Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry Uma K. Aryal * ,y and Andrew R. S. Ross z National Research Council, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 Received 19 June 2009; Revised 7 November 2009; Accepted 9 November 2009 Titanium dioxide metal oxide affinity chromatography (TiO 2 -MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO 2 -MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO 2 -MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO 2 -MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO 2 -MOAC showed higher specificity than immobilized gallium (Ga 3R ), immobilized iron (Fe 3R ), or zirconium dioxide (ZrO 2 ) affinity chromatography for phospho- peptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatog- raphy/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright # 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd. Reversible phosphorylation is one of the most common mechanisms for covalent modification of proteins and is found in as many as one third of eukaryotic gene products. 1,2 Although the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature of this modification. 3 The detection and sequencing of tryptic phosphopeptides derived from such proteins has become an important aspect of biological and biomedical research. However, the prevalence of non-phosphorylated peptides in protein digests has made it necessary to develop efficient separation and enrichment methods for phospho- peptide analysis. Immobilized metal-ion affinity chromatography (IMAC) has been widely used for the selective enrichment of phosphopeptides; 4–6 however, this method is prone to low re- coveries and/or non-specific binding of non-phosphorylated peptides. 7 Metal oxide affinity chromatography (MOAC) using titanium dioxide (TiO 2 ) has recently been proposed as an alternative to IMAC. 8,9 This technique is based on the selective interaction of phosphopeptides with porous TiO 2 microspheres (titanospheres) via bidentate binding at the TiO 2 surface. 10,11 Such interactions arise from the affinity of oxygen in the phosphate groups for metal atoms in the MOAC resin. 3 According to established protocols, peptide mixtures are loaded onto the column under acidic conditions and the bound phosphopeptides eluted in basic solution. 8,9,12 The selectivity of different IMAC and MOAC methods for phosphopeptides has been studied extensively; however, many of the results presented in the literature are either contradictory or of limited practical use. In the case of TiO 2 -MOAC, peptide loading in the presence of 2,5-dihydroxybenzoic acid (DHB) or phthalic acid has been shown to increase selectivity for phosphopepetides; 9,13 however, both compounds are suspected of causing interferences during the analysis of phosphopeptides by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). 12,14,15 A desire to RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 219–231 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4377 *Correspondence to: U. K. Aryal, Pacific Northwest National Laboratory, P.O. Box 999, 902 Battelle Boulevard, Richland, WA 99352, USA. E-mail: [email protected]y Present address: Pacific Northwest National Laboratory, P.O. Box 999, 902 Battelle Boulevard, Richland, WA 99352, USA. z Present address: Fisheries and Oceans Canada, Institute of Ocean Sciences, P.O. Box 6000, 9860 West Saanich Road, Sidney, British Columbia, Canada V8L 4B2. Copyright # 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.
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RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2010; 24: 219–231
) DOI: 10.1002/rcm.4377
Published online in Wiley InterScience (www.interscience.wiley.com
Enrichment and analysis of phosphopeptides under
different experimental conditions using titanium dioxide
affinity chromatography and mass spectrometry
Uma K. Aryal*,y and Andrew R. S. Rossz
National Research Council, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
Received 19 June 2009; Revised 7 November 2009; Accepted 9 November 2009
*CorrespoLaboratoWA 9935E-mail: UyPresentBox 999,zPresentOcean ScBritish C
Titanium dioxide metal oxide affinity chromatography (TiO2-MOAC) is widely regarded as being
more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide
enrichment. However, the widespread application of TiO2-MOAC to biological samples is hampered
by conflicting reports as to which experimental conditions are optimal. We have evaluated the
performance of TiO2-MOAC under a wide range of loading and elution conditions. Loading and
stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for
phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous
reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards
phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal
specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave
incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the
eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO2-MOACwas
also found to be intolerant of many reagents commonly used as phosphatase inhibitors during
protein purification. However, TiO2-MOAC showed higher specificity than immobilized gallium
(Ga3R), immobilized iron (Fe3R), or zirconium dioxide (ZrO2) affinity chromatography for phospho-
peptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatog-
raphy/electrospray ionization tandemmass spectrometry (LC/ESI-MS/MS), which was more efficient
for smaller, singly phosphorylated peptides. Copyright # 2009 Crown in the right of Canada.
Published by John Wiley & Sons, Ltd.
