Chapter 22 - Springer Link

Chapter 22 Protein Profiling and Phosphoprotein Analysis by Isoelectric Focusing Giuseppina Maccarrone and Michaela D. Filiou Abstract Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification. Key words Isoelectric focusing, Immobilized pH gradient, Mass spectrometry, Peptides, Phosphopeptides, Proteome profiling, Phosphoproteome, Synaptosomes, Mouse brain

Abbreviations CID IEF IPG LC-ESI-MS/MS MS pI S T Y

Collision-induced dissociation Isoelectric focusing Immobilized pH gradient Liquid chromatography-electrospray ionization-tandem mass spectrometry Mass spectrometry Isoelectric point Serine Threonine Tyrosine

Anton Posch (ed.), Proteomic Profiling: Methods and Protocols, Methods in Molecular Biology, vol. 1295, DOI 10.1007/978-1-4939-2550-6_22, © Springer Science+Business Media New York 2015

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Giuseppina Maccarrone and Michaela D. Filiou

Introduction Mass spectrometry (MS)-based proteome profiling provides a protein map for the biological material under investigation, facilitating functional and quantitative analyses. Phosphorylation is a post-translational modification that occurs in approximately onethird of all proteins and is involved in many biological processes in health and disease [1]. For proteome and phosphoproteome analysis, effective fractionation methods prior to MS are crucial to ensure high proteome coverage through peptide and phosphopeptide identification. Here, we describe how isoelectric focusing (IEF), which has been traditionally used to separate proteins in the first dimension of 2D-gel electrophoresis, can be used as an effective fractionation method both for peptides and phosphopeptides. Peptide mixtures are loaded onto an immobilized pH gradient (IPG) strip and upon application of electric current, peptides are separated according to their isoelectric point (pI). The unique characteristic of IEF compared to other fractionation methods is the ability to simultaneously analyze peptides and phosphopeptides. This is due to the addition of a negatively charged phosphogroup to a peptide sequence which results in a decrease of its pI. As a consequence, phosphopeptides migrate toward the acidic part of the IPG strip [2]. MS analysis of the whole and the acidic part of the IPG strip allows the investigation of the proteome and the phosphoproteome, respectively, with no additional step for phosphopeptide enrichment. The pI focusing position of each peptide on the IPG strip after IEF can be used to confirm subsequent MS-based peptide identifications and/or validate the presence of post-translational modifications [3–5]. When applied to brain tissue, IEF has been shown to result in increased proteome coverage compared to 1D-SDS gel electrophoresis [6]. In this chapter, we provide a detailed guide to proteome and phosphoproteome analysis by IEF. As study material we use synaptosomes, which are artificially isolated synapses [7], extracted from whole mouse brain. We chose brain synaptosomes for two reasons; (i) their proteome is highly complex (ii) phosphorylation plays a critical role in the regulation of synaptic function and neurotransmission [8]. In this protocol, we describe the following steps: (1) synaptosome enrichment from brain tissue and synaptosome enrichment quality control by Western blot, (2) sample preparation for IEF and IEF, (3) MS, (4) MS raw data analysis for peptide and phosphopeptide identification and assignment of phosphorylation sites. The experimental workflow is shown in Fig. 1. Mouse synaptosome analysis by IEF resulted in the identification of up to 3,000 proteins and 118 phosphoproteins [6].

IEF Peptide and Phosphopeptide Analysis

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Brain tissue

Synaptosome enrichment quality control

Synaptosome extraction

Reduction & carboxymethylation

In-gel digestion

IEF

3.5

4.5 Peptide extraction

Hexane oil extraction

Extracted/cleaned peptides

LC ESI-MS/MS

Database search

Peptide/Phosphopeptide identification

Proteome profiling

Phosphoproteome profiling Manual MS spectra validation Phosphosite assignment

Fig. 1 Experimental set-up of IEF-based analysis of proteome and phosphoproteome from mouse brain synaptosomes

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Materials

2.1 Synaptosome Enrichment and Western Blot for Enrichment Quality Control 2.1.1 Synaptosome Enrichment

1. Homogenization equipment: Branson sonifier 250 (G. Heinemann, Schwaebisch Gmuend, Germany). 2. Centrifugation equipment: 5804R centrifuge (Eppendorf) and L8-70M ultracentrifuge (Beckman Coulter). 3. Complete protease cocktail inhibitor tablets (Roche Diagnostics). 4. Phosphatase inhibitor cocktail II and III (Sigma-Aldrich). 5. 0.32 M sucrose: 2.74 g sucrose in 25 ml distilled water. 6. 0.8 M sucrose: 6.84 g sucrose in 25 ml distilled water. 7. 1.2 M sucrose: 10.26 g sucrose in 25 ml distilled water. 8. Buffer A: 0.32 M sucrose, 4 mM HEPES, complete protease cocktail inhibitor tablets (added according to the manufacturer’s instructions), pH = 7.4. Adjust pH by adding 1 M NaOH.

