enrichment and analysis of phosphopeptides under

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-

peptide analysis.

Immobilized metal-ion affinity chromatography (IMAC)

has been widely used for the selective enrichment of

ndence to: U. K. Aryal, Pacific Northwest Nationalry, P.O. Box 999, 902 Battelle Boulevard, Richland,2, [email protected]: Pacific Northwest National Laboratory, P.O.902 Battelle Boulevard, Richland, WA 99352, USA.address: Fisheries and Oceans Canada, Institute ofiences, P.O. Box 6000, 9860 West Saanich Road, Sidney,olumbia, Canada V8L 4B2.

Copyright # 200

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 (TiO2) has recently been proposed as

an alternative to IMAC.8,9 This technique is based on the

selective interaction of phosphopeptides with porous TiO2

microspheres (titanospheres) via bidentate binding at the

TiO2 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 TiO2-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

9 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.

220 U. K. Aryal and A. R. S. Ross

avoid potential losses during further purification of TiO2-

enriched phosphopeptides prior toMS analysis has led to the

search for alternative ‘non-phosphopeptide excluders’ that

are compatible with both matrix-assisted laser desorption/

ionization (MALDI) and ESI. Jensen and Larsen14 have

proposed glycolic acid as an alternative to DHB or phthalic

acid; however, Sugiyama and co-workers12 have since

reported lower specificity of TiO2-MOAC in the presence

of this reagent. Compared with the extensive literature on

IMAC-based methods, relatively little information is

available regarding optimal conditions for TiO2-MOAC.16

This makes it difficult to predict which method would be

better suited to particular applications, or more compatible

with the reagents used in biological studies. For example,

TiO2-MOAC is known to be more tolerant than IMAC to

certain reagents and buffers,14 but its compatibility with

many of the protease and phosphatase inhibitors used in

protein sample preparation is unknown. This information is

needed to develop faster and more efficient enrichment

protocols that can be applied to a wide range of biological

samples.

To address these uncertainties, we have evaluated the

performance of TiO2-MOAC under different loading and

elution conditions, using a single make and model of

commercially available TiO2 column (NuTipTM NT1TIO,

Glygen Corp., USA) to minimize experimental variability.

We also compared the selectivity of TiO2-MOACwith that of

zirconium dioxide (ZrO2)-MOAC, gallium (Ga3þ)- and iron

(Fe3þ)-IMAC, and evaluated the performance of MALDI-MS

and LC/ESI-MS/MS in detecting the phosphopeptides

isolated using these different affinity techniques.

EXPERIMENTAL

MaterialsAcetonitrile (ACN) and HPLC-grade water were obtained

from Merck (Darmstadt, Germany). Trifluoroacetic (TFA),

formic (FA), phosphoric (PA) and acetic (AA) acids,

ammonium bicarbonate (NH4HCO3), phosphate (NH4H2PO4)

and hydroxide (NH4OH), bovine a- and b-caseins, dithio-

threitol (DTT) and iodoacetamide, mass calibrants (angio-

tensin 1, ACTH clip 1-17 and 18-35), PhosphoProfileTM

gallium silica spin columns (product no. P2873) and PHOS-

selectTM iron affinity gels (product no. P9740) were

purchased from Sigma (St. Louis, MO, USA). Modified

porcine trypsin (sequencing grade) was obtained from

Promega (Madison, WI, USA) and 2,5-dihydroxybenzoic

acid (DHB) from Waters (Milford, MA, USA). Porous

titanium dioxide NuTipsTM (product no. NT1TIO), zirco-

nium dioxide NuTipsTM (NT1ZRO) and empty TopTipsTM

(TT2EMT) were purchased from Glygen Corp. (Columbia,

MD, USA). PepCleanTM C18 spin columns (product no.

89870) were purchased from Pierce (Rockford, IL, USA),

and C18 3M high-performance extraction disks from Empore

(St. Paul, MA, USA).

Preparation of tryptic digestsEach of the model phosphoproteins a-casein and b-casein

(100 picomoles) was dissolved separately in 100mL of 0.1M

NH4HCO3 (pH 8.5) containing 10mM DTT and incubated at

Copyright # 2009 Crown in the right of Canada. Published by John Wiley &

568C for 30min. Each protein was alkylated using 40mM

iodoacetamide for 1 h at room temperature in the dark. The

reaction was quenched by addition of DTT to a final

concentration of 5mM, and the protein digested with trypsin

at 378C overnight using an enzyme/substrate ratio of 1:50

(w/w). Each tryptic digest was dried in a vacuum centrifuge

(model DNA 120; Thermo Savant, Colin Drive, NY, USA),

reconstituted in 100mL of 0.1% aqueous TFA, and desalted

using PepCleanTM C18 spin columns (Pierce) according to the

manufacturer’s instructions. Briefly, each column was

conditioned with 200mL of 50% methanol and equilibrated

with 200mL of 0.5% TFA in 5% ACN, repeating each step

once and centrifuging after each addition. The 100mL of

tryptic digest in 0.1% TFA was loaded, centrifuged, and the

collected solution reloaded and centrifuged again. After

washing twice with 200mL of 0.5% TFA in 5% ACN, the spin

column was eluted twice with 40mL of 70% ACN. The

combined eluate (80mL)was dried, reconstituted in 100mL of

0.1% TFA, and stored at �208C until further use.

