Robust Phosphoproteome Enrichment Using Monodisperse Microsphere

protocol
Robust phosphoproteome enrichment using

monodisperse microspherebased immobilized
titanium (IV) ion affinity chromatography
Houjiang Zhou15, Mingliang Ye2,5, Jing Dong2, Eleonora Corradini1,3, Alba Cristobal1,3, Albert J R Heck1,3,
Hanfa Zou2 & Shabaz Mohammed1,3
1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht,
The Netherlands. 2Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Dalian, China. 3Netherlands Proteomics Centre, Utrecht, The Netherlands. 4Present address: Department of Biochemistry, University of Cambridge,
Cambridge, UK. 5These authors contributed equally to this work. Correspondence should be addressed to A.J.R.H. (a.j.r.heck@uu.nl), H.Z. (hanfazou@dicp.ac.cn) or
S.M. (s.mohammed@uu.nl).
2013 Nature America, Inc. All rights reserved.
Published online 7 February 2013; doi:10.1038/nprot.2013.010
Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be
successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because
of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative
materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microspherebased
immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups
that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides,
with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO2). Here
we present a detailed protocol for the preparation of monodisperse microspherebased Ti4+-IMAC adsorbents and the subsequent
enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples
before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation
of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed.
Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols
provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ~2 weeks to
complete the protocol: 1 week to prepare the Ti4+-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched
sample and 2 d for data analysis.
INTRODUCTION
Protein phosphorylation by kinases and dephosphorylation by
phosphatases are key tools for signaling in cellular networks1,2.
Protein phosphorylation has been estimated to affect ~30% of
a proteome and represents a major regulatory machinery for
many cellular processes3. A fundamental understanding of biological processes and signaling networks at the molecular level
requires the detailed analysis of the phosphorylated proteins being
involved. However, the dynamic nature of signaling networks, the
complexity of the phosphoproteome and the low stoichiometry
of protein phosphorylation pose a serious technical challenge4.
Nowadays, high-throughput protein identification is predominantly performed by MS5. However, this approach often fails to
identify phosphopeptides in the complex peptide mixtures generated by protein digests. Therefore, it has become a crucial step to
specifically isolate subsets of phosphopeptides from a complex
peptide mixture6,7.
To date, a number of approaches have been developed.
Phosphopeptides can be enriched by immunoprecipitation; however, the strategy has only been proven to be highly applicable
to peptides containing phosphotyrosine8,9. Another approach
is to perform chemical coupling, in which the phosphopeptides
are either covalently conjugated to a polymer support and then
released or covalently attached with an affinity tag, followed by
affinity purification10. Currently, the most common strategies
for global comprehensive enrichment involve chromatography.
Strong cation exchange (SCX) chromatography performed at a

low pH has proven to be very popular11. Under these conditions,
peptides are fractionated on the basis of their solution net charge
and the orientation of peptides to the negatively charged chromatographic material. Unlike glutamic and aspartic acid, phosphorylated amino acids are able to retain a negative charge under acidic
(pH 2.7) conditions. This property can be exploited in SCX for the
enrichment of phosphopeptides, which tend to elute earlier and are
thus separated from the majority of nonphosphopeptides12.
Chelation/coordination chemistry was, however, the first truly
successful strategy for phosphopeptide enrichment. It has continually developed over the past few decades and continues to be the
most popular strategy. The general principle involves an immobilized metal oxide or metal ion, which is capable of coordination and, specifically, has a high preference for phosphate groups.
IMAC with Fe3+ or Ga3+ is one of the most-used enrichment
methods for phosphopeptides13,14. The selectivity of conventional
IMAC toward phosphopeptides is based on the high affinity of the
phosphate groups with the metal ions (Fe3+ or Ga3+) bound to
adsorbents through iminodiacetic acid (IDA)13 or nitrilotriacetic
acid (NTA)15 chelating ligand. One of the limitations associated
with the conventional IMAC-based phosphopeptide enrichment
is the nonspecific adsorption resulting from nonphosphorylated
peptides containing multiple acidic amino acids (glutamate
and aspartate)15. To improve the specificity of phosphopeptide
nature protocols | VOL.8 NO.3 | 2013 | 461
protocol
enrichment, an approach that blocks the acidic residues (carboxylic groups of the peptide) using methyl esterification was developed before enrichment16. Metal oxide affinity chromatography
(MOAC6,1720, typically TiO2 (ref. 17)) has become an effective
alternative to IMAC for the enrichment of phosphopeptides. Much
higher selectivity can be achieved using a multifunctional acid
such as 2,5-dihydroxybenzoic acid (DHB)21, lactic acid22 or glycolic acid23 in the loading buffer. However, one weakness common
to MOAC and classical strategies is their poor binding to phosphopeptides that contain multiple basic residues2427.
Metal (IV) phosphate/phosphonate chemistry has emerged
over the past three decades. The coordination of phosphate/
phosphonate group to metal (IV), especially for Zr4+, has now
been extensively illustrated in the literature28,29. Nakayama et al.30
also revealed the coordination of Ti4+ with phosphate groups similar to Zr4+ with phosphate groups using31P NMR. In this case, the
MO6 are octahedrally coordinated by oxygen atoms, with the three
oxygens of each phosphonate bound to three different zirconium
ions31. Notably, the unique coordination of Zr4+ or Ti4+ with the
phosphate/phosphonate group has been shown to have a richness
of applications, including DNA microarrays32, protein microarrays33 and self-assembled monolayers34. We recently developed
a new generation of IMAC for phosphopeptide enrichment3539
by using the unique property of Zr(IV) or Ti(IV) phosphate/
phosphonate chemistry. Figure 1 shows the architecture of the
Ti4+-IMAC adsorbent and the practical principle of phosphopeptide enrichment enabled by Ti4+-IMAC. To improve the specificity
of phosphopeptide enrichment and make it such that there is less
bias toward different types of phosphopeptides such as basophilic
kinase substrates, we have made a few key modifications for Ti4+IMAC technology40,41. The first of these is the use of monodisperse microspheres. Monodisperse microspheres have a uniform
monodisperse size distribution, uniform column packing, uniform flow profile, low column pressure, high column efficiency
and excellent stability in the field of separation sciences42,43. The
monodisperse microspheres we prepared40 not only have stable
chemical-physical properties, such as durability toward strong acid
and alkaline buffer, but also contain a large surface area because of
abundant mesopores in the microspheres and have a hydrophilic
surface that minimizes nonspecific adsorption. Second, a flexible
linker (poly(GMA-co-TMPTMA), where GMA is glycidyl methacrylate and TMPTMA is trimethylol-propane trimethacrylate) is
introduced to increase the spatial distance between the active Ti4+
and the matrix (polystyrene microspheres)40,41. The flexible linker
provides a beneficial spatial orientation for the phosphopeptide
binding by reducing the steric hindrance, which is caused by the
matrix and the bound phosphopeptides. The amino groups in
the linker further improve the hydrophilicity of monodisperse
microspheres. Third, as opposed to commercial IMAC materials
in which the chelating ligands IDA13 or NTA15 for Fe3+/Ga3+IMAC are used, we coupled phosphonate groups for chelation
and immobilization of Ti4+ via coordination between Ti4+ and
the P-O bond. Such immobilization also creates a beneficial structural orientation for the selective binding of phosphopeptides.
We recently showed that the developed Ti4+-IMAC protocol is
capable of enriching phosphopeptides containing multiple basic
residues41. Given the inherent complexity of cellular proteome,
prefractionation is prerequisite to improve the coverage of cellular phosphoproteome. For example, the combination of SCX
462 | VOL.8 NO.3 | 2013 | nature protocols
O
P
O
O
O
O
Ti
O
O
Ti4+
4+
O
P
Phosphopeptide
enrichment
Ti4+
O
O
O
P
Chelating ligand
Flexible linker
Microspheres matrix
Figure 1 | Depiction of the architecture of the Ti4+-IMAC adsorbent and

the principle of phosphopeptide binding with Ti4+-IMAC. The monodisperse
microsphere is used for the preparation of Ti4+-IMAC. The phosphonate
groupterminated monodisperse microspheres are coordinated to titanium
ions; each titanium ion bridges two phosphate oxygen bonds from two
phosphonate groups. The flexible linker between the phosphonate group
and the monodisperse microsphere matrix provides a spatial space, which
enables the reduction of binding steric hindrance. The free coordination site
of titanium ions can be used for phosphopeptide binding.
and IMAC or MOAC has been proven to be an excellent strategy

for phosphoproteome analysis12,4451.
Here we present a detailed protocol for the preparation of
Ti4+-IMAC adsorbents and the subsequent enrichment process.
Furthermore, we discuss the general pitfalls and crucial steps in the
preparation of phosphoproteomic samples before enrichment and,
just as importantly, the subsequent mass spectrometric analysis.
The key points such as lysis, preparation of the chromatographic
system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization are briefly discussed. Finally, we
demonstrate how the protocols provided are appropriate for the
screening of entire phosphoproteomes.
Experimental design
Preparation of immobilized titanium (IV) ion IMAC adsorbents
(Ti4+-IMAC). In this new generation of IMAC, the metal ion, i.e.,
Ti4+, is immobilized onto the solid matrix by using the phosphonate group as the chelating ligand. In principle, any matrix with the
surface modified with phosphonate groups can be used to prepare
Ti4+-IMAC adsorbents. However, to achieve better performance,
the selection of matrix is crucial. To minimize the nonspecific
adsorption, the surface of the matrix should be highly hydrophilic.
As sample loading and elution are performed at extreme pH, the
matrix should have sufficient chemical stability. To ensure fast and
efficient capture and release of phosphopeptides, a matrix with
macroporous structure is preferable. Compared with a matrix of
irregular shape, the microspheres with uniform size are beneficial
and lead to an improvement of the enrichment performance. This
is especially true when the enrichment is performed in column
format. Here we provide a protocol to synthesize a tailored matrix
to prepare Ti4+-IMAC adsorbents (Fig. 2). The porous monodisperse microspheres were prepared by a swelling and polymerization
method similar to the classic method developed in 1980 by Ugelstad
et al.52. First, the monodisperse polystyrene seed microspheres
protocol
a
CH3CH2OH; 70 C, 24 h
Dispersion polymerization
O
O
O
O
Polystyrene
monodisperse
microspheres
Polystyrene monodisperse microspheres
O
O
AIBN
O
n 70 C, 24 h
GMA
m
TMPTMA
O
Poly (GMA-co-TMPTMA)
H2N
NH2
OH
80 C, 3 h
OH
Poly (GMA-co-TMPTMA)
d
OH
OH
N
H
N
H
OH
N
H
N
H
NH2
NH2
Poly (GMA-co-TMPTMA-NH2)
NH2
H3PO4, HCHO
NH2
OH
OH
H
N
H
N
H
N
N
H
H , 100 C, 24 h
Poly (GMA-co-TMPTMA-NH2)
N
H
P O
O
O
P O
O
H
N
N
H
Poly (GMA-co-TMPTMA-PO3H2)
O
P O
O
O
P O
TiCl4, H
RT, 8 h
OH
N
H
H
N
H
N
P O
O
Ti 4+
O
P O
O
profiling. Each step of sample preparation,

including cell lysis, protein digestion and
sample cleanup, must be considered for
optimal performance. Phosphatase inhibitors are required in the lysis buffer in order
to inhibit the activity of phosphatases so
that they stay as close to the original state of
the proteins as possible. Although trypsin
is the most common enzyme for digestion,
other proteases may be a better choice if
there is a need to locate specific phosphorylation sites53 or to improve the phosphoproteome coverage54,55 in general. As
the phosphopeptide enrichment occurs at
the peptide level, the described protocol is
compatible with virtually any protease (e.g.,
trypsin, Lys-C, Lys-N, Arg-C and Glu-C).
Although sample fractionation protocols
are not provided here, the protocol is compatible with a number of chromatographic
techniques, including high-pH reversephase56, SCX41 and hydrophilic interaction
liquid chromatography (HILIC)57.
Phosphopeptide enrichment. The phosphopeptide enrichment is performed with

