robust phosphoproteome enrichment using monodisperse microsphere

8/9/2019 Robust Phosphoproteome Enrichment Using Monodisperse Microsphere

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NATURE PROTOCOLS|VOL.8 NO.3 |2013 |461

INTRODUCTIONProtein phosphorylation by kinases and dephosphorylation byphosphatases are key tools for signaling in cellular networks1,2.Protein phosphorylation has been estimated to affect ~30% ofa proteome and represents a major regulatory machinery formany cellular processes3. A fundamental understanding of bio-logical processes and signaling networks at the molecular levelrequires the detailed analysis of the phosphorylated proteins beinginvolved. However, the dynamic nature of signaling networks, thecomplexity of the phosphoproteome and the low stoichiometryof protein phosphorylation pose a serious technical challenge4.Nowadays, high-throughput protein identification is predomi-nantly performed by MS5. However, this approach often fails to

identify phosphopeptides in the complex peptide mixtures gener-ated by protein digests. Therefore, it has become a crucial step tospecifically isolate subsets of phosphopeptides from a complexpeptide mixture6,7.

To date, a number of approaches have been developed.Phosphopeptides can be enriched by immunoprecipitation; how-ever, the strategy has only been proven to be highly applicableto peptides containing phosphotyrosine8,9. Another approachis to perform chemical coupling, in which the phosphopeptidesare either covalently conjugated to a polymer support and thenreleased or covalently attached with an affinity tag, followed byaffinity purification10. Currently, the most common strategiesfor global comprehensive enrichment involve chromatography.

Strong cation exchange (SCX) chromatography performed at alow pH has proven to be very popular11. Under these conditions,peptides are fractionated on the basis of their solution net chargeand the orientation of peptides to the negatively charged chroma-tographic material. Unlike glutamic and aspartic acid, phosphor-

ylated amino acids are able to retain a negative charge under acidic(pH 2.7) conditions. This property can be exploited in SCX for theenrichment of phosphopeptides, which tend to elute earlier and arethus separated from the majority of nonphosphopeptides12.

Chelation/coordination chemistry was, however, the first trulysuccessful strategy for phosphopeptide enrichment. It has contin-ually developed over the past few decades and continues to be the

most popular strategy. The general principle involves an immo-bilized metal oxide or metal ion, which is capable of coordina-tion and, specifically, has a high preference for phosphate groups.IMAC with Fe3 + or Ga3 + is one of the most-used enrichmentmethods for phosphopeptides13,14. The selectivity of conventionalIMAC toward phosphopeptides is based on the high affinity of thephosphate groups with the metal ions (Fe3 + or Ga3 +) bound toadsorbents through iminodiacetic acid (IDA)13or nitrilotriaceticacid (NTA)15chelating ligand. One of the limitations associatedwith the conventional IMAC-based phosphopeptide enrichmentis the nonspecific adsorption resulting from nonphosphorylatedpeptides containing multiple acidic amino acids (glutamateand aspartate)15. To improve the specificity of phosphopeptide

Robust phosphoproteome enrichment usingmonodisperse microspherebased immobilizedtitanium (IV) ion affinity chromatographyHoujiang 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 Chemistr y, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, ChineseAcademy 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. ([email protected]), H.Z. ([email protected]) orS.M. ([email protected]).

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 besuccessful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary becauseof the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternativematerials 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 groupsthat 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). Herewe present a detailed protocol for the preparation of monodisperse microspherebased Ti4 +-IMAC adsorbents and the subsequentenrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samplesbefore enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparationof 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 protocolsprovided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ~2 weeks tocomplete the protocol: 1 week to prepare the Ti4 +-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enrichedsample and 2 d for data analysis.


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462|VOL.8 NO.3 |2013 |NATURE PROTOCOLS

enrichment, an approach that blocks the acidic residues (carboxy-lic groups of the peptide) using methyl esterification was devel-oped before enrichment16. Metal oxide affinity chromatography(MOAC6,1720, typically TiO2(ref. 17)) has become an effectivealternative to IMAC for the enrichment of phosphopeptides. Muchhigher selectivity can be achieved using a multifunctional acidsuch as 2,5-dihydroxybenzoic acid (DHB)21, lactic acid22or gly-colic acid23in the loading buffer. However, one weakness commonto MOAC and classical strategies is their poor binding to phos-phopeptides that contain multiple basic residues2427.

Metal (IV) phosphate/phosphonate chemistry has emergedover the past three decades. The coordination of phosphate/phosphonate group to metal (IV), especially for Zr4 +, has nowbeen extensively illustrated in the literature28,29. Nakayama et al.30also revealed the coordination of Ti4 + with phosphate groups sim-ilar to Zr4 + with phosphate groups using31P NMR. In this case, theMO6 are octahedrally coordinated by oxygen atoms, with the threeoxygens of each phosphonate bound to three different zirconiumions31. Notably, the unique coordination of Zr4 + or Ti4 + with thephosphate/phosphonate group has been shown to have a richnessof applications, including DNA microarrays32, protein microar-rays33and self-assembled monolayers34. We recently developeda new generation of IMAC for phosphopeptide enrichment3539by using the unique property of Zr(IV) or Ti(IV) phosphate/phosphonate chemistry. Figure 1shows the architecture of theTi4 +-IMAC adsorbent and the practical principle of phosphopep-tide enrichment enabled by Ti4 +-IMAC. To improve the specificityof phosphopeptide enrichment and make it such that there is lessbias toward different types of phosphopeptides such as basophilickinase substrates, we have made a few key modifications for Ti4 +-IMAC technology40,41. The first of these is the use of monodis-perse microspheres. Monodisperse microspheres have a uniformmonodisperse size distribution, uniform column packing, uni-