For each experiment, 1mL of the desalted a-casein digest

was combined with an equal volume of b-casein digest and

98mL of the appropriate loading solution (see below) to

produce a 10 fmol/mL combined digest solution. This was

divided into five 20mL aliquots, each containing 200 fmol of

digest, from which phosphopeptides were subsequently

purified by MOAC or IMAC. Unless otherwise noted,

200 fmol of the combined a- and b-casein digests were used

in all experiments.

Metal oxide affinity chromatography (MOAC)Prior to sample loading, the disposable TiO2 NuTipsTM

(Glygen) were conditioned/equilibrated with 20mL of 1%

TFA in 30% ACN using 10 aspirate/expel (A/E) cycles. To

evaluate the effect of different organic acids on phosphopep-

tide binding, 1mL volumes of both desalted casein digests

were combined in 98mL of 30% ACN containing 0.1, 1 or 5%

AA, FA or TFA, and 20mL of the resulting solution loaded

onto a TiO2 tip using 50A/E cycles. The effect of DHB on

phosphopeptide binding was investigated by loading in 50%

ACN containing 1% TFA, with or without DHB (130mM).

After loading the tips were washed once with 20mL of 1%

TFA in 30% ACN, once with 20mL of 1% TFA in 50% ACN,

twice with 20mL of 1% TFA in 75% ACN, and, finally, twice

with 20mL of HPLC-grade water, using 10A/E cycles each

time. Bound phosphopeptides were eluted with 20mL of

0.4M NH4OH in 30% ACN, using 20A/E cycles. The eluate

was acidified immediately by adding 5mL of aqueous 1%

TFA and analyzed by mass spectrometry (MS).

To investigate the efficiency of different eluants, TiO2 tips

were loaded using 1% TFA in 30% ACN, washed

sequentially with various solutions as described above,

and eluted with 20mL of 0.25M NH4HCO3 (pH 9) in 30%

ACN, 0.4M NH4OH (pH �11) in 30% ACN, 0.1M

NH4H2PO4 (pH 10) in 30% ACN, or 1% PA (with and

without 130mM DHB) in 50% ACN.

The compatibility of TiO2 with reagents commonly used in

protein sample preparation was also investigated by adding

appropriate concentrations of okadaic acid, sodium fluoride,

sodium molybdate, sodium orthovanadate, sodium b-

glycerophosphate, imidazole, calyculin A, phenylmethylsul-

Sons, Ltd. Rapid Commun. Mass Spectrom. 2010; 24: 219–231

DOI: 10.1002/rcm

Evaluation of TiO2 for phosphopeptide enrichment 221

fonyl fluoride (PMSF), Sigma protease inhibitor cocktail

(product no. P9599), or poly(ethylene glycol) to the loading

solution (1% TFA in 30% ACN).

To compare performance using the different MOAC

media, TiO2 and ZrO2 NuTipsTM were equilibrated/

conditioned using 20mL of 1% TFA in 30% ACN and loaded

with 20mL of combined digest in the same solvent, using

50A/E cycles. After washing, as previously described,

bound phosphopeptides were eluted with 20mL of 0.4M

NH4OH in 30% ACN, using 20A/E cycles. All these

experiments were repeated at least three times to confirm

the results.

Immobilized metal-ion affinitychromatography (IMAC)For Ga-IMAC, pre-packed PhosphoProfileTM gallium silica

spin columns (Sigma) were first washed/equilibrated with

50mL of 1% TFA in 30% ACN and then loaded with 20mL of

combined digest in the same solvent, using a pipette to apply

gentle pressure and ensure proper loading of the sample.

After loading, columns were incubated at room temperature

for 15min before centrifuging at 1000 rpm for 1min. The

sample flow-through was then re-loaded, and the incubation

and centrifugation repeated. The columns were washed with

50mL volumes of the same washing solutions used for TiO2-

MOAC, with centrifugation at 1000 rpm for 2min during

each washing step. Bound phosphopeptides were eluted by

loading the columnwith 20mL of 0.4MNH4OH in 30%ACN,

incubating at room temperature for 5min, then centrifuging

at 1000 rpm for 1min. The eluate was immediately acidified

by adding 5mL of 1% aqueous TFA and used forMS analysis.

For Fe-IMAC, PHOS-SelectTM iron affinity gel beads were

carefully stirred until completely and uniformly suspended

in the stabilizing buffer supplied. The 5mL of the resulting

slurry was mixed with 5mL of 1% TFA in 30% ACN and

loaded into empty TopTipsTM (Glygen), using a regular

pipette tip with about 1mm of the end cut off to allow

unrestricted flow and uniform distribution of the suspended

beads. The beads were washed/equilibrated by adding

50mL of 1% TFA in 30% ACN and spun in a microcentrifuge

for 1min at 1000 rpm. This operation was repeated two more

times to ensure complete removal of the stabilizing buffer,

which contains glycerol. The flow-through was discarded

and the Fe-IMAC tips loaded with 20mL of combined digest

in 1% TFA/30% ACN. To ensure proper loading, the tip was

again fitted to a pipette and gentle pressure applied to push

the sample into the gel beads. The tips were incubated at

room temperature for 15min before spinning at 1000 rpm for

1min. The sample flow-through was reloaded and the

incubation and centrifugation process repeated. Using

the same procedure as for Ga-IMAC, the Fe-IMAC columns

were washed, eluted, and the eluate acidified and used for

MS analysis (see below).