2.1.2 Western Blot

1. Electrophoretic equipment: Mini-PROTEAN Tetra Cell (Bio-Rad). 2. Bradforf protein assay dye reagent concentrate (Biorad). 3. Immobilon PVDF membrane (Millipore). 4. ECL Plus reagent kit (GE Healthcare). 5. TBS-T buffer: 137 nM NaCl, 20 mM Tris–HCl, pH = 8.0, 0.05 % Tween 20. 6. Blocking buffer: 5 % carnation instant non-fat dry milk in TBS-T buffer.

2.2

IEF

1. IEF equipment: Protean IEF Cell (Bio-Rad). 2. Lyophilization equipment: Savant Speed Vac plus SC210A concentrator (Thermo Fisher Scientific). 3. 200 mM ammonium bicarbonate, 8 M urea, pH = 8.5. 4. Ammonium bicarbonate. 5. 10 mM dithiothreitol. 6. 50 mM iodoacetamide. 7. Lys-C (Wako). 8. Trypsin (Promega). 9. IPG strips (pH = 3.5–4.5, 18 cm) (GE Healthcare) (see Note 1). 10. Hexane. 11. Mineral oil. 12. OMIX tips (Varian). 13. Vivaspin 5 kDa cartridge (Vivascience).


2.3

ESI-LC-MS/MS

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1. Nanoflow HPLC-2D system (Eksigent) including a trap column Zorbax SB300, 5 mm × 0.3 mm, packing material 5 μm RP-C18 (Agilent Technologies). 2. LTQ Orbitrap XL mass spectrometer (Thermo Fisher). 3. Nano electrospray ionization source (Thermo Fisher). 4. Picofrit self-packed column 75 μm × 15 cm i.d., 10 μm tip (New Objective), packing material 3 μm RP-C18. 5. Acetonitrile. 6. Formic acid.

2.4 MS Data Analysis Software

1. LC-ESI-MS/MS data acquisition software: Xcalibur v. 2.07 (Thermo Fisher). 2. Nano-LC controlling software: LC-Eksigent v.2.09 (Eksigent). 3. Raw MS and MS/MS spectra processing software: BioWorks 3.3.1 (Thermo Fisher). 4. Protein database search engine: BioWorks 3.3.1 and SEQUEST v.28 software (Thermo Fisher).

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Methods

3.1 Synaptosome Enrichment and Western Blot for Enrichment Quality Control 3.1.1 Synaptosome Enrichment

Synaptosome enrichment is performed according to [9] with slight modifications. For optimal results, perfused, snap frozen mouse brain tissue is recommended (see Note 2). 1. Tissue (mg) is homogenized in 10 volumes (μl) of Buffer A by 12–15 up and down strokes. Phosphatase inhibitors II and III are added to Buffer A at a 1:100 v/v ratio each. 2. Homogenates are centrifuged at 1,000 × g for 10 min, 4 °C (S1: supernatant 1; P1: pellet 1 containing nuclei and cell debris). 3. S1 is transferred to a different tube and P1 is resuspended in 10 volumes (μl) of Buffer A (phosphatase inhibitors optional) and centrifuged at 1,000 × g for 10 min, 4 °C (S2: supernatant 2; P2: pellet 2). S1 and S2 fractions are combined into fraction S. 4. S is centrifuged at 17,000 × g for 55 min, 4 °C (S3: supernatant 3; P3: pellet 3 containing membrane fraction with intact synaptosomes). 5. P3 is resuspended in 0.32 M sucrose and laid on top of a discontinuous sucrose density gradient (1 ml 0.32 M/1 ml 0.8 M/1 ml 1.2 M sucrose) (phosphatase inhibitors optional). 6. Sucrose gradient with P3 is ultracentrifuged at 100,000 × g for 2 h, 4 °C.