form flow profile, low column pressure, high column efficiencyand excellent stability in the field of separation sciences42,43. Themonodisperse microspheres we prepared40not only have stablechemical-physical properties, such as durability toward strong acidand alkaline buffer, but also contain a large surface area because ofabundant mesopores in the microspheres and have a hydrophilicsurface that minimizes nonspecific adsorption. Second, a flexiblelinker (poly(GMA-co-TMPTMA), where GMA is glycidyl meth-acrylate and TMPTMA is trimethylol-propane trimethacrylate) isintroduced to increase the spatial distance between the active Ti4 +

and the matrix (polystyrene microspheres)40,41. The flexible linkerprovides a beneficial spatial orientation for the phosphopeptidebinding by reducing the steric hindrance, which is caused by the

matrix and the bound phosphopeptides. The amino groups inthe linker further improve the hydrophilicity of monodispersemicrospheres. Third, as opposed to commercial IMAC materialsin which the chelating ligands IDA13 or NTA15 for Fe3 +/Ga3 +-IMAC are used, we coupled phosphonate groups for chelationand immobilization of Ti4 + via coordination between Ti4 + andthe P-O bond. Such immobilization also creates a beneficial struc-tural orientation for the selective binding of phosphopeptides.We recently showed that the developed Ti4 +-IMAC protocol iscapable of enriching phosphopeptides containing multiple basicresidues41. Given the inherent complexity of cellular proteome,prefractionation is prerequisite to improve the coverage of cel-lular phosphoproteome. For example, the combination of SCX

and IMAC or MOAC has been proven to be an excellent strategyfor phosphoproteome analysis12,4451.

Here we present a detailed protocol for the preparation ofTi4 +-IMAC adsorbents and the subsequent enrichment process.Furthermore, we discuss the general pitfalls and crucial steps in thepreparation of phosphoproteomic samples before enrichment and,

just as importantly, the subsequent mass spectrometric analysis.The key points such as lysis, preparation of the chromatographicsystem for analysis and the most appropriate methods for sequenc-ing phosphopeptides are discussed. Bioinformatics analysis specifi-cally relating to site localization are briefly discussed. Finally, wedemonstrate how the protocols provided are appropriate for thescreening 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 phospho-nate group as the chelating ligand. In principle, any matrix with thesurface modified with phosphonate groups can be used to prepareTi4 +-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, thematrix should have sufficient chemical stability. To ensure fast andefficient capture and release of phosphopeptides, a matrix withmacroporous structure is preferable. Compared with a matrix ofirregular shape, the microspheres with uniform size are beneficialand lead to an improvement of the enrichment performance. Thisis especially true when the enrichment is performed in columnformat. Here we provide a protocol to synthesize a tailored matrixto prepare Ti4 +-IMAC adsorbents (Fig. 2). The porous monodis-perse microspheres were prepared by a swelling and polymerizationmethod similar to the classic method developed in 1980 by Ugelstadet al.52. First, the monodisperse polystyrene seed microspheres

Microspheres matrix

PO

O OP

O

O O

PO

O O

PO

O O

P

OO

O

O

O

Ti4+ Ti

4+Ti

4+

P

O O

O

Phosphopeptide

enrichment

Chelating ligand

Flexible linker

Figure 1|Depiction of the architecture of the Ti4 +-IMAC adsorbent andthe 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.
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using a very basic solution (10% (vol/vol) NH3H2O), which wehave found to be excellent for the recovery of bound material. Insuch high basic conditions, the phosphate group can be lost fromthe peptide, and therefore it is crucial that the eluent be acidifiedimmediately. We suggest adding a volume of 10% (vol/vol) formicacid (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 runningtechnical duplicates can increase the number of phosphopeptidesidentified by another 3040% (refs. 58,59; also see ANTICIPATEDRESULTS for further details). Although SCX fractionation is usedfor reducing the sample complexity, we also recommend usinga longer gradient (3 h) for the enriched phosphopeptides fromthe abundant SCX fractions and short gradient (90 min) for theenriched phosphopeptides from the less-abundant fractions. Inaddition, experimental replicates and label swapping are recom-mended for quantitative studies that link phosphorylation dynam-ics to biological function in order to confirm any observed changesin phosphorylation.