MALDI-MSThe acidified peptide eluates were vacuum-dried, recon-

stituted in 10mL of 10% aqueous TFA and desalted with

STAGE (STop And Go Extraction) tips prepared using

EmporeTM C18 (octadecyl) extraction disks, according to

established procedures.17 The bound peptides were eluted


using 1mL of matrix solution containing 130mM DHB in

10mM (NH4)2HPO4 and 75% ACN and deposited directly

onto the MALDI target plate. MALDI-MS analysis was

performed using a Voyager-DE STR instrument (Applied

Biosystems, Framingham, MA, USA) operating in the

positive ion and reflectron modes with delayed ion

extraction. Close external mass calibration was performed

using angiotensin 1, ACTH clip 1-17 and 18-35. Spectra were

acquired over the m/z range 700–4000 and the results of 200

laser shots combined to produce a single averaged spectrum

for each sample. The efficiency and selectivity of phospho-

peptide binding under different loading and elution

conditions were evaluated by comparing the number, type

(i.e. singly, multiply, or non-phosphorylated) and relative

abundance of peptide ions detected by MALDI-MS.

LC/ESI-MS/MSFor LC/ESI-MS/MS the eluates were dried, reconstituted in

10mL of 1% aqueous TFA, and 6mL injected onto the LC

column. Analysis was performed using a nanoACQUITY

UPLC system (Waters, Milford, MA, USA) interfaced to a

quadrupole time-of-flight (Q-TOF) Ultima Global hybrid

tandem mass spectrometer fitted with a Z-spray nano-ESI

source (Waters, Mississauga, ON, Canada). Chromato-

graphic separation of peptides was accomplished using a

Waters BEH130 C18 column (75mm� 100mm, 1.75mm) and

a flow rate of 400 nL/min. Solvent A was 0.2% FA in water

and solvent B was 0.2% FA in 100% ACN. The peptides were

separated for 55min by increasing the organic content of the

mobile phase linearly from 0% to 45% over 45min and then

to 80% at 46min, holding this concentration until 52min and

then reducing to 1% at 53min. A 5min seal wash with 10%

ACN was also performed after each run. Mass calibration

was performed using the product ion spectrum of Glu-

fibrinopeptide B acquired over the m/z range 50–1900. The

instrument was run in positive ion mode with a source

temperature of 808C. Analysis was carried out using data-

dependent acquisition, during which peptide precursor ions

were detected by scanning from m/z 400–1900 in TOF-MS

mode. Multiply charged (2þ, 3þ, or 4þ) ions rising above pre-

determined threshold intensity were automatically selected

for TOF-MS/MS analysis, and product ion spectra acquired

over the m/z range 50–1900. The absence of singly

charged peptides was subsequently verified by means of

extracted ion chromatograms generated for m/z values

corresponding to every theoretical, singly charged a- and

b-casein peptide ion.

The MS/MS data were converted into a peak list (pkl) file

format using MassLynx v2.15 software (Micromass) and

searched against the NCBInr database using an in-house

Mascot server (v. 2.2). Phosphorylation of peptides identified

as phosphopeptides via database searching was confirmed

by manual inspection of the corresponding MS/MS spectra.

Each validated phosphopeptide MS/MS spectrum was then

reduced to single-charged, monoisotopic, centroided peaks

using MaxEnt 3 (MassLynx v.4.1, Micromass), and in silico

spectral fragmentation of the corresponding, database-

matched phosphopeptide sequence carried out using

BioLynx software. The calculated fragment ion masses were


DOI: 10.1002/rcm


then compared with the experimental values to confirm the

position(s) of the phosphorylated residue(s).

RESULTS AND DISCUSSION

Effect of different organic acids onphosphopeptide bindingDuring affinity purification of phosphopeptides, acidic

loading/washing solutions generally are effective in inhibit-

ing non-specific binding of acidic and/or hydrophobic

peptides, whereas high pH solutions are more efficient in

recovering bound phosphopeptides.6 There have been

several studies to determine optimal sample loading

conditions for TiO2-MOAC;3,8,9,12–14,18,19 however, the infor-

mation presented in the literature remains contradictory. In

view of these uncertainties, we decided to re-evaluate

systematically the effects of different organic acids and

modifiers on TiO2-MOAC selectivity towards phosphopep-

tides.

MALDI-MS analysis of the non-enriched casein digest

detected only three phosphorylated peptide ions (labeled 8,

12 and 13; Fig. 1(a)), and at relatively low intensities.

However, many additional phosphopeptides were detected

following TiO2-MOAC enrichment (Figs. 1(b)–1(d)). As in all

subsequent figures and tables, phosphorylated peptides are

annotated using numbers (1–25) and non-phosphorylated

peptides using letters (A–F) in Fig. 1.