4+
Poly (GMA-co-TMPTMA-PO3H2)
Ti -IMAC
Ti4+-IMAC material loaded in GELoader
4+
spin tips in which liquid manipulation is
|
Figure 2 The scheme of the preparation of Ti -IMAC adsorbents. (a) Synthesis of monodisperse
performed with centrifugation. In this way,
polystyrene microspheres. (b) Synthesis of monodisperse poly(GMA-co-TMPTMA) microspheres.
(c) Amination of monodisperse poly(GMA-co-TMPTMA) microspheres. (d) Coupling of phosphonate
an easy-to-use, clean, reproducible and
groups onto the monodisperse microspheres. (e) Immobilization of titanium ion (IV) on the
time-saving enrichment can be performed.
phosphonate groupfunctionalized monodisperse microspheres.
Depending on the amount of sample, the
Ti4+-IMAC spin tip can also be scaled.
In the cases outlined in this protocol, we
found that the tip capacity corresponded to
are synthesized by dispersion polymerization. Monodisperse
~200 g of nonstimulated K562 cell lysate digest per column,
poly(GMA-co-TMPTMA) microspheres are then prepared using a although this number will change depending on the level of phosseed-swelling suspension polymerization system using polystyrene
phorylation expected in the sample. The efficiency of the enrichmicrospheres as seeds. After polymerization, hydrophilic porous ment of phosphorylated peptides by Ti4+-IMAC can be tested using
monodisperse microspheres are prepared (Fig. 3). The size of the a semicomplex sample originating from tryptic digests of standard
microspheres is increased from the ~4 m of seed microspheres phosphoprotein (-casein and -casein) and nonphosphoprotein
to ~12 m (Fig. 3). To chelate with Ti4+, the resulting poly(GMA- (BSA) or commercial standard phosphopeptides. We recommend that
co-TMPTMA) microspheres are modified with phosphonate
such an experiment be performed in parallel as a control. To achieve
groups. This is achieved by reaction with the epoxide groups on optimal enrichment, a few key points have to be addressed. During
the microspheres as shown in Figure 2c,d. After this reaction, the
sample loading, limiting spinning speed and using a relatively low
phosphonate groups are coupled to the microspheres with a space sample concentration (~1 g l1) are favorable for complete bindarm of nine atoms, which facilitate the accessibility of the phos- ing of phosphopeptides. A high level of trifluoroacetic acid (TFA, 6%
phopeptides. Finally, Ti4+ is immobilized on the monodisperse
(vol/vol)) is used for the sample loading. TFA can effectively protonate
microspheres via chelation with the phosphonate groups; each Ti4+ the acidic residues (aspartic and glutamic acid) and thereby inhibit
ion bridges two phosphate oxygen atoms from two phosphonate
the nonspecific binding of acidic peptides. Longer peptides resulting
groups present on the monodisperse microspheres30,34. The Ti4+- from cleavage sites missed during trypsin digestions are often found
IMAC adsorbent prepared by this way has excellent performance during digestion of any sample. These missed cleavages are more frein phosphopeptide enrichment, as demonstrated below.
quent for phosphopeptides55, as trypsin cannot efficiently access the
cleavage site because there is an intramolecular bond between the
Preparation of control and experimental samples for analysis. positively charged basic residue and the negatively charged phosphate
Sample preparation is a crucial step for the analysis of protein group. We have shown that TFA can reduce the level of intramolecular
phosphorylation. Because of the low abundance and low stoichi- binding and increase enrichment of such phosphopeptides41.
ometry of protein phosphorylation, it is recommended to start with
To remove the nonspecific ionic adsorption, we chose to use 200 mM
at least a few milligrams of protein for large-scale phosphorylation NaCl in one of the washing buffers. The elution is performed by
OH
N
H
OH
N
H
protocol
a
Polystyrene monodisperse
microspheres
20 kV 7,000 2 m
Poly(GMA-co-TMPTA-PO3H2)
monodisperse microspheres
20 kV 7,000 2 m
Poly(GMA-co-TMPTA) monodisperse
microspheres
20 kV 7,000 2 m
4+
Ti -IMAC adsorbents
20 kV 7,000 2 m
Figure 3 | Characterization of the synthesized products by scanning electron

microscopy. The scanning electron microscope imaging can be used to check
whether the product is successfully prepared as desired. (a) Monodisperse
polystyrene seed microspheres. (b) Poly(GMA-co-TMPTA) monodisperse
microspheres. (c) Poly(GMA-co-TMPTA-PO3H2) monodisperse microspheres.
(d) Ti4+-IMAC adsorbents. Magnification is fixed at 7,000.
using a very basic solution (10% (vol/vol) NH3H2O), which we

