein-S2:153-164
R-casein-S2:141-152
R-casein-S2:152-164(153-165)
R-casein-S1:121-134
R-casein-S1:58-73
R-casein-S1:58-73
R-casein-S1:119-134
R-casein-S1:118-134
R-casein-S1:74-94
R-casein-S2:16-36
R-casein-S2:61-85
R-caseinS2:57-95
-casein:48-63
-casein:45-63
-casein:17-40
-casein:16-40
-casein:16-40
R-casein-S1:126-133
R-casein-S1:55-65
R-casein-S1:126-140
R-casein-S1:55-70
R-casein-S1:77-92
R-casein-S2:67-83
R-casein-S2:16-33
-casein:47-52
-casein:47-57
-casein:47-59
-casein:21-36
-casein:21-46
-casein:21-46

trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C

1phos:158
2phos:144,146
1phos:158
1phos:130
1phos:61,63,68a
2phos:61,63,68a
1phos:130
1phos:130
1phos:79
4phos:23,24,25,31
4phos:71,71,73,76
4phos:71,72,73,76
1phos:50
1phos:50
4phos:30,32,33,34
3phos:30,32,33,34a
4phos:30,32,33,34
1phos:130
2phos:61,63
1phos:130
3phos:61,63,68
5phos:79,81,82,83,90
4phos:71,72,73,76
4phos:23,24,25,31
1phos:50
1phos:50
1phos:50
4phos:30,32,33,34
3phos:30,32,33,34a
4phos:30,32,33,34

1465.6047
1538.5902
1593.6997
1659.7868
1846.7179
1926.6842
1950.9451
2079.0401
2719.9055
2745.9923
3007.0221
4717.9274
2060.8211
2431.0430
2965.1571
3041.5000
3121.2582
937.3793
1324.4836
1818.8335
1978.6556
2075.6103
2093.6080
2353.7863
846.3160
1460.5820
1704.6516
2007.6804
3110.4225
3190.3888

w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
Zr, Ti
Zr, Ti
w, Zr, Ti
w, Zr, Ti
Zr
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w
w, Zr, Ti
w
w, Zr, Ti
w
w
w, Zr, Ti
Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti

All possible (known) phosphorylation sites are listed.

ZrO2 enrichment (74 and 31%, respectively). In addition, the 4charge state of peptide 7 is not detected following TiO2 enrichment, nor is the singly phosphorylated peptide 4. These enrichments were performed from 100-pmol aliquots of the same tryptic
digest. Thus, variations in the digestion conditions cannot explain
the absence of peptide 4. We believe these results constitute an
important finding because a plentitude of biological regulatory
mechanisms involve a single phosphorylation event and our results
imply that ZrO2 phosphopeptide enrichment would be preferred
over other isolation strategies in the characterization of such cases.
We do not believe the observed behavior is a result of irreversible
binding of multiply phosphorylated peptides to ZrO2 because we
could still detect such phosphopeptides in the left-over solution
of tryptic peptides. Also, we do not believe the differentiation
between singly and multiply phosphorylated peptides is a result
of our specific enrichment protocol because changing the conditions did not result in varied ratios of, for example, peptides 6
and 7 for R-casein (Figure 1).
The finding that ZrO2 more selectively isolates singly phosphorylated peptides as compared to TiO2 was confirmed with a
tryptic digest of -casein; see Figure 2d-f. Again, the most
abundant peak following ZrO2 enrichment originates from a singly
phosphorylated peptide (the 2- charge state of FQSEEQQQTEDELQDK, corresponding to amino acid residues 48-63 in
-casein (peptide 13 in Table 1) whereas its abundance following
TiO2 enrichment is comparable to the quadruply phosphorylated
peptide RELEELNVPGEIVESLSSSEESITR (peptide 17, triply
deprotonated). The latter peptide displays similar S/N ratios
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Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