To evaluate the binding selectivity of the TiO2 columns for

phosphorylated peptides under different loading conditions,

casein digests were loaded onto TiO2 tips using 0.1, 1 or 5%

AA, FA or TFA in 30% ACN and the bound peptides eluted

using 0.4M NH4OH in 30% ACN (Table 1). When samples

were loaded in 0.1% concentrations of these acids, several

acidic non-phosphorylated peptides were detected in the

eluants. However, the number of non-phosphorylated

peptides decreased as the concentration of acid in the

loading solution increased (Table 1). The number of non-

phosphorylated peptides detected was much lower with

TFA than with either AA or FA. Repeated experiments

confirmed that the ability of these organic acids to inhibit

binding of non-phosphorylated peptides is in the order

TFA> FA>AA.

The effect of TFA concentration on the intensities of two

representative non-phosphorylated peptide peaks A (m/z

1266.8) andD (m/z 1759.1) relative to phosphorylated peptide

peaks was also monitored (Figs. 1(b)–1(d)). The relative

intensities of peaks A and D were reduced when using 1%

rather than 0.1%TFA in the loading buffer, whereas using 5%

TFA resulted in detection of an additional non-phosphory-

lated peptide F (m/z 2315.5). At the same time, multiply

phosphorylated peptide 25 was enhanced in 1% relative to

0.1% TFA but absent from the 5% TFA spectrum. Although

these differences are relatively minor they do suggest that a

concentration of 1% TFA is generally sufficient to achieve

optimum performance. Increasing the proportion of ACN

from 30% to 50% or 75% did not improve TiO2 selectivity for

phosphopeptides (data not shown). To summarize, loading

in 1% TFA/30% ACN followed by stringent washing of the

loaded peptides with 1% TFA in increasing concentrations of

ACN (see Experimental section) gave best results in terms of


phosphopeptide coverage (Fig. 1(c)), and this loading

solution was used for all subsequent experiments.

Effect of different eluants on phosphopeptiderecoveryConventional protocols often use basic solutions to recover

phosphopeptides from affinity columns.20 However, acidic

solutions containing phosphoric acid (PA) or amixture of PA

and DHB have been shown to act as efficient eluants for Fe-

IMAC.15,20,21–23 Similarly, although NH4OH is known to be

an efficient eluant for both IMAC and MOAC,9,24 the

suitability of other bases (e.g. NH4HCO3, NH4H2PO4) for

TiO2-MOAC has yet to be explored. We decided to evaluate

these acidic and basic eluants for the recovery of phospho-

peptides from TiO2 using 1%TFA in 30%ACN as the loading

solution (Fig. 2).

A number of singly phosphorylated and multiply

phosphorylated peptides (peaks 11, 18, 20 and 24/2400) were

detected following elution with PA in 50% ACN (Fig. 2(b));

however, this was not as effective as 0.4M NH4OH in 30%

ACN (Fig. 2(a)). Furthermore, inclusion of DHB significantly

reduced the number of multiply phosphorylated peptides, as

exemplified by the low signal intensity for peak 24 (Fig. 2(c)).

Hence, these acidic eluants were found to be relatively

inefficient for the recovery of phosphopeptides from TiO2.

With regard to basic eluants, 0.4M NH4OH in 30% ACN

(Fig. 2(a)) and 0.1M NH4H2PO4 in 30% ACN (Fig. 2(e)) gave

better recovery of multiply phosphorylated peptides than

did 0.25M NH4HCO3 in 30% ACN (Fig. 2(d)). This is

illustrated by the relative abundance of peak 24 in each

spectrum and the fact that multiply phosphorylated peptide

peaks 18, 19, 23 and 25 were detected after elution with

NH4OH and NH4H2PO4, but not with NH4HCO3. Although

similar results were obtained with NH4OH and NH4H2PO4

we decided to use the former as the eluant for all subsequent

experiments, since optimal conditions for sample loading

had already been determined using 0.4M NH4OH in 30%

ACN as the eluant (Fig. 1).

In order to have practical utility, any enrichment method

must enable the detection of phosphopeptides at concen-

trations typically found in gel spot digests, or in complex

mixtures derived from whole cell lysates or subcellular

fractions.25 Sensitivity was therefore evaluated by loading

100, 50 and 25 fmol of the combined casein digests onto the

TiO2 tips using 1% TFA in 30% ACN, eluting with 0.4M

NH4OH in 30%ACN and analyzing the peptides byMALDI-

MS (data not shown). The majority of casein phosphopep-

tides were detected in all cases.

The sensitivity and specificity of the optimized TiO2-

MOAC method were further evaluated using Arabidopsis

thaliana leaf proteins separated by two-dimensional gel

electrophoresis (2-DE) and visualized by silver staining. Two

gel spots corresponding to isoforms of carbonic anhydrase 1

(CA1), which contains four phosphorylation sites (Aryal et al.

unpublished data; Supplementary Table S1, see Supporting

Information), were excised from the gel, combined, and

subjected to trypsin digestion using a MassPREP digest

station (Micromass, Manchester, UK) according to the

recommended procedure. Half of the digest (�5mL) was

analyzed directly by LC/ESI-MS/MS while the remainder


DOI: 10.1002/rcm

699.0 1359.4 2019.8 2680.2 3340.6 4001.00

2.2E +4

0

20

40

60

80

100

A D

128

135

109

11

1716 2425

7

0

2.2E +4

0

20

40

60

80

100

699.0 1359.4 2019.8 2680.2 3340.6 4001.0

577’

8

9

10

11

12

13

F F’15

161718

1920

2324”

