Determining In Vivo Phosphorylation Sites Using Mass Spectrometry

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Phosphorylation is the most studied protein post?translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High?resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole?cell and tissue extracts. For the first, endogenous or epitope?tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO₂ ) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC?MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole?cell lysate, followed by peptide fractionation by strong cation?exchange chromatography, phosphopeptide enrichment by IMAC or TiO₂ , and LC?MS/MS. Alternatively, the protein lysate can be fractionated by SDS?PAGE, followed by digestion, phosphopeptide enrichment, and LC?MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC?MS/MS. Curr. Protoc. Mol. Biol. 98:18.19.1?18.19.27. © 2012 by John Wiley & Sons, Inc.

Keywords: phosphorylation; mass spectrometry; LC?MS/MS; SCX; IMAC; immunoprecipitation; SDS?PAGE; nano?LC; phosphoproteomics

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Introduction
Basic Protocol 1: Single‐Protein (Protein Complex) Phosphorylation Site Mapping
Alternate Protocol 1: Acetone Precipitation of Protein Samples (for IP Elution with Peptide or Small Molecule)
Support Protocol 1: Phosphopeptide Enrichment Using Protea Biosciences TiO2 Spin Tips
Support Protocol 2: Phosphopeptide Enrichment Using the Phos‐Trap TiO2 Phosphopeptide Enrichment Kit
Basic Protocol 2: Global Phosphorylation Analysis (Ser/Thr/Tyr)
Alternate Protocol 2: SDS‐PAGE Protein Fractionation of Whole‐Cell Extracts
Basic Protocol 3: Phosphotyrosine (pTyr) Site Identification
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Single‐Protein (Protein Complex) Phosphorylation Site Mapping

Materials

Cells/tissue source (e.g., cell line, tumor, extracted bodily tissue)
Lysis/IP buffer (see recipe )
Liquid nitrogen (−196°C)
Bradford protein assay kit (BioRad; also see unit 10.1 )
Antibody targeting protein of interest
Protein A (GE Healthcare, cat. no. 17‐0963‐03) or G (GE Healthcare, cat. no. 17‐0618‐02) agarose beads
1× SDS sample buffer (see recipe )
SDS‐PAGE Tris‐glycine polyacrylamide minigels 10‐well, 1‐mm (10% fixed or 4% to 20% gradient; Lonza, cat. no. 58511)
Tris‐glycine SDS running buffer (1× formulation: 25 mM Tris⋅Cl, 192 mM glycine, 0.1% SDS, pH 8.3)
Coomassie blue stain (15% methanol, 10% acetic acid, 2 g Coomassie Brilliant Blue)
Coomassie destain (15% methanol, 10% glacial acetic acid)
50% (v/v) acetonitrile/H₂ O (LC‐MS grade)
10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate (NH₄ HCO₃ )
100% acetonitrile
55 mM iodoacetamide (IAA) in 100 mM NH₄ HCO₃
100 mM, 50 mM and 20 mM NH₄ HCO₃
25 ng/µl trypsin (see recipe )
2% (v/v) formic acid/40% (v/v) acetonitrile
HPLC buffer A: 99% (v/v) H₂ O, 0.9% acetonitrile, 0.1% formic acid
HPLC buffer B: 100% acetonitrile

Stainless steel mortar with ceramic pestle
Platform rocker with circular motion
15‐ml conical polypropylene centrifuge tubes
Refrigerated centrifuge and microcentrifuge
End‐over‐end rotator
56° and 95°C heat block or water bath
Minigel SDS‐PAGE apparatus (BioRad or other vendor) with power supply
37°C shaking incubator
Nanoflow HPLC: ThermoFisherScientific EASY‐nLC, Waters NanoAcquity, Eksigent NanoLC Ultra (http://www.eksigent.com/), Bruker Nanoflow‐LC, or equivalent
Pico‐Frit packed C₁₈ columns: 75 µm ID × 15 cm length (New Objective, PF7515‐150H002‐3P; http://www.newobjective.com/)
High resolution/high mass accuracy mass spectrometer: ThermoFisherScientific (LTQ‐Orbitrap XL, Velos Pro Orbitrap, Velos Elite Orbitrap, qExactive), Waters Xevo, AB/Sciex 5600, Agilent QTOF 6500 series, Bruker microTOF, or equivalent
Database search engine software: e.g., Sequest (ThermoFisherScientific) or Mascot (Matrix Science; http://www.matrixscience.com/)
Proteomics Browser Software (ThermoFisherScientific or http://www.mcb.harvard.edu/microchem/)
Scaffold PTM Software (Proteome Software, Inc.; http://www.proteomesoftware.com/)

Additional reagents and equipment for protein assay (unit 10.1 ), SDS‐PAGE (unit 10.2 ), and staining of gels (unit 10.6 ),