Considerations for sample preparation relating to quantifica-tion.The Ti4 +-IMAC is also applicable to different quantitativesamples such as stable isotope labeling by amino acids in cellculture (SILAC)60, isotope-coded affinity tags (ICAT)61, isobarictags for relative and absolute quantification (iTRAQ)62, isobarictandem mass tags (TMT)63or dimethyl labeling64. To obtain asuccessful outcome for a quantitative phosphoproteome experi-ment using Ti4 +-IMAC, we recommend some key evaluations foreach sample preparation stage before running the real sample of

interest. First, we recommend checking the sample labeling effi-ciency by analyzing a minute digested sample, 1 g, via a 3-h MSanalysis. Second, it is advisable to check the basal phosphorylationcontext of the sample of interest using Ti4 +-IMAC enrichmentalone. To do this, a small amount of biological sample, such asa few hundred micrograms, is subjected to Ti4 +-IMAC withoutany fractionation, and then the enriched sample is analyzed byperforming a 3-h MS analysis. For example, about 3,000 phos-phopeptides can be identified from unstimulated 100g of humancell lysate (in our case, HeLa and K562 cells, see ANTICIPATEDRESULTS). Third, in order to reduce the quantification variationsof protein bicinchoninic (BCA) assay or Bradford assay, we rec-ommend analyzing (by MS) a small equal amount of the pooledlabeled sample before fractionation. On the basis of this prequan-tification, equal mixing of each labeled sample can be correctlyperformed before fractionation. Fourth, we also recommend usingstandard phosphoprotein digests such as -casein to check thechromatography system.

Evaluation of the liquid chromatography-tandem MS (LC-MS/

MS) system.The performance of the LC-MS/MS system has to beevaluated to determine whether it can be applied to phosphopeptideanalysis and whether it can support large-scale phosphoproteomicanalysis in terms of LC separation and MS. Most LC systems con-tain metal components, which can (and do) adsorb phosphopep-tides. Phosphopeptide losses can be minimized by performingSteps 4752 in the PROCEDURE. With regard to the performanceof the LC-MS system, the phosphoproteome is as complex as theproteome and benefits greatly from improvements in chroma-tographic separations. The choice of the MS/MS fragmentationmethod(s) can also affect phosphopeptide identification and sitelocalization. Certain fragmentation regimes are more appropriatethan others, but it depends on the sequence of the peptide 65. For

example, electron-transfer/capture dissociation is most appropri-ate for peptides containing three charges66,67or more, whereas allmodes of collision-induced dissociation perform sufficiently forpeptides containing two charges68,69.

Determination of phosphorylation sites.Pinpointing the exactphosphorylation site on the basis of the achieved mass spectra isimportant for biological follow-up and for understanding relevantbiological function. To confidently localize the site of phosphoryla-tion, specific diagnostic backbone fragments (also site-determiningions) must be present. Several software tools such as Ascore70, PTMscore47, Mascot delta ion score71and phosphoRS72can be used toautomatically process fragmentation spectra of phosphorylated

peptides and localize phosphorylation site. All these algorithms useMS/MS data in conjunction with the respective peptide sequencesto calculate site probabilities for all potential phosphorylationsites. In our case, we use phosphoRS (freely available from http://cores.imp.ac.at/protein-chemistry/download/ ), which handles allcommonly used fragmentation methods (collision-induced dis-sociation (CID), higher-energy C-trap dissociation (HCD) andelectron-transfer dissociation (ETD)) and data sets with high orlow mass accuracy.

Polystyrene monodisperse

microspheres

Poly(GMA-co-TMPTA) monodisperse

microspheres

Poly(GMA-co-TMPTA-PO3H2)

monodisperse microspheres Ti4+

-IMAC adsorbents

a

c

b

d

20 kV 7,000

20 kV 7,000

2 m 20 kV 7,000 2 m

2 m 20 kV 7,000 2 m

Figure 3|Characterization of the synthesized products by scanning electronmicroscopy. The scanning electron microscope imaging can be used to check

whether the product is successfully prepared as desired. (a) Monodispersepolystyrene seed microspheres. (b) Poly(GMA-co-TMPTA) monodispersemicrospheres. (c) Poly(GMA-co-TMPTA-PO3H2) monodisperse microspheres.(d) Ti4 +-IMAC adsorbents. Magnification is fixed at 7,000.
http://cores.imp.ac.at/protein-chemistry/download/http://cores.imp.ac.at/protein-chemistry/download/http://cores.imp.ac.at/protein-chemistry/download/http://cores.imp.ac.at/protein-chemistry/download/


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MATERIALSREAGENTS

CRITICALWe recommended preparing all the Ti4 +-IMAC buffers beforeenrichment and using freshly prepared Ti4 +-IMAC buffer for phosphopeptideenrichment.