24

m/z

699.0 1359.4 2019.8 2680.2 3340.6 4001.000

20

40

60

80

100 2.2E +4

A

B C

D

E

F

812

13In

tens

ity (%

)In

tens

ity (%

)

699.0 1359.4 2019.8 2680.2 3340.6 4001.00

2.2E +4

0

20

40

60

80

100

5 7

8

11

9 10

12

13

1617 18

1920

2324”24

25

Inte

nsity

(%)

Inte

nsity

(%)

(a)

(b)

(c)

(d)

Figure 1. Effect of TFA concentration on the loading of phosphopeptides during TiO2-

MOAC. Panels show representative MALDI mass spectra obtained from 200 fmol of a

combined a- and b-casein digest (a) before TiO2-MOAC enrichment, and following TiO2-

MOAC enrichment using (b) 0.1%, (c) 1%, or (d) 5% TFA in 30% ACN as the loading solution.

Bound phosphopeptides were eluted with 0.4 M NH4OH (pH 11) in 30% ACN. Phosphory-

lated peptides are annotated using numbers (1–25) and non-phosphorylated peptides using

letters (A–F), as summarized in Table 3 and Supplementary Table 2 (see Supporting

Information), respectively.


was vacuum-dried, re-constituted in 15mL of 1% TFA in 30%

ACN, and then purified by TiO2-MOAC prior to LC/ESI-

MS/MS analysis. Without prior enrichment eleven non-

phosphorylated peptides and four singly phosphorylated


peptides were identified; however, only one non-phosphory-

lated peptide (YMVFACSDSR) was detected following TiO2-

MOAC enrichment, whereas the flow-through fraction

contained all twelve non-phosphorylated CA1 peptides


DOI: 10.1002/rcm

Table 1. Effect of acidic loading conditions on TiO2-MOAC

enrichment of phosphorylated peptides from 200 fmol of a

combined a- and b-casein digest

Loadingsolution acida

Concentration(%) pH

No. of peptides identified(p< 0.05)b,c

Phosphorylated

Non-phosphorylatedSingly Multiply

Acetic acid 0.1 3.2 7 4 71.0 2.8 7 6 65.0 1.2 7 8 1

FA 0.1 2.6 7 5 71.0 2.2 7 7 55.0 1.0 7 8 0

TFA 0.1 2.1 7 8 41.0 1.2 7 10 05.0 0.2 7 10 0

aAll loading solutions contained 30% ACN.b Peptides eluted with 0.4M NH4OH in 30% ACN.cAverage of three independent experiments.


and no phosphopepetides. Furthermore, ion scores for the

identified phosphopeptides were significantly improved in

the enriched sample, presumably due to enhancement of the

peptide ion signal (Supplementary Table S1, see Supporting

Information). Results demonstrate the utility of our TiO2-

MOAC method for detecting phosphopeptides and identify-

ing sites of protein phosphorylation in biological extracts.

Use of non-phosphopeptide excludersLoading TiO2 columns with 1% TFA in 30% ACN provides

high selectivity towards phosphopeptides for relatively

simple samples; however, Jensen and Larsen14 have

suggested that a non-phosphopeptide excluder should be

used to optimize selectivity for the enrichment of phospho-

peptides from complex samples such as cell lysates. DHB

and phthalic acid have been used successfully for this

purpose,9,13 but are thought to inhibit LC/ESI-MS/MS

analysis of the recovered phosphopeptides by contaminating

the LC system and the inlet of the mass spectrometer.12,14,15

Jensen and Larsen14 have recently shown that 1M glycolic

(hydroxyacetic) acid is equally effective as a non-phospho-

peptide excluder, and is compatible with both MALDI-MS

and LC/ESI-MS/MS analysis. However, Sugiyama and co-

workers found that using 300mg/mL (�4M) glycolic acid in

the loading solvent resulted in non-specific binding of

peptides.12 In view of this uncertainty we decided to re-

evaluate the use of DHB and glycolic acid as non-

phosphopeptide excluders for MOAC.

Based on previous investigations involving a- and b-

casein,23 DHB was added to the MOAC loading solution at a

concentration of 130mM,whereas glycolic acidwas included

at 1, 0.5 or 0.25M to investigate whether reducing (rather

than increasing)12 the concentration of this reagent might

enhance specificity. MALDI mass spectra of casein digests

enriched by TiO2-MOAC with DHB or glycolic acid in the

loading solution are shown in Fig. 3. The specificity obtained

using 1% TFAwith 30%ACN alone as the loading solution is

sufficient to eliminate non-phosphorylated peptides from the


spectrum (Fig. 3(a)). DHB appeared to enhance detection of

certain multiply phosphorylated peptides, as illustrated by

increases in the relative intensities of peaks 6 and 24 in the

presence of DHB (Fig. 3(b)). However, the relative intensities

of other peaks such as 8, 10 and 17 were apparently reduced

in the presence of DHB, suggesting that addition of this

reagent to the loading solution offers no significant

advantage.

When 1M glycolic acid was used in the loading solution

several non-phosphorylated peptides (labeled A, B, D, E, and

F/F0) were detected at significant levels (Fig. 3(c)). The

relative abundances of these peptides decreased as the

concentration of glycolic acid decreased from 1 to 0.5 or

0.25M (Figs. 3(c)–3(e)), suggesting that the addition of

glycolic acid to the loading solution actually increased non-

specific binding of peptides to TiO2. No enhancement in the

relative abundance ofmultiply phosphorylated peptides was

observed using glycolic acid.