have found to be excellent for the recovery of bound material. In
such high basic conditions, the phosphate group can be lost from
the peptide, and therefore it is crucial that the eluent be acidified
immediately. We suggest adding a volume of 10% (vol/vol) formic
acid (FA) that is equal to the volume of the base added to neutralize
the solution. In terms of MS analysis, it has been found that running
technical duplicates can increase the number of phosphopeptides
identified by another 3040% (refs. 58,59; also see ANTICIPATED
RESULTS for further details). Although SCX fractionation is used
for reducing the sample complexity, we also recommend using
a longer gradient (3 h) for the enriched phosphopeptides from
the abundant SCX fractions and short gradient (90 min) for the
enriched phosphopeptides from the less-abundant fractions. In
addition, experimental replicates and label swapping are recommended for quantitative studies that link phosphorylation dynamics to biological function in order to confirm any observed changes
in phosphorylation.
Considerations for sample preparation relating to quantification. The Ti4+-IMAC is also applicable to different quantitative
samples such as stable isotope labeling by amino acids in cell
culture (SILAC)60, isotope-coded affinity tags (ICAT)61, isobaric
tags for relative and absolute quantification (iTRAQ)62, isobaric
tandem mass tags (TMT)63 or dimethyl labeling64. To obtain a
successful outcome for a quantitative phosphoproteome experiment using Ti4+-IMAC, we recommend some key evaluations for
each sample preparation stage before running the real sample of
interest. First, we recommend checking the sample labeling efficiency by analyzing a minute digested sample, 1 g, via a 3-h MS
analysis. Second, it is advisable to check the basal phosphorylation
context of the sample of interest using Ti4+-IMAC enrichment
alone. To do this, a small amount of biological sample, such as
a few hundred micrograms, is subjected to Ti4+-IMAC without
any fractionation, and then the enriched sample is analyzed by
performing a 3-h MS analysis. For example, about 3,000 phosphopeptides can be identified from unstimulated 100 g of human
cell lysate (in our case, HeLa and K562 cells, see ANTICIPATED
RESULTS). Third, in order to reduce the quantification variations
of protein bicinchoninic (BCA) assay or Bradford assay, we recommend analyzing (by MS) a small equal amount of the pooled
labeled sample before fractionation. On the basis of this prequantification, equal mixing of each labeled sample can be correctly
performed before fractionation. Fourth, we also recommend using
standard phosphoprotein digests such as -casein to check the
chromatography system.
Evaluation of the liquid chromatography-tandem MS (LC-MS/
MS) system. The performance of the LC-MS/MS system has to be
evaluated to determine whether it can be applied to phosphopeptide
analysis and whether it can support large-scale phosphoproteomic
analysis in terms of LC separation and MS. Most LC systems contain metal components, which can (and do) adsorb phosphopeptides. Phosphopeptide losses can be minimized by performing
Steps 4752 in the PROCEDURE. With regard to the performance
of the LC-MS system, the phosphoproteome is as complex as the
proteome and benefits greatly from improvements in chromatographic separations. The choice of the MS/MS fragmentation
method(s) can also affect phosphopeptide identification and site
localization. Certain fragmentation regimes are more appropriate
than others, but it depends on the sequence of the peptide65. For
example, electron-transfer/capture dissociation is most appropriate for peptides containing three charges66,67 or more, whereas all
modes of collision-induced dissociation perform sufficiently for
peptides containing two charges68,69.
Determination of phosphorylation sites. Pinpointing the exact
phosphorylation site on the basis of the achieved mass spectra is
important for biological follow-up and for understanding relevant
biological function. To confidently localize the site of phosphorylation, specific diagnostic backbone fragments (also site-determining
ions) must be present. Several software tools such as Ascore70, PTM
score47, Mascot delta ion score71 and phosphoRS72 can be used to
automatically process fragmentation spectra of phosphorylated
peptides and localize phosphorylation site. All these algorithms use
MS/MS data in conjunction with the respective peptide sequences
to calculate site probabilities for all potential phosphorylation
sites. In our case, we use phosphoRS (freely available from http://
cores.imp.ac.at/protein-chemistry/download/), which handles all
commonly used fragmentation methods (collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD) and
electron-transfer dissociation (ETD)) and data sets with high or
low mass accuracy.
protocol
MATERIALS
REAGENTS
CRITICAL We recommended preparing all the Ti4+-IMAC buffers before
enrichment and using freshly prepared Ti4+-IMAC buffer for phosphopeptide
enrichment.
Styrene (Alfa Aesar, cat. no. A18481)
Polyvinyl alcohol (PVA; Alfa Aesar, cat. no. 41239)
Polyvinylpyrrolidone (Alfa Aesar, cat. no. A14315)
TMPTMA (Sigma-Aldrich, cat. no. 246840)
GMA (Sigma-Aldrich, cat. no. 151238)
Triton X-100 (98% (vol/vol), for molecular biology, DNase, RNase and
protease free; Acros Organics, cat. no. 327371000)
2, 2-Azobis(2-methylpropanenitrile) (AIBN; Aladdin Reagent,
cat. no. 1138733)
SDS (Aladdin Reagent, cat. no. 1098983)
Toluene (Aladdin Reagent, Chemically pure)
Ethylenediamine anhydrous (Aladdin Reagent, cat. no. 1098404)
Phosphorous acid (Aladdin Reagent, cat. no. 1099867)
Tetrahydrofuran (Aladdin Reagent, cat. no. 1095496)
Hydrochloric acid (HCl; Aladdin Reagent, cat. no. 1042074)
Formaldehyde (Aladdin Reagent, cat. no. 1095084)
Ethanol (Aladdin Reagent, cat. no. 1095113)
Methanol (Biosolve, cat. no. 13680602)
Acetonitrile (Biosolve, cat. no. 012007)
Acetic acid (Merck, cat. no. 1.00063)
TFA (Thermo Scientific, Pierce, cat. no. TS-28904) ! CAUTION TFA
solutions and TFA vapors are toxic; prepare solutions in a fume hood.
FA (Fluka, cat. no. 94318)
High-purity water obtained from a Milli-Q purification system (Millipore)
Urea (Merck, cat. no. 66612)
Ammonium bicarbonate (NH4HCO3; Fluka, cat. no. 09830)
Complete mini EDTA-free cocktail (Roche, cat. no. 11.836.170.001)
PhosphoSTOP phosphatase inhibitor cocktail (Roche,
cat. no. 04.906.845.001)
Sodium orthovanadate (Sigma-Aldrich, cat. no. S6508)
dl-dithothreitol (DTT; Sigma-Aldrich, cat. no. 43815)
Iodoacetamide (IAA; Sigma-Aldrich, cat. no. I6125)
Trypsin (Promega, cat. no. V528A)
Lysyl endopeptidase (Lys-C), MS grade (Wako Chemicals,
cat. no. 129-02541)
BSA (Sigma-Aldrich, cat. no. A2153)
-Casein (Sigma-Aldrich, cat. no. C6780)
-Casein (Sigma-Aldrich, cat. no. C6905)
Ortho-phosphoric acid (Fisher Chemical, cat. no. O/0450/PB08)
Titanium (IV) chloride solution (TiCl4, 0.09 M in 20% (vol/vol) HCl;
Sigma-Aldrich, cat. no. 404985)
Ammonia solution (NH3H2O, 25%; Merck, cat. no. 105432)
Potassium dihydrogen phosphate (KH2PO4; Sigma, cat. no. P5655)
Sodium chloride (NaCl; Sigma, cat. no. S9888)
Sep-Pak solvents (see Reagent Setup)
RP-HPLC solvents (see Reagent Setup)
Ti4+-IMAC buffers (see Reagent Setup)
PBS
EQUIPMENT
Vortex (VWR)
Eppendorf centrifuge 5417R (Eppendorf)
Milli-Q purification system (Millipore)
Sep-Pak C18 cartridges (Waters)
Lyophilizer (Thermo Scientific)
SpeedVac (Thermo Scientific)
Autoflex matrix-assisted laser desorption/ionizationtime of flight mass
spectrometry (MALDI-TOF MS; Bruker)
LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) equipped with
a nanoHPLC system (Agilent)
Q-Exactive quadrupole Orbitrap mass spectrometer (Thermo Scientific)
equipped with an EASY-nLC 1000 system (Thermo Scientific)
Proteome Discoverer software package (Thermo Scientific)
Three-necked round-bottom flasks
Centrifuge tubes
Conical tubes
Kimtech wipes
REAGENT SETUP
Lysis buffer Mix 8 M urea, 50 mM NH4HCO3, 1 mM sodium orthovanadate, 1
tablet of complete mini EDTA-free cocktail and 1 tablet of phosphoSTOP phosphatase inhibitor cocktail per 10 ml of lysis buffer. CRITICAL It is
recommended to prepare all the reagents fresh and to add phosphatase
inhibitor and protease inhibitor tablets just before use. Keep the lysis buffer on ice.
Quality control (QC) sample 1 Use a complex mixture of E. coli digests
(50 ng) for benchmarking test.
QC sample 2 Use a standard protein digest (referred to as protmix)
consisting of 50 fmol of BSA, 50 fmol of -casein and 50 fmol of -casein for
the evaluation of the system for phosphopeptide analysis.
Sample to analyze Use the enriched sample by Ti4+-IMAC from Step 42
directly for LC-MS/MS analysis in order to reduce the sample loss during
vacuum dryness. We recommend using at least 2 mg of protein for large-scale
phosphoproteomic experiments.
Sep-Pak solvents Loading buffer: 2% (vol/vol) acetic acid; washing buffer 1:
0.6% (vol/vol) acetic acid; elution buffer: 80% (vol/vol) acetonitrile and
0.6% (vol/vol) acetic acid. Sep-Pak solvents are freshly prepared.
RP-HPLC solvents Solvent A: 0.6% (vol/vol) acetic acid; solvent B: 80%
(vol/vol) acetonitrile and 0.6% (vol/vol) acetic acid. RP-HPLC solvents are
freshly prepared.
Ti4+-IMAC buffers Loading buffer: 80% (vol/vol) acetonitrile and 6%
(vol/vol) TFA; washing buffer 1: 50% (vol/vol) acetonitrile, 0.5% (vol/vol)
TFA containing 200 mM NaCl; washing buffer 2: 50% (vol/vol) acetonitrile
and 0.1% (vol/vol) TFA; elution buffer 1: 10% (vol/vol) NH3H2O, pH 11.0;
elution buffer 2: 80% (vol/vol) acetonitrile and 2% (vol/vol) FA. All buffers are
freshly prepared. CRITICAL Elution buffer 2 is used to recover the bound
phosphopeptides by C8 plug. Elution buffer 2 will further acidify the eluent and
avoid the removal of phosphates by the alkaline components. CRITICAL To
specifically enrich phosphopeptides from complex digests, it is recommended to
use a high concentration of TFA in loading buffer to protonate the acidic amino
residues and break down the ion binding between the positive amino residues
and the negative phosphate groups. The intrapeptide binding often prevents the
enrichment of phosphopeptide containing multiple basic residues24.
EQUIPMENT SETUP
MS analysis MALDI-TOF MS can be used to characterize the enriched sample
from a semicomplex sample. In our study, MALDI analysis was performed
on a Bruker Autoflex time-of-flight mass spectrometer. The instrument was
equipped with a delayed ion-extraction device and a pulsed nitrogen laser operated at 337 nm, and its available accelerating potential was in the range of 20 kV.
The MALDI uses a ground-steel sample target with 384 spots. The range of laser
energy was adjusted to slightly above the threshold in order to obtain good resolution and good signal-to-noise ratio. We obtained all mass spectra reported
in the positive-ion linear mode with delayed extraction for 50 ns. We obtained
external mass calibration by using two points that bracketed the mass range of
interest. Each mass spectrum was typically summed with 50 laser shots.
LC-MS/MS is used to analyze enriched samples of higher complexity such as
those originating from mammalian cells. For the LC system, it is preferable to use
a system that contains a trap column so as to allow rapid loading and desalting of
large sample volumes. In our case, an Agilent 1100 HPLC system is connected to
the LTQ-Orbitrap Velos mass spectrometer and is equipped with a 100 m 20 mm,
3 m, 120 Reprosil-Pur C18 (Dr Maisch) trapping column and a 50 m
400 mm, 3 m, 120 Reprosil-Pur C18 analytical column, using a vented column
configuration73. Trapping is performed at 5 l min1 for 10 min with RP solvent
A, whereas gradient elution is performed at a column flow rate of ~100 nl min1.
The column effluent is directly introduced into the electrospray ionization (ESI)
source of the MS. All fragmentation techniques (HCD, CID and ETD) are enabled
on the LTQ-Orbitrap Velos. In some cases, we also used a nano-UHPLC Proxeon
Easy-nLC 1000 (Thermo Scientific) connected to an LTQ-Orbitrap Q-Exactive
mass spectrometer. The injected sample was first trapped on a trapping column
(Dr Maisch Reprosil C18, 3 m, 2 cm 100 m) before being separated in an
analytical column (Agilent Zorbax SB-C18, 1.8 m, 35 cm 50 m). Peptides are
chromatographically separated by using a gradient of 60 min, 90 min, 120 min
and 180 min at a column flow rate of ~100 nl min1, respectively . The column
effluent is directly introduced into the ESI source of the MS; HCD fragmentation
is used on the Q-Exactive platform.
protocol
PROCEDURE
Preparation of polystyrene monodisperse microspheres TIMING ~1.5 d
1| Prepare a solution of Triton X-100 (1.14 ml) in ethanol (64 ml) in a 100-ml three-necked round-bottom flask and add
polyvinylpyrrolidone (1.25 g).
CRITICAL STEP Triton X-100 is viscous, so pipette it slowly. Pipette Triton X-100 into the flask containing 64 ml of
ethanol. Pipette the solution several times to ensure that all of the Triton X-100 is washed out of the pipette.
2| Stir the resulting mixture at 100 r.p.m. for about 5 min until the solid is dissolved completely.
3| Dissolve AIBN (0.58 g) into 16 ml of styrene monomer and slowly add the mixture into the above reaction mixture.
! CAUTION Styrene is toxic and volatile. The manipulations should be carried out in a ventilated fume hood.
CRITICAL STEP The mixture of AIBN (0.58 g) and 16 ml of styrene monomer is added into the mixture prepared in Step 2
in 30 min with a constant pressure funnel.
4| Heat the mixture gently in an oil bath maintained at a temperature of 70 C, and stir it gently for 24 h. During this
period, the transparent mixture turns into a milk-like suspension, which indicates that polymerization occurred.
The schematic for the reaction is given in Figure 2a.
CRITICAL STEP Stir the mixture constantly before and during polymerization to ensure that the mixture is homogenous.
This is crucial to prepare monodisperse seed microspheres.
? TROUBLESHOOTING
5| After 24 h of polymerization, transfer this suspension into 50-ml centrifuge tubes. After the centrifugation for 5 min at
10,000g at room temperature (RT, 22 C), the desired polystyrene seed microspheres are obtained as a pellet.
6| Wash the product with 20 ml of ethanol for each tube. Repeat this step twice.
! CAUTION There is a strong irritant smell. The manipulation should be conducted in a ventilated fume hood using proper
nitrile gloves.
CRITICAL STEP Extensive washing using ethanol can efficiently remove all the residual reactants and additives from the
products.
7| Dry down the washed product in a vacuum oven at 100 C to obtain about 11.1 g of white powder. The size of the
polystyrene monodisperse microsphere is about 4 m (Fig. 3a).
CRITICAL STEP At this point, the product can be checked by scanning electron microscopy. The prepared polystyrene
monodisperse microspheres should show property of monodispersity in size of ~4 m.
PAUSE POINT The dried polystyrene seed microsphere can be placed into a sealed tube with a cap and kept at RT for
future use.
Preparation of poly(GMA-co-TMPTMA) monodisperse microspheres TIMING ~2 d
8| Prepare a 200-ml aqueous solution containing 1% (wt/wt) PVA and 0.25% (wt/wt) SDS.
CRITICAL STEP Heating and stirring make it easier to dissolve PVA.
9| Take 15 ml of the solution prepared in Step 8 and add 0.45 g of dried polystyrene seed microspheres.
10| Sonicate the solution for 6 s at 300 W using 60 cycles with 50% duty cycle and transfer the resulting suspension to a
250-ml three-necked round-bottom flask.
11| Prepare an oil-phase solution containing all reagents for polymerization by mixing 6.7 ml of GMA, 6.7 ml of TMPTMA,
0.14 g of AIBN and 16.6 ml of toluene.
! CAUTION GMA, TMPTMA and toluene are toxic and volatile. Work in a ventilated fume hood.
12| Add the prepared oil-phase solution into 150 ml of aqueous solution containing 1% (wt/wt) PVA and 0.25% (wt/wt)
SDS. Sonicate the resulting two-phase mixture for 9 s at 300 W using 90 cycles with a 30% duty cycle to give a
milk-like emulsion.
CRITICAL STEP Ensure that the mixtures of oil-phase and aqueous solutions are completely emulsified. The resulting
homogenous emulsion should consist of micrometer-sized droplets, which could be checked under a microscope. If this is not
achieved, polydisperse microspheres will be synthesized.
protocol
13| Add the oil emulsion to the aqueous polystyrene seed microsphere solution under mechanical stirring at 150 r.p.m. in
the 250-ml three-necked round-bottom flask.
CRITICAL STEP The oil emulsion should be added to the aqueous polystyrene seed microsphere suspension solution.
The reverse will lead to bulk polymerization.
14| To allow the polystyrene seed microspheres to swell in the emulsion, maintain the temperature of the oil bath at 30 C
for 20 h under mechanical stirring at 150 r.p.m.
15| Increase the temperature to 70 C to initiate polymerization. The polymerization is carried out for 24 h, resulting in the
formation of poly(GMA-co-TMPTMA) microspheres. The schematic for the reaction is given in Figure 2b.
? TROUBLESHOOTING
16| Transfer the prepared microspheres to 50-ml centrifuge tubes and wash them with 20 ml of tetrahydrofuran and 20 ml of
acetone for each tube, respectively. Repeat the washing steps twice.
! CAUTION Perform this step in a ventilated fume hood.
CRITICAL STEP The obtained product should be thoroughly washed with tetrahydrofuran to remove residue-swelled
polystyrene and emulsion droplets.
17| Dry the product in a vacuum oven at 100 C to obtain ~7.1 g of white powder. The size of the microspheres is about
12 m (Fig. 3b).
CRITICAL STEP At this point, the product can be checked using scanning electron microscopy. The poly(GMA-co-TMPTMA)
microspheres still maintain high monodispersity. However, there are two differences; the microspheres are larger (at ~12 m
size), and a mesoporous structure on the microspheres surfaces should be observable.
PAUSE POINT The dried poly(GMA-co-TMPTMA) microspheres can be placed into a sealed tube with a cap and kept at RT for
future use.
Preparation of poly(GMA-co-TMPTMA-NH2) monodisperse microspheres TIMING ~5 h
18| Add 7 g of the anhydrous poly(GMA-co-TMPTMA) microspheres to a 250-ml double-necked round-bottom flask charged
with 150 ml of ethylenediamine.
! CAUTION Perform this step in a ventilated fume hood.
19| Keep the reaction temperature at 80 C for 3 h under gentle agitation at 100 r.p.m. This epoxide ring opening by
aminolysis results in the formation of poly(GMA-co-TMPTMA-NH2). The schematic for the reaction is given in Figure 2c.
CRITICAL STEP Ethylenediamine has a few crucial roles for the final IMAC material. First, ethylenediamine introduces a free
amino-terminated group that can be used for the addition of the derivation phosphonate ligand. Second, ethylenediamine
introduces a spacer arm onto the surface of the microspheres and is beneficial to the enrichment of phosphopeptides.
Third, two amino groups in the spacer linker also improve the hydrophilicity of monodisperse material.
20| Transfer the obtained poly(GMA-co-TMPTMA-NH2) microspheres to 50-ml centrifuge tubes and wash each tube with 20 ml
of water and 20 ml of ethanol, respectively. Repeat the washing steps twice.
CRITICAL STEP The microspheres should be thoroughly washed to remove any residual ethylenediamine.
21| Dry down the product in a vacuum oven at 100 C to obtain about 7.8 g of white powder.
PAUSE POINT The dried poly(GMA-co-TMPTMA-NH2) microspheres can be placed in a sealed tube with a cap and kept at RT
for future use.
Preparation of poly(GMA-co-TMPTMA-PO3H2) microspheres TIMING ~1.5 d
22| Add 7 g of poly(GMA-co-TMPTMA-NH2) into 100 ml of water and thoroughly disperse it. To the above solution,
add 5.1 ml of phosphorous acid, 10 ml of HCl (37% (vol/vol)) and 8 ml of formaldehyde successively.
! CAUTION Formaldehyde and HCl are toxic and volatile. Prepare the solutions in a ventilated fume hood.
23| Elevate the reaction temperature to 100 C at 100 r.p.m. with stirring. After 24 h, the desired poly(GMA-co-TMPTMAPO3H2) microspheres are obtained. The schematic for the reaction is given in Figure 2d.
24| Transfer the poly(GMA-co-TMPTMA-PO3H2) microspheres to 50-ml centrifuge tubes and wash each tube with 20 ml of
ethanol and 20 ml of water, respectively. Repeat the washing steps twice.
protocol
25| Dry down the product in a vacuum oven at 100 C to obtain about 8.3 g of light-yellow powder. The size of the
microsphere does not have any visible change (Fig. 3c).
CRITICAL STEP At this point, the products can be checked by scanning electron microscopy. The poly(GMA-co-TMPTMAPO3H2) microspheres should either show the property of monodispersity with ~12 m or the mesoporous structure.
PAUSE POINT The dried poly(GMA-co-TMPTMA-PO3H2) microspheres can be placed in a sealed tube with a cap and kept at
RT for future use.
Preparation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspherebased immobilized titanium (IV) ion affinity