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 andprotease 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) !CAUTIONTFAsolutions 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

EQUIPMENTVortex (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 massspectrometry (MALDI-TOF MS; Bruker)LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) equipped witha 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 flasksCentrifuge tubesConical tubesKimtech wipes

REAGENT SETUP

Lysis buffer Mix 8 M urea, 50 mM NH4HCO3, 1 mM sodium orthovanadate, 1tablet of complete mini EDTA-free cocktail and 1 tablet of phosphoSTOP phos-phatase inhibitor cocktail per 10 ml of lysis buffer.CRITICALIt isrecommended to prepare all the reagents fresh and to add phosphataseinhibitor and protease inhibitor tablets just before use. Keep the lysis buffer on ice.Quality control (QC) sample 1 Use a complex mixture of E. colidigests

(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 forthe evaluation of the system for phosphopeptide analysis.Sample to analyze Use the enriched sample by Ti4 +-IMAC from Step 42directly for LC-MS/MS analysis in order to reduce the sample loss duringvacuum dryness. We recommend using at least 2 mg of protein for large-scalephosphoproteomic 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 and0.6% (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 arefreshly 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) acetonitrileand 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 arefreshly prepared.CRITICALElution buffer 2 is used to recover the boundphosphopeptides by C8 plug. Elution buffer 2 will further acidify the eluent andavoid the removal of phosphates by the alkaline components.CRITICALTospecifically enrich phosphopeptides from complex digests, it is recommended touse a high concentration of TFA in loading buffer to protonate the acidic aminoresidues and break down the ion binding between the positive amino residuesand the negative phosphate groups. The intrapeptide binding often prevents theenrichment of phosphopeptide containing multiple basic residues24.

EQUIPMENT SETUP

MS analysis MALDI-TOF MS can be used to characterize the enriched samplefrom a semicomplex sample. In our study, MALDI analysis was performedon a Bruker Autoflex time-of-flight mass spectrometer. The instrument wasequipped with a delayed ion-extraction device and a pulsed nitrogen laser oper-ated 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 laserenergy was adjusted to slightly above the threshold in order to obtain good reso-lution and good signal-to-noise ratio. We obtained all mass spectra reportedin the positive-ion linear mode with delayed extraction for 50 ns. We obtainedexternal mass calibration by using two points that bracketed the mass range ofinterest. Each mass spectrum was typically summed with 50 laser shots.

LC-MS/MS is used to analyze enriched samples of higher complexity such asthose originating from mammalian cells. For the LC system, it is preferable to usea system that contains a trap column so as to allow rapid loading and desalting oflarge sample volumes. In our case, an Agilent 1100 HPLC system is connected tothe LTQ-Orbitrap Velos mass spectrometer and is equipped with a 100 m 20 mm,

3m, 120 Reprosil-Pur C18 (Dr Maisch) trapping column and a 50 m 400 mm, 3m, 120 Reprosil-Pur C18 analytical column, using a vented columnconfiguration73. Trapping is performed at 5l min1for 10 min with RP solventA, 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 enabledon the LTQ-Orbitrap Velos. In some cases, we also used a nano-UHPLC ProxeonEasy-nLC 1000 (Thermo Scientific) connected to an LTQ-Orbitrap Q-Exactivemass 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 ananalytical column (Agilent Zorbax SB-C18, 1.8 m, 35 cm 50 m). Peptides arechromatographically separated by using a gradient of 60 min, 90 min, 120 minand 180 min at a column flow rate of ~100 nl min 1, respectively . The columneffluent is directly introduced into the ESI source of the MS; HCD fragmentationis used on the Q-Exactive platform.


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PROCEDUREPreparation of polystyrene monodisperse microspheres TIMING~1.5 d1|Prepare a solution of Triton X-100 (1.14 ml) in ethanol (64 ml) in a 100-ml three-necked round-bottom flask and addpolyvinylpyrrolidone (1.25 g).

CRITICAL STEPTriton X-100 is viscous, so pipette it slowly. Pipette Triton X-100 into the flask containing 64 ml ofethanol. 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.!CAUTIONStyrene is toxic and volatile. The manipulations should be carried out in a ventilated fume hood.CRITICAL STEPThe mixture of AIBN (0.58 g) and 16 ml of styrene monomer is added into the mixture prepared in Step 2in 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 thisperiod, 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 STEPStir 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 at10,000gat 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.!CAUTIONThere is a strong irritant smell. The manipulation should be conducted in a ventilated fume hood using propernitrile gloves.CRITICAL STEPExtensive washing using ethanol can efficiently remove all the residual reactants and additives from theproducts.

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 STEPAt this point, the product can be checked by scanning electron microscopy. The prepared polystyrenemonodisperse microspheres should show property of monodispersity in size of ~4 m. PAUSE POINTThe dried polystyrene seed microsphere can be placed into a sealed tube with a cap and kept at RT forfuture use.

Preparation of poly(GMA-co-TMPTMA) monodisperse microspheresTIMING~2 d8|Prepare a 200-ml aqueous solution containing 1% (wt/wt) PVA and 0.25% (wt/wt) SDS.CRITICAL STEPHeating 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 a250-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.

!CAUTIONGMA, 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 amilk-like emulsion.CRITICAL STEPEnsure that the mixtures of oil-phase and aqueous solutions are completely emulsified. The resultinghomogenous emulsion should consist of micrometer-sized droplets, which could be checked under a microscope. If this is notachieved, polydisperse microspheres will be synthesized.


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13|Add the oil emulsion to the aqueous polystyrene seed microsphere solution under mechanical stirring at 150 r.p.m. inthe 250-ml three-necked round-bottom flask.

CRITICAL STEPThe 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 Cfor 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 theformation 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 ofacetone for each tube, respectively. Repeat the washing steps twice.