We also evaluated DHB and glycolic acid as non-

phosphopeptide excluders for ZrO2-MOAC. The addition

of DHB to the loading solution reduced the binding of non-

phosphorylated peptides to ZrO2 columns while again

enhancing the relative abundance of certain multiply

phosphorylated peptides (data not shown). As well, the

use of glycolic acid appeared to increase non-specific binding

of peptides to ZrO2 columns, and gave no enhancement in

the relative abundance of multiply phosphorylated peptides

(data not shown). These observations are in direct contrast

with those of Jensen and Larsen,14 who found 1M glycolic

acid to be an efficient non-phosphopeptide excluder. Our

results complement those obtained by Sugiyama and co-

workers12 who used a single, relatively high concentration

(�4M) of glycolic acid.

Hydroxy acids bind to metal oxides by forming a cyclic

chelate with the metal,12,26,27 whereas phosphate anions do

so by bridging two metal atoms.9 Thus, hydroxy acids such

as DHB should bind to metal oxides more weakly than a

phosphate group but more strongly than the carboxylic

groups of acidic non-phosphorylated peptides. It is unclear

why glycolic (hydroxyacetic) acid should have been

ineffective in displacing non-phosphorylated peptides from

TiO2 during our study. One possibility is that the structure

and retention properties of titania, which are strongly

dependent on the calcination temperature of the beads,28,29

may differ from one study to another. In any event, it appears

that loading samples with glycolic acidmay actually increase

non-specific binding of peptides during MOAC, depending

on the materials and conditions used.

Effect of phosphatase inhibitors onphosphopeptide enrichmentDuring proteomic analysis a number of reagentsmay be used

to solubilize hydrophobic proteins and to inhibit protease

and phosphatase activities. TiO2-MOAC is known to be

compatible with most of the buffers and detergents used in

biological experiments;14 however, the tolerance of TiO2-

MOAC to other reagents, including protease and phospha-

tase inhibitors, has not been widely explored. This infor-

mation would be helpful in deciding what purification steps

might be necessary prior to enrichment by TiO2-MOAC and


DOI: 10.1002/rcm

0699.0 1359.4 2019.8 2680.2 3340.6 4001.0

2.2E +4

0

20

40

60

80

100

A

567

8

9 10

1112

13

F’D 16 17 2024”

24

Inte

nsity

(%)

699.0 1359.4 2019.8 2680.2 3340.6 4001.00

2.2E +4

0

20

40

60

80

100

57

8

9 10

11

12

13

1617 18 19

2023

24” 24

25

Inte

nsity

(%)

699.0 1359.4 2019.8 2680.2 3340.6 4001.000

20

40

60

80

100 2.2E +4

9

A C5 5’

7

8

D1011 12

E E’F

F’

18 2024”

24

13

0 0

2.2E +4

20

40

60

80

100

CA

55’

7

8

9 10

1112

13

24F’F

699.0 1359.4 2019.8 2680.2 3340.6 4001.0

Inte

nsity

(%)

Inte

nsity

(%)

699.0 1359.4 2019.8 2680.2 3340.6 4001.00

2.2E +4

0

20

40

60

80

100

5 7

8

11

9 10

12

13

1617 18

1920

2324”24

25

Inte

nsity

(%)

m/z

(a)

(b)

(c)

(d)

(e)

Figure 2. Recovery of phosphopeptides from TiO2 using different eluants. Panels show repre-

sentative MALDI mass spectra obtained from 200 fmol of a combined a- and b-casein digest

following TiO2-MOAC enrichment and elution with (a) 0.4 M NH4OH in 30% ACN, (b) 1% phosphoric

acid (PA) in 50% ACN, (c) 1% PA and 130 mM DHB in 50% ACN, (d) 0.25 M NH4HCO3 in 30% ACN,

or (e) 0.1 M NH4H2PO4 in 30% ACN. Samples were loaded using 1% TFA in 30% ACN.


MS analysis, especially when using ‘gel-free’ proteomic

approaches.

Sodium fluoride, sodium molybdate, sodium orthovana-

date, sodium b-glycerophosphate, okadaic acid, imidazole


and calyculin A are some of the more commonly used

phosphatase inhibitors, and were included in our study.

These reagents were added to the 1% TFA in 30% ACN

loading solution at concentrations normally used in bio-


DOI: 10.1002/rcm

m/z

899.0 1519.4 2139.8 2760.2 3380.6 4001.00

8397.1

0

20

40

60

80

100

AB

5 7

8

D

910

12

13 E

FF’ 24” 24

Inte

nsity

(%)

899.0 1519.4 2139.8 2760.2 3380.6 4001.000

20

40

60

80

100 8397.1

5 5’6

7

8

910 11

12

13

2023 24”

24

Inte

nsity

(%)

899.0 1519.4 2139.8 2760.2 3380.6 4001.00

8397.1

0

20

40

60

80

100

A BD

56 7

8

91011

12

13

E FE’

F’ 20 24”24

Inte

nsity

(%)

899.0 1519.4 2139.8 2760.2 3380.6 4001.000

20

40

60

80

100 8397.1

567

8

9 10

11 13

151617 1819

2023 24”

24

Inte

nsity

(%)

12

Inte

nsity

(%)

899.0 1519.4 2139.8 2760.2 3380.6 4001.000

20

40

60

80

100 8397.1

57

8

91011

12

13

16

171819

202324”

24

25

25

(a)

(b)

(c)

(d)

(e)

Figure 3. Effect of non-phosphopeptide excluders on phosphopeptide recovery using

TiO2-MOAC. Panels show MALDI mass spectra obtained from 200 fmol of a combined

a- and b-casein peptide digest loaded in a 30% ACN solution of 1% TFA containing (a) no

additives, (b) 130 mM DHB, (c) 1 M, (d) 0.5 M, or (e) 0.25 M glycolic acid. Peptides were

eluted with 0.4 M NH4OH in 30% ACN.