chromatography (Ti4+-IMAC) TIMING ~12 h
26| Incubate 100 mg of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspheres with 20 ml of titanium(IV) chloride
(TiCl4, 0.09 M in 20% (vol/vol) HCl) at RT for 8 h under gentle stirring. The schematic for the preparation of Ti4+-IMAC is
given in Figure 2e.
! CAUTION Titanium (IV) chloride is toxic. Perform this step in a ventilated fume hood.
CRITICAL STEP TiCl4 in 20% HCl is used in order to prevent the formation of hydrated titanium. Gently stir the mixture to
prevent the precipitation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspheres.
27| Transfer the resulting mixture into two 15-ml conical tubes and centrifuge at 20,000g for 5 min at RT, followed by
removal of the supernatant.
28| Wash the residue with 30% acetonitrile containing 0.1% (vol/vol) TFA and centrifuge it at 20,000g for 5 min at RT.
Discard the supernatant. Repeat the process twice.
CRITICAL STEP The free Ti4+ has to be completely removed in order to eliminate competitive binding. In our experience,
three extensive washes (10 ml of 30% acetonitrile/0.1% TFA (vol/vol) for a total of 50 mg material per washing) can
completely remove the free Ti4+.
29| Disperse the well-washed Ti4+-IMAC adsorbents (ca 100 mg) in 5 ml of 30% acetonitrile/0.1% TFA (vol/vol) in each
conical tube.
30| Aliquot the Ti4+-IMAC slurry to a concentration of 10 mg ml1 in each vial and store it at 4 C for further use. The final
product of Ti4+-IMAC keeps the property of monodispersity and porous structure (Fig. 3d).
CRITICAL STEP At this point, the products can be checked by scanning electron microscopy. The final Ti4+-IMAC material
should still possess the property of monodispersity with ~12 m and the mesoporous structure.
PAUSE POINT The prepared Ti4+-IMAC slurry (Ti4+-IMAC: 10 mg ml1 in 30% acetonitrile/0.1% TFA, (vol/vol)) can be
stored at 4 C for several weeks.
Suggested preparation of protein sample and protein digests
31| Follow the steps in options A, B or C for the preparation of digests of standard proteins of -casein and -casein, BSA,
and cell lysates, respectively. Follow option D for the desalting of protein digests.
(A) Preparation of digests of standard proteins of -casein and -casein TIMING ~1 d
(i) Dissolve a standard 1 mg of proteins (-casein and -casein (bovine)) in 1 ml of NH4HCO3 (50 mM, pH 8.2), and then
digest them for any amount of time between 4 and 12 h at 37 C with trypsin at an enzyme-to-protein ratio of
1:100 (wt/wt).
(B) Preparation of digests of standard protein of BSA TIMING ~1 d
(i) Dissolve 1 mg of BSA in 1 ml of denaturing buffer containing 8 M urea and 50 mM NH4HCO3. Add DTT from a 0.2 M
stock to obtain a final concentration of 5 mM and incubate the mixture for 1 h at 37 C with gentle agitation.
CRITICAL STEP DTT is used to reduce the disulfide bonds of BSA.
(ii) Bring the protein solution to RT and add IAA to a final concentration of 10 mM. Incubate the solution at RT for 30 min
in the dark.
(iii) Add DTT from 0.2 M stock to obtain a final concentration of 5 mM and incubate the solution for 30 min at RT in the dark.
CRITICAL STEP This step is recommended in order to stop overalkylation.
(iv) Dilute the protein solution five times with 50 mM NH4HCO3 to reduce the urea to <2 M.
(v) Add trypsin at an enzyme-to-protein ratio of 1:100 and incubate the mixture for 12 h at 37 C.
(vi) Quench the digestion by acidification with TFA to 1% (vol/vol).
(C) Suggested preparation of cell lysate TIMING ~2 d
(i) Cell lysis (iiii, takes 3 h). Wash cells (at least 5 106 cells) with ice-cold PBS, and then add 1 ml of lysis buffer.
protocol
CRITICAL STEP Phosphatase and protease inhibitors in the lysis buffer are required in order to get the most
accurate snapshot of the protein state. Furthermore, phosphatases can become rampant during lysis, reducing
phosphorylation without inhibitors.
(ii) Remove cell debris by centrifugation at 20,000g for 15 min at 4 C.
(iii) Perform a protein assay to determine the protein concentration.
CRITICAL STEP If lysates are to be stored, immediately freeze the lysates using liquid nitrogen and then store them
at 80 C for longer.
(iv) Preparation of digests of cell lysates (ivxi, takes 1 d). Reduce 600 l of lysate sample (2 mg in total) by adding 15 l
of DTT from a 200 mM stock solution to a final concentration of 5 mM for 1 h at 37 C with gentle agitation.
(v) Bring the protein solution to RT and add IAA to obtain a final concentration of 10 mM. Incubate the solution at RT for
30 min in the dark.
(vi) Add DTT to a final concentration of 5 mM to quench unreacted iodoacetamide; incubate the mixture at RT for 30 min
with gentle agitation.
CRITICAL STEP This step is recommended to stop overalkylation.
(vii) Add Lys-C from a 10-ng l1 stock at a 1:100 enzyme-to-protein ratio (wt/wt) and incubate the solution for 4 h at 37 C.
(viii) Dilute the sample solution four times with 50 mM NH4HCO3 to reduce the urea to <2 M and the protein concentration
to ~1 g l1.
(ix) Add trypsin at an enzyme-to-protein ratio of 1:100 for 12 h at 37 C.
(x) Quench the digestion by acidification with TFA to 1% (vol/vol) on ice.
(xi) Centrifuge the mixture at 2,500g for 5 min at RT and remove the pellet.
? TROUBLESHOOTING
(D) Peptide desalting TIMING ~1 h
(i) Condition the Sep-Pak C18 cartridge with 2 ml of acetonitrile and equilibrate it with 2 1 ml of 0.6% (vol/vol)
acetic acid.
(ii) Load the acidified tryptic peptide digests and wash them with 2 1 ml of 0.6% (vol/vol) acetic acid.
(iii) Elute the desalted peptides with 2 350 l of 80% acetonitrile/0.6% acetic acid (vol/vol).
(iv) Lyophilize the desalted peptides almost to dryness.
CRITICAL STEP To completely recover the bound peptides, the elution step should take at least 10 min.
CRITICAL STEP To avoid complete dryness and sample loss, do not leave the samples for extended periods of time in
the speed vacuum.
PAUSE POINT It is recommended to take small aliquots of digests and analyze them by LC-MS/MS to check the digests.
Phosphopeptide enrichment using Ti4+-IMAC GELoader spin tips by centrifugation TIMING ~3 to 4 h
CRITICAL The level of phosphorylation observed for each biological sample type is unique, and therefore we recommend
evaluating the capacity of Ti4+-IMAC GELoader spin tip for the sample of interest. We suggest that a series of enrichments
with differing amounts of sample of interest be performed (e.g., at levels of 50 g, 100 g, 200 g, 500 g and 1 mg).
The capacity of Ti4+-IMAC GELoader spin tips can be determined by plotting the detected phospho-PSMs, and specificity
can be determined by analyzing 50 g of sample from each enrichment. We have found that the capacity of a Ti4+-IMACloaded GELoader spin tip for unstimulated K562 cell lysate digest is ~200 g and that the specificity of phosphopeptide
enrichment is ~90% (Fig. 4). Notably, although a different centrifuge is used to perform the Ti4+-IMAC GELoader spin tip
enrichment, we recommend optimizing the centrifugation speed such that the flow rate for phosphopeptide enrichment
process is ~3 l min1.
32| Freshly prepare the loading, washing and elution buffers.
! CAUTION TFA solutions are toxic. Prepare the solutions in a fume hood.
33| Prepare a constricted GELoader tip using Empore C8 material in a manner similar to the preparation of a StageTip74.
Punch out a small C8 disk using a fused silica capillary (534 m inner diameter (i.d.), 665 m outer diameter (o.d.)).
The disk sticks at the end of the capillary tube and can be transferred into a GELoader tip. Push the disk out of the capillary
using a second capillary (365 m o.d.) and fix it in the tapering of a GELoader tip using a 365-m (o.d.) fused silica
capillary. Cut the column at the open end, ~0.2 cm away from C8 plug. Wash the C8 plug using 20 l of methanol.
34| Prepare the Ti4+-IMAC-loaded GELoader spin tip. Vortex the Ti4+-IMAC slurry obtained from Step 5 and then pipette out
50 l of slurry onto the GELoader spin tip. Inset the Ti4+-IMAC GELoader spin tip into an Eppendorf tube using the adaptor
and pack the Ti4+-IMAC microcolumn at 100g. Figure 5 shows the scheme of assembly of a Ti4+-IMAC-loaded GELoader
spin tip.
protocol
CRITICAL STEP To properly pack the Ti4+-IMAC, a spinning