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7. Synaptosomal fraction is extracted from the 0.8 M/1.2 M sucrose interphase (see Note 3), diluted at a 1:1 v/v ratio in distilled water and ultracentrifuged at 164,000 × g for 60 min, 4 °C (S4, P4). 8. P4 is dissolved in 20 μl distilled water and stored at −20 °C (see Note 4). 3.1.2 Western Blot

1. Protein content is estimated by Bradford protein assay. 2. An aliquot of each synaptosomal fraction is loaded onto a 12.5 % SDS polyacrylamide gel. 3. Electrotransfer of protein extracts onto an Immobilon PVDF membrane is performed for 1 h at 100 V. Membranes are blocked overnight with blocking buffer, followed by incubation with the selected primary antibody for 1.5–2 h at room temperature, washing twice with TBS-T and 1 h incubation at room temperature with the corresponding secondary antibody (see Note 5). 4. Immune complexes are detected by ECL Plus reagent kit.

3.2

IEF

1. Synaptosomal proteins in distilled water are dissolved to a final concentration of 200 mM ammonium bicarbonate, 8 M urea, pH 8.5 (final volume of 100 μl) (see Note 6). 2. Sample is reduced with 10 mM dithiothreitol for 1 h at 37 °C and then alkylated in the dark by 50 mM iodoacetamide for 30 min at room temperature. 3. Urea concentration is reduced to 2 M by using a Vivaspin 5 kDa cartridge according to the manufacturer’s instructions. The sample is washed three times with 200 mM ammonium bicarbonate, pH 8.5, and concentrated to a final sample volume of 100 μl. The sample is diluted by the addition of 100 μl distilled water in a total volume of 200 μl (see Note 7). 4. The sample is digested with 24 μg endoproteinase Lys-C overnight at room temperature followed by overnight digestion with 24 μg trypsin at 37 °C (see Note 8). 5. Distilled water and urea are added to a final volume of 300 μl and a concentration of 2.5 M, respectively. 6. The sample is loaded on the IPG strip, left to be absorbed for 1 h at room temperature and then covered with mineral oil. 7. The rehydration runs for 12 h at constant voltage (50 V). 8. After rehydration is completed, electrode wicks wet with deionized water are placed at the basic and acidic ends of the IPG strip. 9. IEF is performed following the voltage/current steps according to the manufacturer’s instructions for the IPG strip used (see Note 9).


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10. After IEF completion, the IPG strip is removed from the tray and mineral oil is drained by vertically holding the IPG strip. 11. The IPG strip is cut in 4 mm pieces. Each gel piece is transferred in a sample tube prefilled with 100 μl 5 % formic acid. 12. Peptides from each gel piece are extracted with 50 μl 5 % formic acid three times (15 min vortexing and 15 min sonication) and the three supernatants are combined. To remove the residual mineral oil from the combined supernatant, the peptide extracts are overlaid with hexane, mixed briefly and the upper part of the organic solvent is discarded. The aqueous phase is kept and the oil extraction with hexane (50 μl) is repeated three times. After oil extraction, the aqueous phase is kept and lyophilized (see Note 10). 13. The resulting pellet is oil extracted with 10 μl hexane and left to dry overnight. The following day, pellets are dissolved in 25 μl 5 % formic acid, desalted with OMIX tips according to the manufacturer’s instructions and lyophilized (see Note 11). 3.3

ESI-LC-MS/MS

1. Lyophilized peptides from each IPG strip fraction are dissolved in 6 μl 1 % formic acid. 2. LC-ESI-MS/MS analysis is performed with a nanoflow HPLC-2D system coupled online to an LTQ-Orbitrap mass spectrometer via a nano ESI. The mass spectrometer is operated in the positive ion mode data-dependent scan acquisition using Xcalibur. Full scans are recorded in the Orbitrap mass analyzer (resolution-FWHM 60000) at a mass/charge (m/z) range of 380–1,600 in profile mode. The MS/MS analysis of the five most intense peptide ions per scan is recorded in the LTQ mass analyzer in centroid mode (top five method). Additional MS conditions: spray voltage 1.9–2.1 kV; normalized collision energy 35 %; dynamic exclusion 120 s; activation q = 0.25 and activation time 30 ms. Detailed MS conditions are provided in [6]. 3. From each IPG strip fraction, 3 μl are loaded onto an in-house packed column and analyzed with a 2.5 h gradient (washing with 0.1 % formic acid for 20 min and elution with 95 % acetonitrile/0.1 % formic acid from 2 to 45 % at a flow rate of 200 nl/min).