following both techniques, again indicating that its lower relative


yield following ZrO2 enrichment is not due to poorer recovery
from the column.
The observed difference in phosphopeptide binding selectivity
between zirconia and titania may be related to their different
surface chemistry: First, zirconia is a stronger Lewis acid than
titania,31 which could contribute to an enhanced binding of singly
phosphorylated peptides. Second, the coordination numbers of
Zr and Ti are different in the crystalline forms typically present
in chromatographic materials, six for Ti and seven for Zr,31 which
could result in different binding properties for singly and multiply
phosphorylated peptides. However, because the surface properties
of zirconia and titania are not well understood,24 these suggestions
are rather speculative at this point.
ZrO2 and TiO2 Phosphopeptide Enrichment from Glu-C
Digests of Phosphoproteins. Regnier and co-workers have
recently shown that the use of Glu-C rather than trypsin for
phosphoprotein digestion prior to IMAC phosphopeptide enrichment significantly reduces nonspecific binding of acidic nonphosphorylated peptides.32 This behavior is a result of the specificity
of Glu-C, which can cleave a protein (depending on the digestion
conditions) at the C-terminal side of acidic amino acid residues
(Asp and Glu). Thus, in theory, Glu-C digestion results in only
one acidic amino acid residue per peptide (not counting the
(31) Grun, M.; Kurganov, A. A.; Schacht, S.; Schuth, F.; Unger, K. K. J.
Chromatogr., A 1996, 740, 1-9.
(32) Seeley, E. H.; Riggs, L. D.; Regnier, F. E. J. Chromatogr., A 2005, 817,
81-88.

Figure 2. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of tryptic digests of R- and -casein obtained prior to phosphopeptide
enrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH
2), washed with water, and eluted in 0.5% piperidine (pH 11.5). Highly selective enrichment of singly phosphorylated peptides (7 and 13, see
Table 1) is observed following ZrO2 enrichment. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding
amino acid residue numbers and isoform (for R-casein). *, noise.

phosphopeptides), hence reducing nonspecific binding. We explored the use of Glu-C digestion prior to ZrO2 and TiO2
enrichment; see Figure 3. For R-casein (Figure 3a-c), Glu-C
digestion resulted in lower overall (i.e., with or without enrichment) phosphopeptide signal as compared to trypsin digestion
(see Table 2). With TiO2, the enrichment factor (i.e., the increase
in relative phosphopeptide signal) following Glu-C digestion was
about the same (2.5-fold) as for the R-casein trypsin digest
whereas a higher enrichment factor (4-fold relative increase)
was observed with ZrO2 for the Glu-C digest (compared to 2.5fold relative increase for the trypsin digest). From repeated
experiments, the error of the tabulated selectivity values was
estimated to be 8%. Again, one additional singly phosphorylated
peptide (peptide 20 in Table 1) was detected following ZrO2
enrichment as compared to TiO2 enrichment. However, several
multiply phosphorylated peptides (peptides 22-24 in Table 1)
detected prior to TiO2 and ZrO2 treatment were not observed
following the use of metal oxide microtips, rendering trypsin
digestion the preferred strategy for R-casein.
For -casein, the enrichment factors following both TiO2 and
ZrO2 treatment were similar for the Glu-C and trypsin digests (see
Table 2). Additional evidence for the selective enrichment of singly
phosphorylated peptides with ZrO2 is evident from Figure 3e and
f: Following both ZrO2 and TiO2 enrichment, the most abundant
species correspond to the 2- charge state of singly phosphorylated
KFQSEEQQQTEDE (peptide 27 in Table 1). However, the relative
abundance is 64% following ZrO2 treatment and 44% following TiO2
treatment. Again, TiO2 shows higher selectivity than ZrO2 for a