speed of 100g is preferable. To avoid high back pressure, the
maximal amount of Ti4+-IMAC should be limited to 500 g
for a 20-l GELoader tip. However, the Ti4+-IMAC spin tip
can be also scaled up by using 250-l pipette tips.
No. of unique phosphopeptides
3,000
Specificity
No. of identified
phosphopeptides
2,500
100
80
2,000
60
1,500
40
1,000
20
500
0
100
250
500
1,000
Enrichment specificity (%)
Figure 4 | The capacity of the assembled Ti4+-IMAC GELoader spin tip

prepared as described in Steps 33 and 34 with 500 g of packing material.
Different amounts of unstimulated K562 cell lysate digests were subjected
to enrichment, followed by LC-MS analysis. The graph represents the number
of unique phosphopeptide identifications (blue bars, left y axis) and the
specificity of phosphopeptide enrichment (red line, right y axis).
Amount of sample (g)
? TROUBLESHOOTING
35| Equilibrate the Ti4+-IMAC GELoader spin tip by adding loading buffer onto the GELoader spin tip; centrifuge it at 100g for
5 min at RT. Figure 5c shows the enrichment manipulation using Ti4+-IMAC GELoader spin tip in combination with centrifugation.
36| Next, dissolve the sample in 100 l of loading buffer.
CRITICAL STEP To obtain efficient enrichment and complete binding, it is recommended that the concentration of peptides
be ~1 g l1. To track the enrichment quality, it is also recommended to do a parallel enrichment using a standard sample
such as an -casein digest.
Figure 5 | Flowchart briefly indicating the

preparation and enrichment of a sample by
Ti4+-IMAC. (a) Recommended procedure for
the enrichment of phosphopeptides. Once
the sample is digested (and fractionated), we
suggest a sample cleanup with reversed phase
based solid-phase extraction (SPE) before
enrichment. It is recommended that a sample
consisting of a known phosphopeptide pool
(control) be treated in parallel to the sample
of interest. (b) A detailed schematic
of the assembly of Ti4+-IMAC GELoader spin
tip. (c) A graphical outline of all the steps
performed during the enrichment process
using Ti4+-IMAC GELoader spin tips and
centrifugation. ACN, acetonitrile.
protocol
37| Load the sample by applying 2 50 l of the sample onto the Ti4+-IMAC spin tip. During the loading step, keep the
remaining sample on ice. Collect the flow-through containing nonphosphopeptides and immediately dry it down in a vacuum
for further use.
CRITICAL STEP Centrifuge using the slow spinning speed at ~50g for at least 30 min at RT in order to achieve complete binding.
38| Wash the Ti4+-IMAC spin tip by applying 50 l of washing buffer 1 onto the Ti4+-IMAC spin tip via moderate centrifugation at ~170g (~3 l min1) for ~17 min at RT.
39| Wash the Ti4+-IMAC spin tip by applying 50 l of washing buffer 2 onto the Ti4+-IMAC spin tip to remove the salt via
moderate centrifugation at ~170g (~3 l min1) for ~17 min at RT.
40| Wipe the outside tip using ethanol-wetted Kimtech wipes.

41| Pipette 35 l of 10% (vol/vol) FA into the new Eppendorf tube, and then elute the bound phosphopeptides using 20 l
of 10% (vol/vol) ammonia via slow centrifugation at ~100g (~1 l min1) for 20 min at RT.
CRITICAL STEP Phosphopeptides can be strongly bound onto the Ti4+-IMAC material via the strong interaction of
phosphate groups on phosphopeptides and the immobilized Ti4+. Hence, 10% (vol/vol) NH3H2O (pH ~11.0) is used to completely recover the bound phosphopeptides.
42| Elute the sample with 2 l of 80% acetonitrile/2% FA (vol/vol) into the same tube.
CRITICAL STEP It is possible that some phosphopeptides are retained by the C8 plug; the use of elution buffer 2 ensures
complete elution of highly hydrophobic peptides. Add 3 l of 100% FA to further acidify the samples, and then vortex and
briefly spin down the samples.
PAUSE POINT Samples can be dried down using a SpeedVac and then stored at 20 C for several weeks. Be aware that the
drying process may result in sample loss.
43| The obtained samples can be analyzed by MS. For MALDI-TOF MS analysis (see Box 1 for a method to do this), samples
should be dried. For LC-MS/MS analysis, samples can be directly loaded onto the LC and analyzed by LC-MS/MS.
? TROUBLESHOOTING
QC of the LC-MS/MS system TIMING ~2.5 h
44| Evaluate the complete LC-MS/MS setup using a standard complex sample, i.e., cell lysate and protmix consisting of
phosphopeptides and nonphosphopeptides.
CRITICAL STEP The MS analysis of a standard complex sample tests the LC separation paramaters such as chromatography,
peptide elution time and LC system pressure, as well as the performance of MS such as instrumental sensitivity and peptide
fragmentation. We highly recommend the recently published protocol by Kocher et al.75, in which the authors present an
excellent protocol to benchmark the nanoLC-MS/MS setup.
45| Check the LC-MS/MS system using protmix (20 fmol l1) consisting of BSA tryptic digest, -casein tryptic digest and
-casein tryptic digest without enrichment.
Box
1 | Sample analysis using MALDI-TOF MS TIMING ~30 min
MALDI-TOF MS analysis
Re-dissolve the dried phosphopeptides (PROCEDURE Step 47) with a MALDI matrix of 2 l of DHB solution (25 mg ml1 in
70% (vol/vol) acetonitrile) containing 1% (wt/vol) H3PO4 and directly spot 0.5 l on the MALDI target for analysis.
CRITICAL 1% H3PO4 additive in the MADLI matrix can enhance the MS signal of phosphopeptides84.
Data analysis
To perform data analysis of the recorded MS spectra by MALDI-TOF MS, MALDI MS spectra are exported by the MALDI-TOF MS software
provided by the vendor. The representative MS peaks in MALDI MS spectra should be carefully annotated. If commercial standard
phosphopeptides with known molecular weight are used for evaluation of enrichment performance of Ti4+-IMAC, all detected peptides
should be compared with the standard phosphopeptides and then annotated in the MALDI MS spectra. The MS signal of phospho
peptides should also be compared with and without enrichment to check the enrichment recovery. If semicomplex peptide samples,
such as tryptic digests of standard phosphoproteins (-casein and -casein) and nonphosphoproteins (BSA), are often used for the
evaluation of the enrichment specificity, the obtained MALDI MS spectra should be compared with the expected phosphopeptide peaks.
protocol
46| To test whether the LC-MS
system is compatible for phospho
peptide analysis, inject a protmix
sample. It consists of BSA tryptic
digest, -casein tryptic digest and
-casein tryptic digest, with a final
peptide concentration of 20 fmol l1
corresponding to an initial protein
concentration of 20 fmol l1 for each
protein. A total of 2.5 l corresponding to 50 fmol of sample is injected.
The sample should be analyzed using
a short gradient; we opt to use a
45-min analysis. The level of protmix
will depend on the sensitivity of the
LC-MS system used. The amount used
here should be sufficient for an orbitrap level of instrumentation coupled to
a nanoLC system.
Table 1 | Phosphopeptides detected from a semicomplex mixture consisting of -casein