Reversible phosphorylation is one of the most common
mechanisms for covalent modification of proteins and is
found in as many as one third of eukaryotic gene products.1,2
Although the number of cellular phosphoproteins is
relatively high, the phosphorylated residues themselves
are generally of low abundance due to the sub-stoichiometric
nature of this modification.3 The detection and sequencing of
tryptic phosphopeptides derived from such proteins has
become an important aspect of biological and biomedical
research. However, the prevalence of non-phosphorylated
peptides in protein digests has made it necessary to develop
efficient separation and enrichment methods for phospho-
a Final volume of loading solution adjusted to 20mL followingaddition of reagent.bAll samples loaded in 1%TFA/30% ACN.c Peptides enriched from 200 fmol of a combined a- and b-caseindigest.d Peptides detected using MALDI-MS.
Evaluation of TiO2 for phosphopeptide enrichment 227
logical experiments, as summarized in Table 2. This table
also lists the number of phosphorylated and non-phos-
phorylated peptides detected following TiO2-MOAC enrich-
ment in the presence of these reagents. Examples of the
MALDI mass spectra from which these results were derived
are shown in Fig. 4. By way of comparison, Fig. 4(a) shows
the control spectrum obtainedwithout including any of these
reagents in the loading solution.
Several of the reagents tested were found to be incompa-
tible with TiO2 affinity chromatography. The most dramatic
effect was observed with sodium molybdate and sodium b-
glycerophosphate, which completely removed specificity
towards phosphopeptides, as indicated by the detection of
only two phosphorylated peptides (peaks 8 and 12) but
several additional non-phosphorylated peptides (Figs. 4(d)
and 4(e)). Sodium orthovanadate also had a negative impact
on specificity, as illustrated by the number of non-
phosphorylated peptides co-purified with phosphopeptides
(Fig. 4(f)). Although TiO2-MOAC appears to be somewhat
tolerant of sodium fluoride (Fig. 4(b)) its presence again
reduced specificity and recovery of certain singly (peaks 5, 7,
8) and multiply phosphorylated peptides (peaks 24 & 25)
when compared with the control (Fig. 4(a)). However,
okadaic acid appeared to have negligible impact on the
performance of TiO2-MOAC (Fig. 4(c)).
Copyright # 2009 Crown in the right of Canada. Published by John Wiley &
The effect of adding other reagents to the loading solution
was also investigated. These included imidazole, calyculin
A, PMSF, poly(ethylene glycol) (PEG), and a protease
inhibitor cocktail from Sigma (product no. P9599) containing
Kweon and Hakansson,24 however, we did not find ZrO2 to
be better than TiO2 for recovery of singly phosphorylated
peptides.
Fe-IMAC appeared to favor the enrichment of multiply
phosphorylated peptides when compared with Ga-IMAC,
which showed balanced recovery of singly and multiply
phosphorylated peptides (Figs. 5(c) and 5(d)). However,
both IMAC methods appeared less selective than TiO2-
MOAC, since non-phosphorylated peptides observed in the
MALDI-MS spectra of IMAC-purified samples were not
observed in TiO2-enriched samples. Nevertheless, relative
signal intensities for multiply phosphorylated peptides
were higher for IMAC than for TiO2-MOAC eluants, as
shown by the corresponding MALDI mass spectra
(Figs. 5(a), 5(c) and 5(d)), supporting previous claims that
multiply phosphorylated peptides are recovered more
efficiently using IMAC methods.9 For example, peaks 20,
24 and 25were generally of higher abundance in IMAC than
in TiO2 spectra, whereas peaks 15 and 22 were detected by
MALDI-MS for both IMAC methods but not for TiO2.
Similarly, the tetra-phosphorylated peptide 21 was detected
by LC/ESI-MS/MS in both Ga- and Fe-IMAC eluants but
not TiO2 (Table 3). This could be due to the strength of the
interaction between multiply phosphorylated peptides and
Sons, Ltd. Rapid Commun. Mass Spectrom. 2010; 24: 219–231
DOI: 10.1002/rcm
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Figure
4.
Eff
ect
of
phosp
ha
tase
inhib
itors
on
phosphopeptide
purification
usin
gT
iO2-M
OA
C.
Panels
show
MA
LD
Im
ass
spectr
aof
phosphopeptides
purified
from
200
fmolo
fa
com
bin
eda
-andb
-casein
dig
estlo
aded
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in30%
AC
Nw
ith
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es,
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oly
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)10
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cero
phosphate
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mort
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.P
eptides
were
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ted
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NH
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Hin
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AC
N.