Alternate Protocol 1: Acetone Precipitation of Protein Samples (for IP Elution with Peptide or Small Molecule)

Acetone
Protein sample
1 µg/µl TPCK‐modified trypsin prepared in 50 mM acetic acid
5% (w/v) trifluoroacetic acid (TFA)
ZipTip binding and wash buffer: 0.1% (w/v) trifluoroacetic acid (TFA)
ZipTip elution buffer: 0.1% (w/v) trifluoroacetic acid/40% acetonitrile

Acetone‐compatible tubes (polypropylene only)
pH paper
C₁₈ ZipTips (Millipore, cat. no. ZTC18S096)
0.5‐ml microcentrifuge tubes or 12 × 32 mm autosampler vials (National Scientific, cat. no. C4000‐87)

Support Protocol 1: Phosphopeptide Enrichment Using Protea Biosciences TiO2 Spin Tips

Peptide sample
Protea TiO₂ SpinTips Sample Prep Kit (Protea Biosciences) including:
- SpinTip adapers
- TiO₂ Reconstitution and Wash Solution 1
- TiO₂ Elution Solution
2‐ml microcentrifuge tubes

Support Protocol 2: Phosphopeptide Enrichment Using the Phos‐Trap TiO2 Phosphopeptide Enrichment Kit

Materials

Peptide sample
Phos‐Trap TiO₂ phosphopeptide enrichment kit (PerkinElmer) including:
- Magnetic beads
- Binding buffer
- Wash buffer
- Elution buffer

Magnet (bench top, for microcentrifuge tubes)
12 × 32 mm autosampler vials (National Scientific, cat. no. C4000‐87)

Basic Protocol 2: Global Phosphorylation Analysis (Ser/Thr/Tyr)

Materials

Cells of interest, in log phase
Urea lysis buffer (see recipe )
Liquid nitrogen (−196°C)
Bradford protein assay kit (BioRad; also see unit 10.1 )
45 mM DTT: mix 180 µl of 1.25 M DTT (19.25 g/100 ml) with 5 ml HPLC‐grade water; add to sample at ∼1/10 dilution (e.g., 0.5 ml DTT per 5‐ml sample)
110 mM IAA: Dissolve 209 mg iodoacetamide (IAA) in 10 ml HPLC‐grade water; add to sample at ∼1/10 dilution
Sequencing grade modified trypsin (100 µg/vial; Worthington, cat. no. LS02122)
50 mM ammonium bicarbonate (NH₄ HCO₃ )
20%, 10%, 1%, 0.1% (v/v) trifluoroacetic acid (TFA)
0.1% TFA/40% acetonitrile
SCX buffer A: 7 mM KH₂ PO₄ , pH 2.65 in 30% (v/v) acetonitrile
SCX buffer B: 7 mM KH₂ PO₄ /350 mM KCl, pH 2.65 in 30% (v/v) acetonitrile
IMAC (PHOS‐Select Iron affinity gel, Sigma, cat. no. P9740)
IMAC binding buffer (40% acetonitrile (v/v), 25 mM formic acid, H₂ O)
IMAC elution buffer A (50 mM K₂ HPO₄ /NH₄ OH, pH 10.0)
IMAC elution buffer B (500 mM K₂ HPO₄ , pH 7)
Methanol
40% (v/v) acetonitrile/0.5% acetic acid
1% (v/v) formic acid

Stainless steel mortar with ceramic pestle
Bath sonicator
Centrifuge
56°C water bath or heat block
pH paper
Sep‐Pak C₁₈ cartridges 6‐cc/500 mg (Waters, WAT036790) for whole digested lysate
Strong cation exchange (SCX) column: PolySULFOETHYL A 250 × 9.4 mm; 5 µm pore size; 200 Å (PolyLC, 259‐SE0502)
Nanoflow HPLC: ThermoFisherScientific EASY‐nLC, Waters NanoAcquity, Eksigent NanoLC Ultra (http://www.eksigent.com/), Bruker Nanoflow‐LC, or equivalent
1‐ml sample loop
High‐flow‐rate HPLC (offline SCX fractionation): e.g., Agilent 1200, Michrom Paradigm MG4 (Bruker), Shimadzu Prominence
15‐ml conical centrifuge tubes
Sep‐Pak C₁₈ cartridges 3‐cc/50 mg (Waters, WAT054960) for fractionated sample (after SCX); one cartridge for each fraction (12 SCX fractions = 12 Sep‐Pak 3‐cc/50‐mg cartridges)
0.5‐ml microcentrifuge tubes
Empore 3M C₁₈ material (http://www.shop3m.com/)
Cutter device (Hamilton, Needle Kel‐F hub (KF), point style 3, gauge 16, cat. no. 90516; plunger assembly N, RN, LT, LTN for model 1702 (25 µl), cat. no. 1122‐01
200‐µl pipet tips for StageTip preparation
12 × 32 mm autosampler vials (National Scientific, cat. no. C4000‐87)
High‐resolution/high mass accuracy mass spectrometer: ThermoFisherScientific (LTQ‐Orbitrap XL, Velos Pro Orbitrap, Velos Elite Orbitrap, qExactive), Waters Xevo, AB/Sciex 5600, Agilent QTOF 6500 series, Bruker microTOF, or equivalent

Additional reagents and equipment for protein assay (unit 10.1 )

NOTE: For SCX buffers, organic solvents affect the pH reading. The pH adjustments for SCX buffers A and B (see above) should be performed before the addition of acetonitrile.