3.4

Data Analysis

3.4.1 Proteome Profiling

1. MS raw files are searched against a decoy mouse database utilizing BioWorks and SEQUEST. Search parameters are as follows: peptide mass tolerance, 20 ppm; fragment ion mass tolerance, 1 Da; enzyme, trypsin; missed cleavage sites, up to two; only tryptic peptides allowed; static modification, cysteine carboxyamidomethylation; variable modification, methionine oxidation.

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2. Protein and peptide identifications are filtered using the following parameters: Delta CN value (cross correlation normalized) is 0.08; X correlation values versus ion charge state 1.9 (+1), 2.7 (+2), 3.5 (+3). Minimum distinct peptides per protein: 2. 3.4.2 Phosphoprotein Analysis

1. For phosphoprotein identification, steps 1 and 2 of Subheading 3.4.1 are performed by including in the search parameters serine (S), threonine (T) and tyrosine (Y) phosphorylation as variable modifications (see Note 12). 2. The algorithm used for database search provides a probability score which is indicative of the reliability of the phosphopeptide identification. 3. Besides the probability score, MS/MS spectra should be manually interrogated to confirm the identification of a phosphorylation site in a peptide sequence. Phosphopeptide hits resulting from the protein database search are manually checked for the 98, 49, or 33 amu mass-shifted peaks for single, double, or triple charged ions, respectively. These m/z signals result from the neutral loss of phosphoric acid (H3PO4) from the precursor and respective b- and y-type fragment ions during the collision-induced dissociation (CID)-MS/MS fragmentation process. Due to the low stoichiometry of phosphorylation, spectra with a 98, 49, or 33 amu mass-shift loss signal of the precursor ion that is greater than 0.4 % of the base peak are considered valid phosphopeptide hits (see Note 13). 4. In case of multiple potential phosphorylation sites for a given peptide, phosphorylation site assignment depends exclusively on the observation of neutral loss of H3PO4 (98 Da) in the sequence of b- or y-type ions in one of S, T, or Y residues that could be phosphorylated (see Note 14). An example of manual phosphosite assignment is shown in Fig. 2.

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Notes 1. There are different options for IPG lengths and pH ranges according to the application of interest. For high amounts of starting material to be loaded, longer IPG strips are more appropriate. For phosphorylation studies, IPG strips with a pH range toward more acidic values are recommended [2, 10]. 2. A minimum amount of approx. 30 mg brain tissue is required to obtain a synaptosomal fraction. When studying brain regions with lower tissue weight, pooling is necessary to study the synaptosomal proteome. 3. The crude synaptosome fraction at the interface of the 1.2 M/0.8 M sucrose gradient appears as a “white cloud.”

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IEF Peptide and Phosphopeptide Analysis y*6

EGDGSATTDAAPATSPK

680.39

100 95 90 85 80

Relative Abundance

75 70 65 60 55 50 45 40 30 25 20

313.12

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y6-98

y3-98 y*3

y*

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b15-98

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y*7

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y9*

751.33

y*11

1038.55

*

937.44

y5*

583.24

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b

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976.37

706.76

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y11-98

1041.50

1314.58

b14

1139.57

y12

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y*14

b*15

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y*15

1210.71 1297.56

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Fig. 2 ESI-MSMS spectrum of Neuromodulin phosphorylated peptide EDGSATTDAAPATSPK. The single- and double-charged b- and y-fragment ions identified by SEQUEST algorithm are shown. The phosphorylation site at S15 is unambiguously determined by the loss of the phosphoric acid (H3PO4) from the y3 ion (y3-98) and by the absence of the phosphoric acid loss peak from the b14 ion fragment. Phosphorylation at S15 is ascertained by multiple fragment ions, e.g. y3-98, y6-98, y11-98, b15-98. The phosphorylation site is underlined in the peptide sequence. b- and y-fragment ions are highlighted in blue and red, respectively. Peaks marked by asterisk (*) denote phosphorylated fragments