!CAUTIONPerform this step in a ventilated fume hood.CRITICAL STEPThe obtained product should be thoroughly washed with tetrahydrofuran to remove residue-swelledpolystyrene 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 STEPAt 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 msize), and a mesoporous structure on the microspheres surfaces should be observable.

PAUSE POINTThe dried poly(GMA-co-TMPTMA) microspheres can be placed into a sealed tube with a cap and kept at RT forfuture use.

Preparation of poly(GMA-co-TMPTMA-NH2) monodisperse microspheres TIMING~5 h18|Add 7 g of the anhydrous poly(GMA-co-TMPTMA) microspheres to a 250-ml double-necked round-bottom flask chargedwith 150 ml of ethylenediamine.!CAUTIONPerform 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 byaminolysis results in the formation of poly(GMA-co-TMPTMA-NH2). The schematic for the reaction is given in Figure 2c.CRITICAL STEPEthylenediamine has a few crucial roles for the final IMAC material. First, ethylenediamine introduces a freeamino-terminated group that can be used for the addition of the derivation phosphonate ligand. Second, ethylenediamineintroduces 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 mlof water and 20 ml of ethanol, respectively. Repeat the washing steps twice.

CRITICAL STEPThe 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 POINTThe dried poly(GMA-co-TMPTMA-NH2) microspheres can be placed in a sealed tube with a cap and kept at RTfor future use.

Preparation of poly(GMA-co-TMPTMA-PO3H2) microspheres TIMING~1.5 d22|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.

!CAUTIONFormaldehyde 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-TMPTMA-PO3H2) 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 ofethanol and 20 ml of water, respectively. Repeat the washing steps twice.


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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 themicrosphere does not have any visible change (Fig. 3c).CRITICAL STEPAt this point, the products can be checked by scanning electron microscopy. The poly(GMA-co-TMPTMA-PO3H2) microspheres should either show the property of monodispersity with ~12 m or the mesoporous structure. PAUSE POINTThe dried poly(GMA-co-TMPTMA-PO3H2) microspheres can be placed in a sealed tube with a cap and kept atRT for future use.

Preparation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspherebased immobilized titanium (IV) ion affinitychromatography (Ti4 +-IMAC) TIMING~12 h26|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 Ti

4 +-IMAC isgiven in Figure 2e.!CAUTIONTitanium (IV) chloride is toxic. Perform this step in a ventilated fume hood.CRITICAL STEPTiCl4in 20% HCl is used in order to prevent the formation of hydrated titanium. Gently stir the mixture toprevent the precipitation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspheres.

27|Transfer the resulting mixture into two 15-ml conical tubes and centrifuge at 20,000gfor 5 min at RT, followed byremoval of the supernatant.

28|Wash the residue with 30% acetonitrile containing 0.1% (vol/vol) TFA and centrifuge it at 20,000gfor 5 min at RT.Discard the supernatant. Repeat the process twice.

CRITICAL STEPThe 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 eachconical tube.

30|Aliquot the Ti4 +-IMAC slurry to a concentration of 10 mg ml 1in each vial and store it at 4 C for further use. The finalproduct of Ti4 +-IMAC keeps the property of monodispersity and porous structure (Fig. 3d).CRITICAL STEPAt 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 POINTThe prepared Ti4 +-IMAC slurry (Ti4 +-IMAC: 10 mg ml 1in 30% acetonitrile/0.1% TFA, (vol/vol)) can bestored at 4 C for several weeks.

Suggested preparation of protein sample and protein digests31|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 STEPDTT 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 minin 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 STEPThis step is recommended in order to stop overalkylation. (iv) Dilute the protein solution five times with 50 mM NH4HCO3to reduce the urea to


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CRITICAL STEPPhosphatase and protease inhibitors in the lysis buffer are required in order to get the mostaccurate snapshot of the protein state. Furthermore, phosphatases can become rampant during lysis, reducingphosphorylation without inhibitors.

(ii) Remove cell debris by centrifugation at 20,000gfor 15 min at 4 C. (iii) Perform a protein assay to determine the protein concentration.

CRITICAL STEPIf lysates are to be stored, immediately freeze the lysates using liquid nitrogen and then store themat 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 lof 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 for30 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 minwith gentle agitation.

CRITICAL STEPThis step is recommended to stop overalkylation. (vii) Add Lys-C from a 10-ng l 1stock 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 NH4HCO3to reduce the urea to


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CRITICAL STEPTo properly pack the Ti4 +-IMAC, a spinning

speed of 100gis preferable. To avoid high back pressure, themaximal amount of Ti4 +-IMAC should be limited to 500 gfor a 20-l GELoader tip. However, the Ti4 +-IMAC spin tipcan be also scaled up by using 250-l pipette tips.

?TROUBLESHOOTING

35|Equilibrate the Ti4 +-IMAC GELoader spin tip by adding loading buffer onto the GELoader spin tip; centrifuge it at 100gfor5 min at RT. Figure 5cshows 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 STEPTo obtain efficient enrichment and complete binding, it is recommended that the concentration of peptidesbe ~1 g l 1. To track the enrichment quality, it is also recommended to do a parallel enrichment using a standard sample

such as an -casein digest.