Alternate Protocol 2: SDS‐PAGE Protein Fractionation of Whole‐Cell Extracts

Materials

Digested and purified sample pellet ( protocol 5 , step 24)
Lysis/IP buffer (see recipe )
1 M Tris base (pH not adjusted)
Phosphotyrosine P‐Tyr‐100 mouse antibody (mAb), Sepharose conjugated (Cell Signaling Technology, cat. no. 9419)
P‐Tyr‐100 elution buffer: 0.15% (v/v) trifluoroacetic acid (TFA)
100% acetonitrile
0.1% (v/v) trifluoroacetic acid (TFA)/40% acetonitrile
0.1% (v/v) trifluoroacetic acid

Bath sonicator
End‐over‐end rotator
Gel‐loading pipet tips
Refrigerated centrifuge
C₁₈ ZipTips (Millipore, cat. no. ZTC18S096)
Nanoflow HPLC: ThermoFisherScientific EASY‐nLC, Waters NanoAcquity, Eksigent NanoLC Ultra (http://www.eksigent.com/), Bruker Nanoflow‐LC, or equivalent

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Figures

Figure 18.19.1 Flowchart describing the sequential steps for identifying phosphorylation sites from single proteins or immunopurified simple protein complexes using tandem mass spectrometry. Describes an optional protocol for enriching phosphopeptides from digestion mixtures.

View Image

Figure 18.19.2 (A ) Example of an SDS‐PAGE minigel purification of protein(s) from different biological conditions after immunoprecipitation (IP). (B ) Amino acid coverage map showing the tryptic peptides sequenced by LC‐MS/MS in dark green and the detected phosphorylation sites highlighted in magenta. Light green highlights oxidation, an in vitro processing artifact. Ideally, for successful phosphopeptide mapping of a protein, amino acid coverage should exceed ∼80%. Phosphopeptides can be enriched by using TiO₂ or IMAC, and additional proteolytic enzymes can be used for digestion to increase amino acid coverage.

View Image

Figure 18.19.3 Flowchart describing the sequential steps for identifying global phosphorylation sites from cell and tissue lysate using fractionation, phosphopeptide enrichment, and tandem mass spectrometry. Describes method for identification of Ser/Thr/Tyr phosphorylation as well as pTyr isolation.

View Image

Figure 18.19.4 (A ) Example of an MS/MS fragmentation spectrum of the phosphorylated peptide sequence GpS PEFPGMVTDQGSR at the first serine residue. Notice the dominant neutral loss of phosphoric acid from the precursor ion and sequence‐specific fragment ions, including phosphate losses. (B ) Software such as GraphMod or ASCORE can be used to help identify the site specificity in a phosphopeptide. In this example for the phosphopeptide sequence KIpS TEDINK, the first S residue is the correct modification site. This is especially useful when adjacent or multiple STY residues are present on a phosphopeptide.

View Image

Figure 18.19.5 Example of the typical results of global phosphorylation site identification from (A ) SCX peptide fractionation followed by IMAC phosphopeptide enrichment and subsequent LC‐MS/MS analysis or (B ) SDS‐PAGE protein fractionation followed by trypsin digestion, TiO₂ phosphopeptide enrichment, and LC‐MS/MS. (C ) Data acquired by tandem mass spectrometry is searched against protein databases and results are validated to a false discovery rate (FDR) ≤1%.

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Videos

Literature Cited

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Determining In Vivo Phosphorylation Sites Using Mass Spectrometry

Abstract

Table of Contents

Materials

Basic Protocol 1: Single‐Protein (Protein Complex) Phosphorylation Site Mapping

Alternate Protocol 1: Acetone Precipitation of Protein Samples (for IP Elution with Peptide or Small Molecule)

Support Protocol 1: Phosphopeptide Enrichment Using Protea Biosciences TiO2 Spin Tips

Support Protocol 2: Phosphopeptide Enrichment Using the Phos‐Trap TiO2 Phosphopeptide Enrichment Kit

Basic Protocol 2: Global Phosphorylation Analysis (Ser/Thr/Tyr)

Alternate Protocol 2: SDS‐PAGE Protein Fractionation of Whole‐Cell Extracts

Figures

Videos

Literature Cited