Absorption of the interphase can be performed by a syringe and is the step that introduces the highest variability in the synaptosome enrichment protocol. 4. The sample solubility can be increased by adding some drops of 1 M NaOH. 5. For synaptosome enrichment quality control, specific antibodies for pre- and post- synaptic proteins can be used for assessing the enrichment of both pre- and post-synaptic fractions. As a negative control, a nuclear-specific antibody can be used (nuclei should not be present in the synaptosomal fraction). As positive controls commercially available synaptosome preparations can be used. For optimal monitoring of the enrichment procedure aliquots of all centrifugation steps can be loaded and analyzed. For a representative example see [6]. 6. The protein amount loaded on the IPG strip depends on its length and pI range as well as sample type. We recommend following the guidelines of the IPG strip manufacturer. For

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synaptosome protein extracts we load 300 and 200 μg protein onto IPG strips, 17 and 11 cm (pH 3.5–4.5), respectively. 7. Urea concentration should be decreased in order to avoid the inhibition of trypsin activity. The maximum total volume that can be loaded on the IPG strip is provided in the guidelines of the IPG strip manufacturer. The dilution volume should be accordingly calculated. 8. The use of Lys-C and trypsin enhances the number of proteolytic peptides with Lys and Arg at the C-terminus for highly complex protein mixtures. A combination of different restriction enzymes can be used according to sample and application requirements. 9. The advantage of the automated program is that after IEF completion the electric current is kept at a constant value of 50 μA, thus maintaining the resolution of the pI-based peptide separation. 10. The peptide extraction by formic acid is very efficient due to the low percentage and thin layer of polyacrylamide in the IPG strip. The removal of the mineral oil from the peptide extracts is crucial to obtain high-quality MS data and avoid polymers. Hexane extraction does not have a negative impact on peptide extraction efficacy [2]. 11. Desalting/filtering prior to mass spectrometry analysis is to our experience required so as to eliminate salts and other ionsuppressor compounds which reduce the efficiency of the ionization process and result in low intensive peaks, compromising the peptide/protein identification. 12. Under normal conditions, phosphorylation generally occurs more often on S and T residues. Phosphorylation on Y residues accounts for less than 1 % of the O-phosphorylated residues in a protein sequence. 13. As in this protocol no phosphoprotein enrichment step precedes phosphopeptide analysis, the number of identified phosphopeptides is lower compared to phosphoproteome-targeted analyses due to the low stoichiometry of phosphorylation in a peptide mixture. Strategies exclusively directed to phosphopeptide analysis may include a phosphoprotein enrichment step prior to IEF, as previously described [11]. 14. It should be noted that the unambiguous assignment of phosphorylation sites in phosphopeptides with multiple potential phosphorylation sites can be hindered by gas-phase phosphate group rearrangement reactions occurring under CID-MS/MS conditions [12].


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Acknowledgments This work is funded by the Max Planck Society. M.D.F. is supported by a grant from the Deutsche Forschungsgemeinschaft (FI 1895/1-1). We thank Chris Turck for useful comments. References 1. Cohen P (2001) The role of protein phosphorylation in human health and disease. The Sir Hans Krebs Medal Lecture. Eur J Biochem 268:5001–5010 2. Maccarrone G, Kolb N, Teplytska L, Birg I, Zollinger R, Holsboer F, Turck CW (2006) Phosphopeptide enrichment by IEF. Electrophoresis 27:4585–4595 3. Cargile BJ, Bundy JL, Freeman TW, Stephenson JL Jr (2004) Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification. J Proteome Res 3:112–119 4. Uwaje NC, Mueller NS, Maccarrone G, Turck CW (2007) Interrogation of MS/MS search data with an pI Filter algorithm to increase protein identification success. Electrophoresis 28:1867–1874 5. Xie H, Bandhakavi S, Roe MR, Griffin TJ (2007) Preparative peptide isoelectric focusing as a tool for improving the identification of lysine-acetylated peptides from complex mixtures. J Proteome Res 6:2019–2026 6. Filiou MD, Bisle B, Reckow S, Teplytska L, Maccarrone G, Turck CW (2010) Profiling of mouse synaptosome proteome and phosphoproteome by IEF. Electrophoresis 31: 1294–1301 7. Schrimpf SP, Meskenaite V, Brunner E, Rutishauser D, Walther P, Eng J, Aebersold R,

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