Copyright # 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2010; 24: 219–231

DOI: 10.1002/rcm


Table 2. Effect of different reagents on the enrichment of

casein phosphopeptides using TiO2- and ZrO2-MOACa

Added reagentb

No. of peptides identifiedc,d

Phosphorylated

Non-phosphorylatedSingly Multiply

TiO2-MOAC

None 7 10 020mg/mL DHB 7 10 01M glycolic acid 7 4 5500mM glycolic acid 7 6 3250mM glycolic acid 7 6 220mM sodium fluoride 6 11 42mM sodium molybdate 2 0 32mM sodium orthovanadate 7 10 610mM sodiumb-glycerophosphate

3 0 4

2mM okadaic acid 7 10 1100mM imidazole 7 5 1100 nM calyculin A 7 6 040mM PMSF 7 5 11% PEG 7 11 10.01% Sigma protease inhibitorcocktail (product # P9599)

7 7 1

ZrO2-MOAC

None 7 4 220mg/mL DHB 7 5 01M glycolic acid 7 1 6500mM glycolic acid 7 4 3250mM glycolic acid 7 4 2

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.


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)).


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

4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), bestatin,

pepstatinA, E-64, leupeptin and 1,10-phenanthroline, which

are used routinely for plant protein extractions. The results of

these experiments are again summarized in Table 2. We

included PEG in our investigation because it has been found

to improve detection of low-abundance proteins by selec-

tively precipitating RuBisCo from plant protein extracts.30–33

It is also used to purify plasma membrane proteins by

aqueous two-phase partitioning.27 Our results show that

TiO2 affinity chromatography is somewhat tolerant of most

reagents investigated, but that the presence of imidazole,

PMSF, calyculin A or the Sigma protease inhibitor cocktail

reduces the number and relative intensities of multiply

phosphorylated peptide peaks. The removal of certain

additives prior to TiO2-MOAC may, therefore, be necessary

to achieve optimum performance.

Comparison of TiO2-MOAC with otherphosphopeptide enrichment methodsBoth MALDI-MS and LC/ESI-MS/MS data were used to

compare TiO2-MOAC with ZrO2-MOAC, Ga-IMAC and Fe-

IMAC for phosphopeptide enrichment, and a complete

summary of the phosphorylated a- and b-casein peptides

detected using each technique is provided in Table 3. In all

cases, samples were loaded in 1% TFA/30%ACN and eluted

with 0.4M NH4OH in 30% ACN. Both MS techniques

detected more multiply phosphorylated peptides in the TiO2

eluant than in the ZrO2 eluant, although the number of singly

phosphorylated peptides was similar in each case (Table 3).

These results are in keeping with previous studies that also

found TiO2-MOAC to be more efficient than ZrO2-MOAC at

recovering multiply phosphorylated peptides.14,24 Unlike

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


DOI: 10.1002/rcm

0

5387

.0

020

40

60

80

10

0 69

9.0

13

59

.42

01

9.8

26

80

.23

34

0.6

40

01

.0

5A

8E9 1011

12

131516

1718

1920

2324

25B

69

9.0

13

59

.42

01

9.8

26

80

.23

34

0.6

40

01

.00

538

7.0

020

40

60

80

10

0

A

CD

8

12

13E’

Intensity (%)

69

9.0

13

59

.42

01

9.8

26

80

.23

34

0.6

40

01

.00

5387

.0

020

40

60

80

10

0

AC

57

8 D9 1011

12

13E

F16

17 1819 20

2324

” 2425

0

5387

.0

020

40

60

80

10

0

B

D

1213

69

9.0

13

59

.42

01

9.8

26

80

.23

34

0.6

40

01

.0

Intensity (%)

0

5387

.0

020

40

60

80

10

0 69

9.0

13

59

.42

01

9.8

26

80

.23

34

0.6

40

01

.0

57

8

D91011

12

13

1516

1718

19 202324

”24 25

069

9.0

1359

.420

19.8

2680

.233

40.6

4001

.0

5387

.0

020406080100

57

8

11

910

12

13

161718

1920

2324

”24

25

Intensity (%)

(a)

(b)

(d)

(c)

(e)

(f)

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

usin

g1%

TFA

in30%

AC

Nw

ith

(a)

no

additiv

es,

(b)

20

mM

sodiu

mfluoride,(c

)2

mM

okadaic

acid

,(d

)2

mM

sodiu

mm

oly

bdate

,(e

)10

mM

sodiu

mb

-gly

cero

phosphate

,or

(f)

2m

Msodiu

mort

hovanadate

.P

eptides

were

elu

ted

with

0.4

M

NH

4O

Hin

30%

AC

N.