multiply phosphorylated peptide (quadruply phosphorylated LNVPGEIVESLSSSEE, peptide 28 in Table 1): The 2- charge state
of this peptide is detected at a 27% relative abundance following
TiO2 enrichment versus 15% for ZrO2. Also, its triply deprotonated
form is only observed following TiO2 enrichment.
Sensitivity of ZrO2 and TiO2 Microtip Phosphopeptide
Enrichment. All spectra presented above were obtained following
enrichment of 100 pmol of a phosphoprotein digest employing
microtips containing 50 g of ZrO2 or TiO2. Table 3 shows the
selectivity of these microtips with decreasing amounts of an
R-casein tryptic digest (50 and 25 pmol, respectively). Again, the
error of these values was found to be 8%. It is clear that, for
R-casein, the phosphopeptide selectivity is not compromised when
the sample amount is decreased. However, because our mass
spectrometer had not been optimized for high sensitivity (e.g.,
we did not use nanospray or an ion funnel for improved ion
transmission), we obtained poor S/N values with lower than 25
pmol of sample.
To allow utilization of the ZrO2 microtips for sample amounts
compatible with proteomic applications, it will be necessary to
scale down the size of these columns. An initial attempt to half
the amount of metal oxide to 25 g resulted in improved S/N
ratios of phosphopeptides from a tryptic digest of 1 pmol of
R-casein (Figure 4). These experiments were performed with a
digest different from the one used for the experiments shown in
Figures 1 and 2. Thus, the relative abundance of the detected
phosphopeptides is somewhat different. However, again, singly
phosphorylated peptides (peptides 4 and 7) are more abundant
Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

1747

Figure 3. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of Glu-C digests of R- and -casein obtained prior to phosphopeptide
enrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH
2), washed with water, and eluted in 0.5% piperidine (pH 11.5). For R-casein, poorer phosphopeptide signal is observed as compared to trypsin
digestion (Figure 2) whereas similar performance is seen for -casein with selective enrichment of a singly phosphorylated peptide (27, see
Table 1) following ZrO2 treatment. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding amino acid
residue numbers. *, noise.
Table 2. Selectivitya (%) of ZrO2 and TiO2 Microtips for
Phosphopeptide Enrichment of Proteolytic Digests of
r- and -Casein

without enrichment
ZrO2 enrichment
TiO2 enrichment
a

trypsin
digest of
R-casein

trypsin
digest of
-casein

Glu-C
digest of
R-casein

Glu-C
digest of
-casein

27
67
62

22
61
65

8
31
22

22
62
77

Table 3. Selectivitya (%) of 50-g ZrO2 and TiO2


Microtips for Phosphopeptide Enrichment of a Tryptic
Digest of r-casein as a Function of Sample Amount

without enrichment
ZrO2 enrichment
TiO2 enrichment
aDefined

100 pmol

50 pmol

25 pmol

27
67
62

29
85
77

29
83
74

as relative phosphopeptide signal; see text.

Defined as relative phosphopeptide signal; see text.

than the doubly phosphorylated peptide 6. All three peptides are


more abundant following enrichment with the smaller as compared to the larger column. However, it should be noted that the
phosphopeptide selectivity was compromised at 1 pmol, even with
the 25-g tip: Several singly deprotonated nonphosphorylated
peptides as well as some unidentified peaks were observed.
Calculated selectivity values were 41% for the 25-g tip and 36%
for the 50-g tip whereas values as high as 80% were obtained at
higher sample amounts (Table 3).
ZrO2 Phosphopeptide Enrichment for Positive Ion Mode
Mass Spectrometry. All data discussed above were obtained in
negative ion mode. Although negative ionization generally provides higher sensitivity for phosphopeptides, many phosphoproteomics experiments are performed in positive ion mode because
the dissociation behavior of peptide cations is much better
1748 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