and BSA by MALDI-TOF-MS analysis.
No. of
phosphorylation sites
[M+H]+ (Da)
TVDMEpSTEVF
1,238.08
TVD[Mo]EpSTEVFT
1,254.52
TVDMEpSTEVFTK
1,466.97
TVD[Mo]EpSTEVFTK
1,482.61
EQLpSTpSEENSKK
1,538.31
VPQLEIVPNpSAEER
1,660.79
YLGEYLIVPNpSAEER
1,832.83
DIGpSEpSTEDQAMEDIK
1,927.89
DIGpSEpSTEDQA[Mo]EDIK
1,943.89
10
YKVPQLEIVPNpSAEER
1,951.09
No.
Peptide sequence
47| Check the signal of phosphopep11

KKYKVPQLEIVPNpSAEERL
1
tides at m/z 1,031, 651, 698 and
733 described in Table 1. On orbitrap
12
NTMEHVpSpSpSEESIIpSQETYK
4
systems operating at flows of
~250 nl min1, the signal of each
13
VNELpSKDIGpSEpSTEDQAMEDIK
3
phosphopeptide peak should be
14
Q*MEAEpSIpSpSpSEEIVPNpSVEAQK
5
about 2533% (in height) when compared with the main nonphosphopep15
QMEAEpSIpSpSpSEEIVPNpSVEAQK
5
tide peaks. This value is dependent
16
Q[Mo]EAEpSIpSpSpSEEIVPNpSVEAQK
5
on the quality of the digest and the
ionization conditions for the LC-MS
17
NANEEEYSIGpSpSpSEEpSAEVATEEVK
4
system in question. If the protmix run
18
NANEEEYpSIGpSpSpSEEpSAEVATEEVK
5
does not pass the ratio of 2533%
The
in
the
peptide
number
refers
to
the
fact
that
the
peptide
is
phosphorylated.
criteria, the system requires condition[Mo], Oxidation on methionine; Q*: Pyroglutamylation on the N-terminal Gln, pS: phosphorylated residue.
ing (Steps4853).
CRITICAL STEP To evaluate the
performance of LC-MS/MS toward phosphopeptide analysis, a protmix consisting of
phosphopeptides and nonphosphopeptides (QC sample 2) is used for QC.
2,080.00
2,619.04
2,678.02
2,703.75
2,720.35
2,736.05
3,008.22
3,087.93
Conditioning of the LC system TIMING ~ 6 to 7 h

CRITICAL These steps are required if the results of protmix from Step 51 do not match the 25%33% threshold, or if the
phosphopeptides peptides from protmix are missed.
48| Dilute protmix with 10% (vol/vol) FA into 2 fmol l1.
49| Inject 25 l of diluted protmix using the 10-min loading method. The injection volume is dependent on the size of the
sample loop.
50| Next, inject 40 l of 1% (vol/vol) phosphoric acid to coat the metal/silica parts of the LC using the 10-min loading
method.
51| Next, inject 40 l of 10% (vol/vol) FA to remove phosphoric acid using the 10-min loading method.
52| Inject 20 fmol of BSA digests and analyze them using a 45-min gradient.
protocol
53| Run the diluted protmix again using a 45-min gradient until the signal of phosphopeptides passes the 2533% criteria.
Otherwise, repeat the conditioning until the ratio of phosphopeptide to peptide no longer changes. It can take anywhere
from 1 to 10 cycles.
LC-MS/MS analysis TIMING variable
54| Run the sample. With regard to the complexity of the enriched sample and the sensitive analysis of the enriched phosphopeptides, optimal gradient and replicate analyses are generally required to increase the coverage of phosphoproteome.
In detail, a longer gradient is recommended for both the enriched sample without prefractionation and the enriched sample
from the abundant fractions while prefractionation is being used. A short gradient is applicable for the enriched sample from
less-abundant fractions to increase the sensitivity.
55| To analyze the generated data from a large-scale experiment by LC-MS/MS analysis, export the tandem mass spectra
using MS software such as Bioworks 3.2, Mascot Distiller and others.
56| Search the tandem mass spectra against an appropriate database, e.g., SwissProt, using an appropriate search algorithm,
e.g., Mascot76 (http://www.matrixscience.com/), Sequest77, X!Tandem78 and Andromeda79. Specify which enzyme was used
to digest the sample. Set carbamidomethyl on cysteine as a fixed modification. Set oxidation on methionine (M);
phosphorylation on serine (S), threonine (T) and tyrosine (Y); and protein N-terminal acetylation as variable modifications.
If quantitative analysis is being performed, set metabolic labeling or chemical labeling as variable modifications. A targetdecoy database searching strategy is enabled to evaluate the false-discovery rates (FDRs) at the peptide level in large-scale
phosphoproteomic experiments80. Notably, a percolator-based algorithm has been proven to be an excellent program for
discriminating between the positive and negative matches by integrating a number of features81. Examples of these features
include Mascot score, precursor mass error, fragment mass error, the number of variable modifications and so on. To pinpoint the phosphorylation site localization, phosphoRS is used to calculate the site probability of each possible site in the
identified phosphopeptide sequence72. In this protocol, we used the Proteome Discoverer software package to process the
data; Proteome Discoverer integrates many features such as peak list extraction, database searching, percolator for FDR and
phosphoRS for phosphorylation site localization.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 2.
Table 2 | Troubleshooting table.
Step
Problem
Possible reason
Solution
Irregular solids observed in

the milk-like suspension for
the preparation of
monodisperse polystyrene
seed microspheres
Excessive AIBN (AIBN is the initiator

for free-radical polymerization)
Reduce the amount of AIBN added
Impurity of styrene monomer
Purification of styrene monomer by vacuum

distillation
The two-phase mixture is not fully

emulsified
Increase the swelling time
Polystyrene seed microspheres are not

fully swollen in the emulsion
Reduce the amount of AIBN added
Excessive oil phase
Reduce the level of the oil phase
The lysate is too concentrated
Dilute the lysate to a protein concentration of

~1 g l1
15
31C(xi)
Polydisperse microspheres
observed for preparation of
poly(GMA-co-TMPTMA)
monodispherse microspheres
Large pellet forms after

centrifugation of acidified
digests
(continued)
protocol
Table 2 | Troubleshooting table (continued).
Step
Possible reason
Solution
Inefficient digestion
Check the activity of protease enzyme and ensure

the optimal conditions for protease enzyme
digestion (such as urea concentration <2 M and
pH 7.58.5)
Large back-pressure observed

in the Ti4+-IMAC GELoader tip
Overpacked C8 plug
Check the back-pressure when equilibrating

the Ti4+-IMAC adsorbent before enrichment.
Alternatively, use the lower-back-pressure Ti4+IMAC GELoader tip
Small gap in the Ti4+-IMAC

GELoader tip
Improper packing
Use a higher centrifugation speed for packing
No or a lower-than-expected
level of phosphopeptides
observed in the elution
Insufficient starting material
Increase the amount of sample. The exact amount

generally depends on the natural phosphorylation
content of the sample of interest and MS sensitivity. With regard to the Orbitrap mass spectrometer,
we recommend starting with a few mg if performing multidimensional chromatography and ~200 g
for a single-dimension analysis (e.g., LC-MS)
Contaminants such as EDTA, free

metals, ammonium bicarbonate or
phosphate species inhibit
phosphopeptide binding
Desalt enriched samples before enrichment
Failure to recover the bound

phosphopeptides
Use the fresh alkaline buffer (10% NH3.H2O) to

elute the bound phosphopeptides
Eluent solution insufficiently acidified
Lower the pH of the loading solution to <3.0 by

adding FA or AA (not TFA)
High pH; phosphate group partially

blocked by ion interaction
Add enough TFA into loading buffer before

enrichment
Peptides bound to plastics, which

caused substantial peptide loss
Avoid total dryness of the enriched

phosphopeptide sample
Phosphopeptides adsorbed onto the

metal/silica parts of the HPLC system
Condition the HPLC system (see Steps 4853)
Loading of sample was performed a

high flow rate
Collect the flow-through and reload it using a

slower centrifugation speed
Limited capacity of Ti4+-IMAC GELoader

tip
Scale up the Ti4+-IMAC GELoader tip or split the

sample and load it onto several GELoader tips
Ti4+-IMAC adsorbent is old
Ti4+-IMAC adsorbents need to be stored at 4 C.