0

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0

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100 250 500 1,000

No. of unique phosphopeptides Speci ficity

No.ofidentified

phosphopeptides

Enrichmentspecificity(%)

Amount of sample (g)

Figure 4|The capacity of the assembled Ti4 +-IMAC GELoader spin tipprepared 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, leftyaxis) and the

specificity of phosphopeptide enrichment (red line, rightyaxis).

Figure 5|Flowchart briefly indicating the

preparation and enrichment of a sample byTi4 +-IMAC. (a) Recommended procedure forthe 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 schematicof the assembly of Ti4 +-IMAC GELoader spin

tip. (c) A graphical outline of all the stepsperformed during the enrichment process

using Ti4 +-IMAC GELoader spin tips and

centrifugation. ACN, acetonitrile.


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37|Load the sample by applying 2 50 l of the sample onto the Ti4 +-IMAC spin tip. During the loading step, keep theremaining sample on ice. Collect the flow-through containing nonphosphopeptides and immediately dry it down in a vacuumfor further use.

CRITICAL STEPCentrifuge using the slow spinning speed at ~50gfor 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 centrifuga-tion at ~170g(~3 l min 1) 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 viamoderate centrifugation at ~170g(~3 l min 1) 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 lof 10% (vol/vol) ammonia via slow centrifugation at ~100g(~1 l min 1) for 20 min at RT.CRITICAL STEPPhosphopeptides can be strongly bound onto the Ti4 +-IMAC material via the strong interaction ofphosphate groups on phosphopeptides and the immobilized Ti4 + . Hence, 10% (vol/vol) NH3H2O (pH ~11.0) is used to com-pletely recover the bound phosphopeptides.

42|Elute the sample with 2 l of 80% acetonitrile/2% FA (vol/vol) into the same tube.CRITICAL STEPIt is possible that some phosphopeptides are retained by the C8 plug; the use of elution buffer 2 ensurescomplete elution of highly hydrophobic peptides. Add 3 l of 100% FA to further acidify the samples, and then vortex andbriefly spin down the samples.

PAUSE POINTSamples can be dried down using a SpeedVac and then stored at 20 C for several weeks. Be aware that thedrying process may result in sample loss.

43|The obtained samples can be analyzed by MS. For MALDI-TOF MS analysis (see Box 1for a method to do this), samplesshould 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 ofphosphopeptides and nonphosphopeptides.CRITICAL STEPThe 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 peptidefragmentation. We highly recommend the recently published protocol by Kocher et al.75, in which the authors present anexcellent protocol to benchmark the nanoLC-MS/MS setup.

45|Check the LC-MS/MS system using protmix (20 fmol l 1) 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 analysisRe-dissolve the dried phosphopeptides (PROCEDURE Step 47) with a MALDI matrix of 2 l of DHB solution (25 mg ml 1in70% (vol/vol) acetonitrile) containing 1% (wt/vol) H3PO4and directly spot 0.5 l on the MALDI target for analysis.

CRITICAL1% H3PO4additive in the MADLI matrix can enhance the MS signal of phosphopeptides84.

Data analysisTo 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 theevaluation of the enrichment specificity, the obtained MALDI MS spectra should be compared with the expected phosphopeptide peaks.


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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 anywherefrom 1 to 10 cycles.

LC-MS/MS analysis TIMINGvariable54|Run the sample. With regard to the complexity of the enriched sample and the sensitive analysis of the enriched phos-phopeptides, 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 samplefrom the abundant fractions while prefractionation is being used. A short gradient is applicable for the enriched sample fromless-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 spectrausing 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!Tandem78and Andromeda79. Specify which enzyme was usedto 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 target-

decoy database searching strategy is enabled to evaluate the false-discovery rates (FDRs) at the peptide level in large-scalephosphoproteomic experiments80. Notably, a percolator-based algorithm has been proven to be an excellent program fordiscriminating between the positive and negative matches by integrating a number of features81. Examples of these featuresinclude Mascot score, precursor mass error, fragment mass error, the number of variable modifications and so on. To pin-

point the phosphorylation site localization, phosphoRS is used to calculate the site probability of each possible site in theidentified phosphopeptide sequence72. In this protocol, we used the Proteome Discoverer software package to process thedata; Proteome Discoverer integrates many features such as peak list extraction, database searching, percolator for FDR andphosphoRS for phosphorylation site localization.

?TROUBLESHOOTINGTroubleshooting advice can be found in Table 2.

TABLE 2 |Troubleshooting table.

Step Problem Possible reason Solution

4 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

15 Polydisperse microspheres

observed for preparation of

poly(GMA-co-TMPTMA)

monodispherse microspheres

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

31C(xi) Large pellet forms after

centrifugation of acidified

digests

The lysate is too concentrated Dilute the lysate to a protein concentration of

~1 g l 1

(continued)
http://www.matrixscience.com/http://www.matrixscience.com/


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TABLE 2 |Troubleshooting table (continued).