Copyright # 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd. Rapid Commun. Mass S
228 U. K. Aryal and A. R. S. Ross
m/z
pectrom. 2010; 24: 219–231
DOI: 10.1002/rcm
Evaluation of TiO2 for phosphopeptide enrichment 229
TiO2, which may require a more stringent elution protocol
than used in this comparative study.34 However, TiO2-
MOAC appeared to show even greater selectivity in our
study than previously observed.9 For example, LC/ESI-
MS/MS detected just two non-phosphorylated peptides
(peaks A and D) in TiO2-MOAC eluates whereas four non-
phosphorylated peptides were detected in IMAC-enriched
samples (Supplementary Table S2, see Supporting Infor-
mation). Non-selective binding of peptide D (m/z 1760) to
TiO2 and IMAC columns has been reported else-
where.6,7,9,14 To summarize, TiO2-MOAC showed higher
specificity than Ga-IMAC, Fe-IMAC or ZrO2-MOAC for
phosphopeptide enrichment.
Comparison of MALDI-MS and LC/ESI-MS/MSfor phosphopeptide analysisLC/ESI-MS/MS analysis of the unpurified casein digest
matched a total of twelve peptides (four phosphorylated,
eight non-phosphorylated) to the first (aS1) sub-unit of a-
Table 3. Detection of phosphorylated casein peptides by MALDI-M
ZrO2-MOAC, Ga-IMAC or Fe-IMAC
aaS1 and aS2 refer to the first and second subunits of a-casein, respectivebpI calculated according to Bjellqvist et al.36cHydrophobicity (BB index) determined according to Bull and Breese.37dSamples were loaded in 1% TFA in 30% ACN and eluted with 0.4M Ncombined a- and b-casein digests.e1, e2, e3 and e4 Methionine-oxidized forms of peptides 5, 7, 11 and 16, respfPeptide 6 sequence according to Larsen et al.9gPeptide 14 sequence according to Hsieh et al.38 with the N-terminal glutahPeak due to metastable loss of phosphate from peptide 25.iVariant of b-casein with an N-terminal glycine residue, according to Jens
Copyright # 2009 Crown in the right of Canada. Published by John Wiley &
casein, eleven peptides (five phosphorylated, six non-
phosphorylated) to the second (aS2) sub-unit, and five
peptides (two phosphorylated, three non-phosphorylated) to
b-casein (data not shown). When purified using the TiO2-
MOAC, nine peptides (seven phosphorylated, two non-
phosphorylated) were matched to aS1, eight peptides (all
phosphorylated) to aS2, and three phosphopeptides to b-
casein (Table 3 and Supplementary Table 2, see Supporting
Information). Hence, TiO2 enrichment increased the number
of matched phosphopeptides as well as their confidence
levels (ion scores) when compared with the unextracted
samples. On the other hand, no non-phosphorylated
peptides were detected in TiO2-MOAC-enriched samples
using MALDI-MS (Fig. 1(b)). This difference could be due to
the inherent sensitivity of LC/ESI-MS/MS and of the Q-TOF
instrument, which is greater than that of the instrument used
for MALDI-MS analysis. It is important to note that the two
non-phosphorylated peptides (A and D) detected by LC/
ESI-MS/MS in TiO2-enriched samples were also the most
S and LC/ESI-MS/MS following enrichment by TiO2-MOAC,
ly. b-C represents peptides of b-casein.
H4OH in 30% ACN. Each column was loaded with 200 fmol of the
ectively.
mine cyclized to pyroglutamic acid.
en and Larsen.14
Sons, Ltd. Rapid Commun. Mass Spectrom. 2010; 24: 219–231
DOI: 10.1002/rcm
Inte
nsity
(%)
699.0 1359.4 2019.8 2680.2 3340.6 4001.000
20
40
60
80
100 1.1E +4
A57 7’
8
910
12
11 13
18 2024” 24
699.0 1359.4 2019.8 2680.2 3340.6 4001.000
20
40
60
80
100
B
1.1E +4
5 7
89 10
11
12
13
1516
171819
2024”
2223
24
25
Inte
nsity
(%)
Inte
nsity
(%)
m/z699.0 1359.2 2019.4 2679.6 3339.8 4000.0
00
20
40
60
80
100 1.1E +4
A 7C
9
11
12
13 1516
1718
192022
24” 24
25
23
Inte
nsity
(%)
699.0 1359.4 2019.8 2680.2 3340.6 4001.00
1.1E +4
0
20
40
60
80
100
5 7
811
9 10
12
13
1617 18
1920
23 24”2425
(a)
(b)
(c)
(d)
Figure 5. Comparison of TiO2-MOAC with other phosphopeptide enrichment methods.