When performance is decreased, replace them with
a new batch. We recommend using a complex cell
lysate such as un-stimulated HeLa as quality
control. Do not attempt to recover material by
EDTA stripping and recharging with TiCl4 after it
has been exposed to high pH conditions
Loading solution not sufficiently

acidified
Lower pH of loading solution to <3.0 by adding

TFA
The sample loading buffer was at

pH <3.0
Ensure that pH is <3.0 by increasing the TFA

concentration
34
Problem
Phosphopeptides observed in
the flow-through
43
Nonphosphopeptides observed
in elution
(continued)
protocol
Table 2 | Troubleshooting table (continued).
Step
Problem
Possible reason
Solution
Too much Ti4+-IMAC was used for the

amount of peptides
Reduce the amount of Ti4+-IMAC adsorbent
Nonspecific peptides are highly

hydrophobic
Increase the amount of acetonitrile in loading

buffer to 80% (vol/vol)
Nonspecific peptides are highly acidic
Increase the TFA concentration in the loading

buffer or add 200 mM NaCl to washing buffer 1
Insufficient washing
Increase number of washes
Peptide residues remain on the outside

of the tip
Clean the outside of the tip using an appropriate

organic solvent before elution
TIMING
Steps 17, preparation of polystyrene monodisperse microspheres: ~1.5 d
Steps 817, preparation of poly(GMA-co-TMPTMA) monodisperse microspheres: ~2 d
Steps 1825, preparation of poly(GMA-co-TMPTMA-NH2) monodisperse microspheres: ~5 h
Steps 2630, preparation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspherebased immobilized titanium (IV) ion
affinity chromatography (Ti4+-IMAC): ~12 h
Step 31A, preparation of digests of standard proteins of -casein and -casein: ~1 d
Step 31B, preparation of digests of standard protein of BSA: ~1 d
Step 31C, suggested preparation of cell lysate: ~2 d
Step 31D, peptide desalting: ~1 h
Steps 3243, phosphopeptide enrichment using Ti4+-IMAC GELoader spin tips by centrifugation: ~3 to 4 h
Steps 4447, QC of the LC-MS/MS system: ~2.5 h
Steps 4853, conditioning of the LC system: ~6 to 7 h
Steps 5456, LC-MS/MS analysis: variable; MS analysis time depends on the sample complexity and the collected fractions
Box 1, MALDI-TOF MS analysis: ~30 min plus data analysis time
ANTICIPATED RESULTS
We illustrate the design of Ti4+-IMAC and the enrichment principle of phosphopeptides by Ti4+-IMAC in Figure 1, and we
provide the scheme of the experimental workflow in Figure 5a. To provide a simple, easy-to-use, reproducible and clean
enrichment method, Ti4+-IMAC, we used loaded GELoader spin tips in combination with centrifugation for the following
experiments (Fig. 5b,c). Figure 5b shows the assembly of Ti4+-IMAC-loaded GELoader spin tip.
To demonstrate the specificity of phosphopeptide enrichment using Ti4+-IMAC, we used a semicomplex sample (-casein
tryptic digests/BSA tryptic digests: 1 pmol/100 pmol or 1 pmol/1,000 pmol). Clearly, direct analysis of the semicomplex
peptide mixture (-casein tryptic digests/BSA tryptic digests: 1/100) by MALDI MS generates a spectrum with many dominant nonphosphopeptides (Fig. 6a). After enrichment, the MALDI MS spectrum is dominated by 16 phosphopeptides (listed
in Table 1) originating from 250 fmol of -casein and very few nonphosphopeptides (marked by stars; Fig. 6b). Because
of the fact that phosphopeptides are often less abundant than nonphosphorylated peptides, and because phosphopeptide
enrichment is often compromised by increased sample complexity, we created a more real phosphorylation sample by adding
1,000 times more BSA digest to the same amount of -casein (250 fmol), i.e., 1 pmol casein: 1,000 pmol BSA. The MALDI
results of this semicomplex sample show that good specificity of phosphopeptide enrichment can be achieved by Ti4+-IMAC.
Figure6c shows a MALDI MS spectrum that is similar to that of the mixture with casein/BSA at 1 pmol:100 pmol.
To further evaluate the performance of Ti4+-IMAC for a higher-complexity sample, we used an unfractionated K562 sample.
We enriched total of 2 mg of desalted K562 sample using Ti4+-IMAC. We analyzed the enriched sample on a nanoLC-MS/MS
platform consisting of the UHPLC instrument (EASY-nLC 1000) connected to a Q-Exactive quadrupole orbitrap mass spectrometer. Figure 7a shows a typical 2-h chromatogram for an enriched sample corresponding to 125 g of K562 sample.
We also investigated the effects of separation time and analytical reproducibility on the phosphopeptide identification.
Figure 7b shows the results, including the number of unique phosphopeptides, unique phosphorylation sites and phosphoproteins, as well as the specificity of enrichment. Clearly, we observe an increasing number of identified phosphopeptides
after increasing the separation time. In a single run, we were able to identify more than 3,000, 4,500, 5,600 and 6,000
unique phosphopeptides with 60-min, 90-min, 120-min and 180-min separation times, respectively. The combined data set
protocol
a
6,000
1,600
Direct analysis
1:100
1,400
5,000
1,200
8
Intensity
Intensity
4,000
3,000
1,000
800
9
600
2,000
15
16
400
1,000
200
0
1,000
1,400
1,800
2,200
m/z
2,600
2
4
1
3 5
0
1,000
3,000
1,400
12 14
13
10
11
1,800
17 18
**
2,200
2,600
3,000
m/z
c 1,600
1:1,000
1,400
1,200
Intensity
Figure 6 | The MALDI mass spectrometric analyses of a standard peptide

mixture (consisting of varying amounts of -casein and BSA digests) with
and without enrichment. (a) Mass spectrum of the peptide mixture at a
molar ratio of 1:100 (-casein to BSA digest) without enrichment.
(b) Mass spectrum of the peptide mixture at a molar ratio of 1:100
(-casein to BSA digest) after enrichment. (c) Mass spectrum of the
peptide mixture at a molar ratio of 1:1,000 (-casein to BSA digest) after
enrichment. *, nonphosphopeptides. , phosphopeptides.
800
600
400
200
3 4
0
1,000
*
*
1,400
14 15
16
13
12
17 18
10
**
11
1,800
2,200
m/z
2,600
3,000
of analytical triplicates resulted in a total of 4,329 unique phosphopeptides within a 60-min separation time, 6,387 unique
phosphopeptides within a 90-min separation time, 8,179 unique phosphopeptides within a 120-min separation time and
11,200 unique phosphopeptides within a 180-min separation time (Fig. 7b). Approximately 13% of phosphopeptides were
multiple phosphorylated peptides from individual analysis. The specificity of phosphopeptide enrichment by Ti4+-IMAC is
as high as ~90% using short separation time and ~80% using a long gradient without prefractionation. Moreover, we found
that the overlap between analytical duplicate was between 70% and 80% for each gradient (Fig. 8ad). Notably, running
replicate of the enriched K562 sample from Step 42 helps increase the number of phosphopeptide identifications by
another 3040%, as also shown in other reports58,59, and generally increases the coverage of the phosphoproteome. Similar
to previous reports59,75, we also observed a marked increase of phosphopeptide identifications with increasing separation
time (Fig.8e). In turn, fractionation or multidimensional separations combined with Ti4+-IMAC are needed for resolving the
complexity of cellular phosphoproteome.
a 100
95
No. of phosphoproteins
No. of unique phosphosites
Specificity
No. of unique phosphopeptides
12,000
100
10,000
80
8,000
60
6,000
40
4,000
20
60
70
Time (min)
80
90
100
110
120
60 min
120 min
90 min
Cumulative
50
Cumulative
40
Cumulative
30
Cumulative
20
2,000
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
10
Percentage
Relative abundance
1,000
180 min
Figure 7 | LC-MS analysis of an unstimulated K562 cell lysate digest subjected to Ti4+-IMAC spin tips using a UHPLC system coupled to a Q-Exactive and
differing gradient lengths. (a) A typical 2-h LC-MS analysis chromatogram at a level of 125 g of starting material. (b) A graph representing the number
of unique phosphopeptides, phosphosites and phosphoproteins (bars, left y axis) and the specificity of phosphopeptide enrichment (purple line, right y
axis). A number of gradients were applied (60-min, 90-min, 120-min and 180-min gradient) in triplicate, while maintaining 125 g of starting material
for each experiment.
protocol
187
Experiment 3: 3,052
552
3,179
Experiment 1: 6,415
674
313
Experiment 3: 4,557
Cumulative of triplicate analysises of 60-min,

90-min and 180-min MS analysis time
Triplicate of 60 min: 4,329
293
1,267
463
896
180-min MS analysis time
878
391
526
Experiment 1: 5,675
Experiment 2: 5,835
455
Experiment 2: 4,570
2,105
585
752
30
Experiment 2: 3,218
349
1
34
Experiment 1: 4,874
3,438
1,001
1,015
486
614
736
3,798
1,065
1,157
668
Experiment 3: 5,838
Experiment 3: 6,237
319
25
2
Experiment 1: 3,100
Experiment 2: 6,267
a 60-min MS analysis time b
3,465
4,281
1,327
3,135
Inidividual fraction identifications
The Ti4+-IMAC method can also be combined with multidimensional chromatographic strategies41,56,82. To further exemplify
the performance of Ti4+-IMAC for large-scale phosphoproteomic analysis, we combined the well-established low-pH SCX
chromatography12,54 with Ti4+-IMAC for the analysis of dimethyl-labeled MCF-7 samples. A total of 9,117 unique phosphorylation
sites on 9,678 unique phosphopeptides were identified from a total of 400 g of dimethyl-labeled MCF-7 sample (Fig. 9a).
Notably, all identified phosphopeptides
were across all SCX fractions. Within
a 3,000
10,000
this data set, ~13% of phosphopepHCD
CID
tides were multiply phosphorylated
9,000
ETD
Unique phosphorylation sites
peptides. The majority of phos2,500
8,000
phopeptides can be divided into three
categories: zero net-charged, singly
7,000
charged and multiply charged phos2,000
phopeptides. The distribution of the
6,000
identified phosphopeptide is consistent
1,500
5,000
with the charge-based SCX separation.
Multiply phosphorylated peptides are
4,000
dominant in the early fraction. Singly
1,000
phosphorylated peptides with neu3,000
tralized charge are dominant in the
2,000
middle SCX fraction. Positively charged
500
phosphopeptides with multiple basic
1,000
residues are most identified in the later
0
0
SCX fractions. Strikingly, over 58% of
the total unique phosphorylation
Fraction
sites were identified from the late
b 100
SCX fractions corresponding to the
1~
3
4
5
6
7
9~ 8
11
12
13
14
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Cumulative identifications
90
80
70
60
HCD
50
CID
40
ETD
30
20
10
3
4
5
6
7
9~ 8
11
12
13
14
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
1~
Percentage (%)
Figure 8 | Venn diagram analysis of analytical triplicate MS analyses using varying gradient lengths and the accumulative results of triplicate 60-min,
90-min, 120-min and 180-min gradients. (a) Triplicate analysis using a 60-min gradient. (b) Triplicate analysis using a 90-min gradient. (c) Triplicate
analysis using a 120-min gradient. (d) Triplicate analysis using a 180-min gradient. (e) Cumulative results of increasing the gradient from 60 min to 180 min.
Fraction
Figure 9 | SCX/Ti4+-IMAC approach applied

to a triple dimethyl-labeled MCF-7 sample.
(a) Bar diagram representing the individual
phosphopeptides identified per fraction, and
the fragmentation method by which each
fraction was identified. The accumulation
of unique phosphorylation sites resolved by
fraction is plotted as a dotted line using the
right-hand y axis. (b) The contribution of
phosphopeptides identified by HCD, CID or ETD
in each fraction. HCD identifications are shown
in blue. CID identifications are shown in red. ETD
identifications are shown in green.
protocol
Relative abundance
b5
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
b2 b3 b4 b5
100%
P
b1198
b7b8 b9 b10
HQDGLPYIDDSPSSSPHLSSK
b7
y13 y12 y11y10 y9 y8 y7 y6 y5 y4 y3 y2