Inefficient digestion Check the activity of protease enzyme and ensure

the optimal conditions for protease enzyme

digestion (such as urea concentration


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TIMINGSteps 17, preparation of polystyrene monodisperse microspheres: ~1.5 dSteps 817, preparation of poly(GMA-co-TMPTMA) monodisperse microspheres: ~2 d

Steps 1825, preparation of poly(GMA-co-TMPTMA-NH2) monodisperse microspheres: ~5 hSteps 2630, preparation of poly(GMA-co-TMPTMA-PO3H2) monodisperse microspherebased immobilized titanium (IV) ionaffinity chromatography (Ti4 +-IMAC): ~12 hStep 31A, preparation of digests of standard proteins of -casein and -casein: ~1 dStep 31B, preparation of digests of standard protein of BSA: ~1 dStep 31C, suggested preparation of cell lysate: ~2 dStep 31D, peptide desalting: ~1 hSteps 3243, phosphopeptide enrichment using Ti4 +-IMAC GELoader spin tips by centrifugation: ~3 to 4 hSteps 4447, QC of the LC-MS/MS system: ~2.5 hSteps 4853, conditioning of the LC system: ~6 to 7 hSteps 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 RESULTSWe illustrate the design of Ti4 +-IMAC and the enrichment principle of phosphopeptides by Ti4 +-IMAC in Figure 1, and weprovide the scheme of the experimental workflow in Figure 5a. To provide a simple, easy-to-use, reproducible and cleanenrichment method, Ti4 +-IMAC, we used loaded GELoader spin tips in combination with centrifugation for the followingexperiments (Fig. 5b,c). Figure 5bshows the assembly of Ti4 +-IMAC-loaded GELoader spin tip.

To demonstrate the specificity of phosphopeptide enrichment using Ti4 +-IMAC, we used a semicomplex sample (-caseintryptic digests/BSA tryptic digests: 1 pmol/100 pmol or 1 pmol/1,000 pmol). Clearly, direct analysis of the semicomplexpeptide mixture (-casein tryptic digests/BSA tryptic digests: 1/100) by MALDI MS generates a spectrum with many domi-nant nonphosphopeptides (Fig. 6a). After enrichment, the MALDI MS spectrum is dominated by 16 phosphopeptides (listedin Table 1) originating from 250 fmol of -casein and very few nonphosphopeptides (marked by stars; Fig. 6b). Becauseof the fact that phosphopeptides are often less abundant than nonphosphorylated peptides, and because phosphopeptideenrichment 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 MALDIresults of this semicomplex sample show that good specificity of phosphopeptide enrichment can be achieved by Ti4 +-IMAC.

Figure 6cshows 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/MSplatform consisting of the UHPLC instrument (EASY-nLC 1000) connected to a Q-Exactive quadrupole orbitrap mass spectrom-

eter. Figure 7ashows 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 7bshows the results, including the number of unique phosphopeptides, unique phosphorylation sites and phospho-proteins, as well as the specificity of enrichment. Clearly, we observe an increasing number of identified phosphopeptidesafter increasing the separation time. In a single run, we were able to identify more than 3,000, 4,500, 5,600 and 6,000unique phosphopeptides with 60-min, 90-min, 120-min and 180-min separation times, respectively. The combined data set

TABLE 2 |Troubleshooting table (continued).


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


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of analytical triplicates resulted in a total of 4,329 unique phosphopeptides within a 60-min separation time, 6,387 uniquephosphopeptides 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 weremultiple phosphorylated peptides from individual analysis. The specificity of phosphopeptide enrichment by Ti4 +-IMAC isas high as ~90% using short separation time and ~80% using a long gradient without prefractionation. Moreover, we foundthat 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 byanother 3040%, as also shown in other reports58,59, and generally increases the coverage of the phosphoproteome. Similarto previous reports59,75, we also observed a marked increase of phosphopeptide identifications with increasing separationtime (Fig. 8e). In turn, fractionation or multidimensional separations combined with Ti4 +-IMAC are needed for resolving thecomplexity of cellular phosphoproteome.

0

1,000

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Intensity

Direct analysis

b

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Intensity

m/zm/z

m/z

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* **

1:100

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1:1,000

Figure 6|The MALDI mass spectrometric analyses of a standard peptidemixture (consisting of varying amounts of -casein and BSA digests) with

and without enrichment. (a) Mass spectrum of the peptide mixture at amolar 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 thepeptide mixture at a molar ratio of 1:1,000 (-casein to BSA digest) after

enrichment. *, nonphosphopeptides. , phosphopeptides.

10 20 30 40 50 60 70 80 90 100 110 120Time (min)

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60 min 90 min 120 min 180 min

No. of phosphoproteins No. of unique phosphosites No. of unique phosphopeptides Specificity

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 anddiffering 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 numberof unique phosphopeptides, phosphosites and phosphoproteins (bars, leftyaxis) and the specificity of phosphopeptide enrichment (purple line, righty

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.