understood than that of peptide anions.33,34 However, the major


fragmentation pathway of serine- and threonine-phosphorylated
peptides in both positive and negative modes is loss of phosphoric
acid rather than backbone cleavage,3,4,6 and thus, their sequencing
can be difficult. Phosphate loss is not observed to a large extent
in electron capture dissociation (ECD) of phosphopeptides,35-37
and that technique, which requires positive ion mode operation,
(33) Ewing, N. P.; Cassady, C. J. J. Am. Soc. Mass Spectrom. 2001, 12, 105116.
(34) Bowie, J. H.; Brinkworth, C. S.; Dua, S. Mass Spectrom. Rev. 2002, 21,
87-107.
(35) Stensballe, A.; Norregaard-Jensen, O.; Olsen, J. V.; Haselmann, K. F.;
Zubarev, R. A. Rapid Commun. Mass Spectrom. 2000, 14, 1793-1800.
(36) Shi, S. D.-H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty,
F. W. Anal. Chem. 2001, 73, 19-22.
(37) Chalmers, M. J.; Hakansson, K.; Johnson, R.; Smith, R.; Shen, J.; Emmett,
M. R.; Marshall, A. G. Proteomics 2004, 4, 970-981.

Figure 4. Negative mode ESI FT-ICR mass spectra (8 scans) from


1 pmol of a trypsin digest of R- casein obtained following phosphopeptide enrichment with a 50-g ZrO2 microtip (a) and a 25-g ZrO2
microtip (b). Downscaling the column size results in increased
sensitivity. Identified nonphosphorylated peptides are labeled with
their corresponding amino acid residue numbers and R-casein
isoform. *, noise.

can therefore facilitate phosphopeptide sequencing and localize


sites of phosphorylation. However, ECD also requires high signalto-noise ratios of precursor ions, and thus, enrichment of phosphopeptides may be crucial to its applicability in phosphoproteomics.
Figure 5 shows positive ion mode ESI-FT-ICR mass spectra
obtained prior to and following ZrO2 enrichment of a tryptic digest
of R-casein. Only three singly phosphorylated peptides (peptides
1, 4, and 7 in Table 1) are observed without enrichment (Figure
5a) whereas one more charge state of peptide 7 and four additional
phosphopeptides are detected following ZrO2 enrichment (Figure
5b). Three of these additional phosphopeptides (peptides 2, 3, and
6 in Table 1), of which two are doubly phosphorylated, were also
observed in negative ion mode (Figure 2). However, the fourth
additional phosphopeptide (labeled 31 in Figure 5b) was identified
as the 2+ charge state of the singly phosphorylated peptide
NMAINPSKENLCSTFCK, corresponding to amino acid residues
40-56 in the S2 form of R-casein. No peptides including that
region of the protein were observed in negative ion mode. There
are also several peaks (mainly singly charged) in Figure 5b that
could not be identified as either phosphorylated or nonphosphorylated peptides from R-casein; however, R-casein phosphopeptides
dominate the enriched spectrum. The relative phosphopeptide
signal increased a factor of 3 following enrichment.

Figure 5. Positive mode ESI FT-ICR mass spectra (4 scans) from


100 pmol of a tryptic digest of R-casein obtained prior to phosphopeptide enrichment (a) and following ZrO2 phosphopeptide enrichment
(b). Phosphopeptides are labeled with numbers identified in Table 1,
except for peptide 31 (singly phosphorylated NMAINPSKENLCSTFCK), which was not observed in negative ion mode. The relative
phosphopeptide signal increased a factor of 3 following enrichment.

CONCLUSIONS
In our hands, both zirconium oxide and titanium oxide
microtips provide highly selective phosphopeptide enrichment
from proteolytic peptide mixtures. However, the zirconium oxide
columns possess a unique selectivity for singly phosphorylated
peptides whereas titanium oxide selectively enriches multiply
phosphorylated peptides. Thus, these two column materials have
complementary properties and the choice of phosphopeptide
enrichment strategy can be tailored according to the specific
application. For R- and -casein investigated here, the use of Glu-C
rather than trypsin for proteolytic digestion did not appear to
provide an advantage. Current commercially available 50-g
microtips are limited to sample amounts above 1 pmol although
further miniaturization of these columns appears promising.
ACKNOWLEDGMENT
This work was supported by the Searle Scholars Program and
the University of Michigan. We also thank Ashok Shukla for
valuable discussions and for providing the 25-g microtips.

Received for review December 18, 2005. Accepted


January 23, 2006.
AC0522355

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