98 y11y12y13
+80 y11y12y13
Diagnostic ions: y11
98 y11y12y13
+80 y11y12y13
b1198
Ion score: 64
[M+3H-98]3+
y3
y
phosphopeptides containing multiy
y -98
4+
y
ple basic residues. Clearly, Ti -IMAC
b
y -98
y
y -98
b -98
y
provides efficient enrichment of not
b
y
y
b
y
y
-98
y
only multiple phosphorylated peptides
y
b
y -98
b
y
y
y -98
y
and singly phosphorylated peptides but
b
b
also the phosphopeptides containing
200
400
600
800
1,000
1,200
1,400
1,600
1,800
2,000
multiple basic residues. Ti4+-IMAC is
m/z
therefore a robust and relatively unbi50% 50%
b
y
P
P
ased technology for enriching a wide
100
b3b5 b6 b7b16++ b9b1098
b3 b5
95
range of phosphopeptide types.
b3b5+80
90
Figure 9b shows the contribution to
85
SASSDTSEELNSQDSPPK
the unique phosphopeptide identifica80
y15 y14y13y12y11y10y9 y8 y7 y6 y5 y4 y3 y2
75
tions per fraction broken down by the
70
Ion score: 112
activation technique. HCD and CID
65
Missing diagnostic ions
60
are most effective for the earlier SCX
55
fraction containing lower net-charged
50
phosphopeptides and the phosphopep45
40
tides carrying a 2+ charge. For the late
y
35
y
SCX fractions, ETD clearly outperformed
30
25
HCD and CID, contributing 76% of the
y
y
20
y
y
phosphopeptide identifications. Hence,
y
b
-98
b
-98
15
b -116
b -98
y
b
the complementary fragmentation
10
b -116 y b -116
y
b
y
b
y
b -98
y -18
5
b -98
b -98
y
techniques are highly recommended to
b
0
visualize the map of cellular phospho200
400
600
800
1,000
1,200
1,400
1,600
1,800
m/z
proteome.
A sample enriched by Ti4+-IMAC can
also be subjected to multidimensional
chromatography. We recently showed that Ti4+-IMAC enrichment can be followed by high-pH RP fractionation. By applying
this strategy, 9,719 phosphorylation sites in 2,998 proteins from human liver were identified. By performing the enrichment
first, the user markedly reduces the level of sample preparation. Recently, Gerber et al. showed that enrichment before SCX is
a viable strategy83.
In addition to the identification of phosphopeptides by MS, determination of the exact site of phosphorylation is highly
desired. Almost 16% of the amino acids in the current full NCBI database (20082710) are serine, threonine or tyrosine.
Therefore, the chance that a given peptide contains more than one potential phosphorylation is quite high. As a result,
phosphorylation site localization is highly dependent on the presence of site-determining ions within the obtained MS/MS
spectra. Recently, a new probability-based site localization software called phosphoRS (freely available; http://cores.imp.
ac.at/protein-chemistry/download/) has been developed for automatic data interpretation of MS/MS spectra, assigning and
calculating individual site probabilities for all potential phosphorylation sites. PhosphoRS can be also used in conjunction
with all commonly used fragmentation methods and data sets with high or low mass accuracy. To exemplify how to localize
a phosphorylation site, we give examples of phosphosite localization using the phosphoRS algorithm72. Figure 10 provides
two examples of phosphorylation site localization using phosphoRS. In Figure 10a, a phosphopeptide is confidently identified with a mascot score of 61. Seven potential phosphorylation sites are present in the sequence: Y7, S11, S13, S14, S15,
S19 and S20. Fragment ions b2 to b10 have masses corresponding to the fragment ions that are unmodified, indicating that
these peptide fragments are not phosphorylated. Therefore, Y7 can be excluded as phosphorylation sites. Examining the
C-terminal fragments, y2y10 also correspond to nonmodified peptide sequences. This excludes S13, S14, S15, S19 and S20,
which are not phosphorylated, and leaves S11 as the only option. This is further confirmed by the detected y11, y12 and
y13, which originate from phosphorylation-containing peptide fragments. Finally, some fragments (b11, y11, y12 and y13)
++
10
++
15
++
12
13
++
11
++
13
10
12
13
10
11
++
11
11
12
Relative abundance
Figure 10 | Phosphorylation site localization.

Indicated on the peptide sequence are the
fragment ions that were found, including ions
that lost 98 Da or were 80 Da heavier. Sitedetermining ions are marked as diagnostic
ions. (a) A HCD spectrum of a phosphopeptide
for which the exact site of phosphorylation is
confidently localized. (b) A HCD spectrum of a
phosphopeptide for which the exact site could
not be unambiguously determined.
++
6
12
10
11
16
13
++
10
14
15
protocol
are also observed that have lost the phosphate groups, supporting the notion that the phosphorylation site is again S11.
The algorithm phosphoRS generates fragment ion lists (in a manner similar to PTMScore and Ascore) that cover all permutations for the possible location of the site for that sequence. The algorithm then scores each fragment ion list. The magnitude
of the best score and the difference of that score against the next best score are then equated to a confidence level in the
site localization. In this case, the site is clear and thus the software reports a 100% site probability of S11. In Figure 10b,
a fragmentation spectrum results in confident identification of a phosphopeptide with a mascot score of 112. However, there
are six possible serine residues that might be phosphorylated within this sequence. Fragment ions y2y15 indicate that
the phosphorylation site is not on S4, S7, S12 and S15. Ion b3 indicates that two options exist, S1 or S3, which might be
phosphorylated. However, no site-determining ions exist that resolve the final ambiguity. Again, phosphRS makes all permutations, but now there are two possible fragment ion series (corresponding to two different potential sites). Consequently,
phosphoRS reports a 50% site probability for S1 and S3, respectively. Therefore, the two sites should be taken into account
for the biologically relevant follow-up. In a large-scale phosphoproteomic experiment that consists of over 10,000
phosphopeptides, we have come to expect that ~80% of the phosphorylation site can be localized using phosphoRS.
Acknowledgments This work was supported in part by the PRIME-XS project

with the grant agreement number 262067, funded by the European Union 7th
Framework Program; The Netherlands Proteomics Centre, embedded in the
Netherlands Genomics Initiative; the Netherlands Organization for Scientific
Research (NWO) with the VIDI grant (700.10.429); the Creative Research Group
Project by the National Natural Sciences Foundation of China (21021004); and a
China State Key Basic Research Program grant (2012CB910101, 2013CB911202).
AUTHOR CONTRIBUTIONS H. Zhou, M.Y., S.M. and H. Zou designed the studies.
H. Zhou performed the phosphoproteomic experiments and analyzed the data.
J.D. carried out the synthesis experiment. E.C. and A.C. assisted in the
Q-Exactive experiments. All authors discussed experimental results. A.J.R.H.,
H. Zou and S.M. supervised the project and wrote the manuscript with
H. Zhou and M.Y.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Published online at http://www.nature.com/doifinder/10.1038/nprot.2013.010.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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Robust Phosphoproteome Enrichment Using Monodisperse Microsphere

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Robust phosphoproteome enrichment using

2013 Nature America, Inc. All rights reserved.

Published online 7 February 2013; doi:10.1038/nprot.2013.010

Strong cation exchange (SCX) chromatography performed at a

2013 Nature America, Inc. All rights reserved.

Figure 1 | Depiction of the architecture of the Ti4+-IMAC adsorbent and

and IMAC or MOAC has been proven to be an excellent strategy

Polystyrene monodisperse microspheres

2013 Nature America, Inc. All rights reserved.

profiling. Each step of sample preparation,

Phosphopeptide enrichment. The phosphopeptide enrichment is performed with

nature protocols | VOL.8 NO.3 | 2013 | 463

2013 Nature America, Inc. All rights reserved.

Figure 3 | Characterization of the synthesized products by scanning electron

using a very basic solution (10% (vol/vol) NH3H2O), which we

464 | VOL.8 NO.3 | 2013 | nature protocols

2013 Nature America, Inc. All rights reserved.

2013 Nature America, Inc. All rights reserved.

2013 Nature America, Inc. All rights reserved.

2013 Nature America, Inc. All rights reserved.

Preparation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspherebased immobilized titanium (IV) ion affinity

468 | VOL.8 NO.3 | 2013 | nature protocols

2013 Nature America, Inc. All rights reserved.

CRITICAL STEP To properly pack the Ti4+-IMAC, a spinning

No. of unique phosphopeptides

Enrichment specificity (%)

Figure 4 | The capacity of the assembled Ti4+-IMAC GELoader spin tip

Amount of sample (g)

2013 Nature America, Inc. All rights reserved.

Figure 5 | Flowchart briefly indicating the

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40| Wipe the outside tip using ethanol-wetted Kimtech wipes.

nature protocols | VOL.8 NO.3 | 2013 | 471

2013 Nature America, Inc. All rights reserved.

Table 1 | Phosphopeptides detected from a semicomplex mixture consisting of -casein

47| Check the signal of phosphopep11

Conditioning of the LC system TIMING ~ 6 to 7 h

472 | VOL.8 NO.3 | 2013 | nature protocols

2013 Nature America, Inc. All rights reserved.

Irregular solids observed in

Excessive AIBN (AIBN is the initiator

Reduce the amount of AIBN added

Impurity of styrene monomer

Purification of styrene monomer by vacuum

The two-phase mixture is not fully

Increase the swelling time

Polystyrene seed microspheres are not

Reduce the amount of AIBN added

Excessive oil phase

Reduce the level of the oil phase

The lysate is too concentrated

Dilute the lysate to a protein concentration of

Large pellet forms after

nature protocols | VOL.8 NO.3 | 2013 | 473

Check the activity of protease enzyme and ensure

Large back-pressure observed

Check the back-pressure when equilibrating

Small gap in the Ti4+-IMAC

Use a higher centrifugation speed for packing

Insufficient starting material

Increase the amount of sample. The exact amount

Contaminants such as EDTA, free

Desalt enriched samples before enrichment