Panels show MALDI mass spectra for 200 fmol of a combined a- and b-casein digest
enriched for phosphopeptides using (a) TiO2-MOAC, (b) ZrO2-MOAC, (c) Ga-IMAC, and
(d) Fe-IMAC. In all cases, samples were loaded using 1% TFA in 30% ACN and eluted with
0.4 M NH4OH in 30% ACN.
230 U. K. Aryal and A. R. S. Ross
abundant peptides detected byMALDI-MS in the unpurified
digest (see Fig. 1(a)). Regardless of which MS technique was
used, fewer non-phosphorylated peptides were detected in
TiO2-MOAC-enriched samples than in any other extracts
(Supplementary Table S2, see Supporting Information),
further confirming TiO2-MOAC as themost selectivemethod
for phosphopeptide enrichment.
Copyright # 2009 Crown in the right of Canada. Published by John Wiley &
With regard to phosphopeptide analysis, more singly and
doubly phosphorylated peptides were detected using LC/
ESI-MS/MS than MALDI-MS (Table 3). In fact, with the
exception of peptide 9 all singly or doubly phosphorylated
peptides observed using MALDI-MS were also detected by
LC/ESI-MS/MS, while peptides 1–4 and 14 were only
detected using LC/ESI-MS/MS. In contrast, more peptides
Sons, Ltd. Rapid Commun. Mass Spectrom. 2010; 24: 219–231
DOI: 10.1002/rcm
Evaluation of TiO2 for phosphopeptide enrichment 231
carrying three or more phosphate groups were detected
using MALDI-MS than with LC/ESI-MS/MS. For example,
multiply phosphorylated peptides 15, 17–21, and 25 were
only observed using MALDI-MS whereas the only multiply
phosphorylated peptide detected by LC/ESI-MS/MS alone
was peptide 21 (a missed-cleavage product incorporating
peptide 19). Larsen et al.9 and Gruhler et al.35 also reported a
bias against the detection of multiply phosphorylated
peptides when using LC/ESI-MS/MS. Using our TiO2-
MOAC enrichment method, we were able to detect more
phosphorylated peptides (including peptides 160, 22 and 24)
than reported by others using LC/ESI-MS/MS,9 and with
less material (200 fmol of combined a- and b-casein digests),
confirming the high efficiency of the TiO2 tips. LC/ESI-MS/
MS was more efficient in detecting singly phosphorylated
peptides than MALDI-MS for all four enrichment methods,
whereas the opposite was true for multiply phosphorylated
peptides (Table 3).
CONCLUSIONS
TiO2-MOAC using porous titanium (NuTipTM) media
provides efficient and highly selective enrichment of
phosphopeptides from protein digests. Optimal selectivity
was achieved by loading peptides with 1% TFA in 30% ACN
and washing sequentially with acidic (1% TFA) solutions of
increasing organic content (30, 50 and 75% ACN). Glycolic
acid was found to be ineffective as a non-phosphopeptide
excluder in the loading solution. With the exception of
okadaic acid, TiO2-MOAC was found to be intolerant of
many commonly used phosphatase inhibitors, which has
implications for phosphoproteomic studies that do not
involve gel separation prior to MOAC. Base elution in
0.4M NH4OH or 0.1M NH4H2PO4 gives efficient and
balanced recovery of singly and multiply phosphorylated
peptides and is compatible with MALDI-MS and LC/ESI-
MS/MS, both of which may be necessary to ensure detection
of all phosphopeptides recovered using TiO2-MOAC. Ga-
IMAC and Fe-IMAC gave better recovery of multiply
phosphorylated peptides than MOAC under the experimen-
tal conditions, but were less specific than TiO2 for
phosphopeptide enrichment.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
AcknowledgementsWe thank Doug Olson and Steve Ambrose of the Mass
Spectrometry and Protein Research Group at NRC-PBI for
technical support. We also thank Dr. Joan Krochko for valu-
able suggestions and Dr. Randy Purves for reviewing the
manuscript. The Visiting Fellow and Research Associate
positions awarded to UKA by, respectively, the Natural
Science and Engineering Research Council (NSERC) of
Canada and NRC-PBI are also gratefully acknowledged.
Copyright # 2009 Crown in the right of Canada. Published by John Wiley &
Funding for protein mass spectrometry equipment was pro-
vided by the Saskatchewan Provincial Government and the
National Research Council of Canada, fromwhich this article
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