Copyright # 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd. Rapid Commun. Mass S


m/z

pectrom. 2010; 24: 219–231

DOI: 10.1002/rcm


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


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


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.


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.


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


DOI: 10.1002/rcm


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.


Funding for protein mass spectrometry equipment was pro-

vided by the Saskatchewan Provincial Government and the

National Research Council of Canada, fromwhich this article

is contribution number 50149.

REFERENCES

1. Ahn NG, Resing KA. Nat. Biotechnol. 2001; 19: 317.2. McLachlin DT, Chait BT. Curr. Opin. Chem. Biol. 2001; 5: 591.3. MazanekM,Mituloviæ G, Herzog F, Stingl C, Hutchins JRA,

Peters J-M, Mechtler K. Nat. Protocols 2007; 2: 1059.4. Anderson L, Porath JO. Anal. Biochem. 1986; 154: 250.5. Kokubu M, Ishihama Y, Sato T, Nagasu T, Oda Y. Anal.

Chem. 2005; 77: 5144.6. Posewitz MC, Tempst P. Anal. Chem. 1999; 71: 2883.7. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross

MM, Shabanowitz J, Hunt DF, White FM. Nat. Biotechnol.2002; 20: 301.

8. Pinske MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ. Anal.Chem. 2004; 76: 3935.

9. Larsen MR, Thingholm TE, Jensen ON, Roepstroff P, Jør-gensen TJD. Mol. Cell. Proteomics 2005; 4: 873.

10. Connor PA, McQuillan AJ. Langmuir 1999; 15: 2916.11. Dobson KD,McQuillan AJ.Acta Part AMol. Biomol. Spectrosc.

2000; 56: 557.12. SugiyamaN,Masuda T, Shinoda K, Nakamura A, TomitaM,

Ishihama Y. Mol. Cell. Proteomics 2007; 6: 1103.13. Thingholm TE, Jorgensen TJD, Jensen ON, Larsen MR. Nat.

Protocols 2006; 1: 1929.14. Jensen SS, Larsen MR. Rapid Commun. Mass Spectrom. 2007;

21: 3635.15. Imanishi SY, Kochin V, Eriksson JE. Proteomics 2007; 7: 174.16. Cantin GT, Shock TR, Park SK, Madhani HD, Yates JR III.

Anal. Chem. 2007; 79: 4666.17. Rappsilber J, Ishihama Y, MannM. Anal. Chem. 2003; 75: 663.18. Klemm C, Otto S, Wolf C, Haseloff RF, Beyermann M,

Krause E. J. Mass Spectrom. 2006; 41: 1623.19. Thingholm TE, Jensen ON, Robinson PJ, Larsen MR. Mol.

Cell. Proteomics 2008; 7: 661.20. Hart SR, Waterfield MD, Burlingame AL, Cramer R. J. Am.

Soc. Mass Spectrom. 2002; 13: 1042.21. Stensballe A, Jensen ON. Rapid Commun. Mass Spectrom.

2004; 18: 1721.22. Kocher T, Allmaier G, Wilm M. J. Mass Spectrom. 2003; 38:

131.23. Aryal UK, Olson DJH, Ross ARS. J. Biomol. Techniques 2008;

19: 296–310.24. Kweon HK, Hakansson K. Anal. Chem. 2006; 78: 1743.25. Ross ARS. Methods Enzymol. 2007; 423: 549–572.26. Tani K, Ozawa M. J. Liq. Chrom. Rel. Technol. 1999; 22: 843.27. Tunesi S, Anderson M. J. Phys. Chem. 1991; 95: 3399.28. Tani K, Miyamoto E. J. Liq. Chrom. Rel. Technol. 1999; 22:

857.29. Tani K, Kitada M, Tachibana M, Koizumi H, Kiba T. Chro-

matographia 2003; 57: 409.30. Kim ST, Cho KS, Jang YS, Kang KY. Electrophoresis 2001; 22:

2103.31. Xi J, Wang X, Li S, Zhou X, Yue L, Fan J, Hao D. Phytochem-

istry 2006; 67: 2341.32. Kwon SJ, Choi EY, Seo JB, Park OK. Mol. Cells 2007; 24: 268.33. Kim SG, Kim ST, Kang SY, Wang Y, Kim W, Kang KY. Plant

Cell Rep. 2008; 27: 363.34. Imanishi SY, Kochin V, Ferraris SE, de Thonel A, Pallari H-

M, Corthals GL, Eriksson JE. Mol. Cell. Proteomics 2007; 6:1380.

35. Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faerge-man NJ, Mann M, Jensen ON. Mol. Cell. Proteomics 2005; 4:310.

36. Bjellqvist B, Basse B, Olsen E, Celis JE. Electrophoresis 1994;15: 529.

37. Bull HB, Breese K. Arch. Biochem. Biophys. 1974; 161: 665.38. Hsieh HC, Sheu C, Shi FK, Li DT. J. Chromatogr. A 2007; 1165:

128.


DOI: 10.1002/rcm

enrichment and analysis of phosphopeptides under

Documents

john wiley

ion afnity chromatography

100in ten sity

achieve optimum performance

ensure proper loading

multiply phosphorylated peptides

supplementary table s1

supplementary table s2