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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 SCXchromatography12,54with Ti4 +-IMAC for the analysis of dimethyl-labeled MCF-7 samples. A total of 9,117 unique phosphorylationsites 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. Withinthis data set, ~13% of phosphopep-tides were multiply phosphorylatedpeptides. The majority of phos-phopeptides can be divided into threecategories: zero net-charged, singlycharged and multiply charged phos-phopeptides. The distribution of theidentified phosphopeptide is consistent

with the charge-based SCX separation.Multiply phosphorylated peptides aredominant in the early fraction. Singlyphosphorylated peptides with neu-tralized charge are dominant in themiddle SCX fraction. Positively chargedphosphopeptides with multiple basicresidues are most identified in the laterSCX fractions. Strikingly, over 58% of

the total unique phosphorylationsites were identified from the lateSCX fractions corresponding to the

349

585455

341

2,105

187

305

Experiment 1:3,100

Experiment 3: 3,052

Experiment2:3,2

18

60-min MS analysis time 120-min MS analysis time

878

1,015

1,001

896 463

486

3,438

Experiment 1: 5,675

Exp

eriment2:5,8

35

Experiment 3: 5,838

Experiment 1: 6,415

1,267

1,065 1,157

736 614

668

3,798

Experiment 3: 6,237

Experiment2:6,2

67

90-min MS analysis time

Experiment2:4,5

70

Experiment 1: 4,874

Experiment 3: 4,557

752

526674

313

391

552

3,179

a cb 180-min MS analysis timed

4,2811,327

293

252

319

3,465

3,135

Triplicate of 60 min: 4,329

Triplicate of 180 min: 11,200

Triplicateo

f120min:8,1

79

Cumulative of triplicate analysises of 60-min,

90-min and 180-min MS analysis timee

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) Triplicateanalysis 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.

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9~111213141617 1819202122232425262728293031323334353637383940

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a

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Inidividualfractio

n

identifications

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identifications

Figure 9|SCX/Ti4 +-IMAC approach appliedto a triple dimethyl-labeled MCF-7 sample.

(a) Bar diagram representing the individualphosphopeptides 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-handyaxis. (b) The contribution ofphosphopeptides 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.
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phosphopeptides containing multi-ple basic residues. Clearly, Ti4 +-IMACprovides efficient enrichment of notonly multiple phosphorylated peptidesand singly phosphorylated peptides butalso the phosphopeptides containingmultiple basic residues. Ti4 +-IMAC istherefore a robust and relatively unbi-ased technology for enriching a wide

range of phosphopeptide types.Figure 9bshows the contribution tothe unique phosphopeptide identifica-tions per fraction broken down by theactivation technique. HCD and CIDare most effective for the earlier SCXfraction containing lower net-chargedphosphopeptides and the phosphopep-tides carrying a 2 + charge. For the late

SCX fractions, ETD clearly outperformedHCD and CID, contributing 76% of thephosphopeptide identifications. Hence,the complementary fragmentationtechniques are highly recommended tovisualize the map of cellular phospho-proteome.

A sample enriched by Ti4 +-IMAC canalso be subjected to multidimensional

chromatography. We recently showed that Ti4 +-IMAC enrichment can be followed by high-pH RP fractionation. By applyingthis strategy, 9,719 phosphorylation sites in 2,998 proteins from human liver were identified. By performing the enrichmentfirst, the user markedly reduces the level of sample preparation. Recently, Gerber et al.showed that enrichment before SCX isa viable strategy83.

In addition to the identification of phosphopeptides by MS, determination of the exact site of phosphorylation is highlydesired. 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/MSspectra. 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 andcalculating individual site probabilities for all potential phosphorylation sites. PhosphoRS can be also used in conjunctionwith all commonly used fragmentation methods and data sets with high or low mass accuracy. To exemplify how to localizea phosphorylation site, we give examples of phosphosite localization using the phosphoRS algorithm72. Figure 10providestwo examples of phosphorylation site localization using phosphoRS. In Figure 10a, a phosphopeptide is confidently identi-fied 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 thatthese peptide fragments are not phosphorylated. Therefore, Y7 can be excluded as phosphorylation sites. Examining theC-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)

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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. Site-

determining ions are marked as diagnostic

ions. (a) A HCD spectrum of a phosphopeptidefor which the exact site of phosphorylation is

confidently localized. (b) A HCD spectrum of aphosphopeptide for which the exact site could

not be unambiguously determined.
http://cores.imp.ac.at/protein-chemistry/download/http://cores.imp.ac.at/protein-chemistry/download/http://cores.imp.ac.at/protein-chemistry/download/http://cores.imp.ac.at/protein-chemistry/download/


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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 permuta-tions for the possible location of the site for that sequence. The algorithm then scores each fragment ion list. The magnitudeof the best score and the difference of that score against the next best score are then equated to a confidence level in thesite 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, thereare 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 bephosphorylated. However, no site-determining ions exist that resolve the final ambiguity. Again, phosphRS makes all permu-tations, 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 accountfor the biologically relevant follow-up. In a large-scale phosphoproteomic experiment that consists of over 10,000phosphopeptides, 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 projectwith the grant agreement number 262067, funded by the European Union 7thFramework Program; The Netherlands Proteomics Centre, embedded in theNetherlands Genomics Initiative; the Netherlands Organization for Scientific

Research (NWO) with the VIDI grant (700.10.429); the Creative Research GroupProject by the National Natural Sciences Foundation of China (21021004); and aChina 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 theQ-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 financialinterests.

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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