Identification of Phosphorylation-Dependent Interaction Partners of the

Jun 23, 2010 - the Adapter Protein ADAP using Quantitative Mass Spectrometry: ... ADAP (adhesion and degranulation promoting adapter protein) plays an...
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Identification of Phosphorylation-Dependent Interaction Partners of the Adapter Protein ADAP using Quantitative Mass Spectrometry: SILAC vs 18O-Labeling Sabine Lange, Marc Sylvester,† Michael Schu ¨ mann, Christian Freund, and Eberhard Krause* Leibniz-Institut fu ¨ r Molekulare Pharmakologie, Robert-Ro¨ssle-Str. 10, 13125 Berlin, Germany Received April 1, 2010

The immune adapter protein ADAP (adhesion and degranulation promoting adapter protein) plays an important role in integrin-dependent migration and adhesion processes as a consequence of T cell stimulation. ADAP undergoes multiple phosphorylation events during T cell receptor (TCR) or chemokine receptor stimulation. The role of individual phosphotyrosines for protein complex formation and the regulation of cellular adhesion are still under debate. Here, we use peptide pull-down assays and quantitative mass spectrometry to identify interaction partners of site-specifically phosphorylated ADAP sequences. Phosphotyrosine peptide motifs covering Y595, Y625, and Y771 and the corresponding nonphosphorylated sequences were covalently coupled to agarose beads and incubated with Jurkat T cell lysates. For unambiguous differentiation between phosphorylation-specific and nonspecific protein interaction, we employed two different isotope labeling techniques: stable isotope labeling of amino acids in cell culture (SILAC) and enzymatic 18O-labeling, both in combination with high-resolution mass spectrometry. In addition to previously known SH2 domain-based interactions of ADAP with SLP76, we identified novel ADAP interaction partners – such as the Ras GTPase activating protein – which belong to the larger TCR proximal signaling complex. The results show that both isotope labeling techniques are well suited for distinguishing phosphorylation-specific peptide-protein interactions from the background. Keywords: quantitative proteomics • mass spectrometry • tyrosine phosphorylation • protein interactions • SILAC • 18O-labeling

Introduction Phosphorylation is an ubiquitous modification of proteins, involved in many signal transduction processes such as cell differentiation, proliferation, energy storage, and apoptosis.1 Protein-protein interactions in intracellular signaling events are often mediated by short, unstructured peptide sequences bearing phosphorylated amino acid residues. Examples of this are phosphotyrosine-containing sequences in signaling molecules. Most of these are assigned to unstructured regions of proteins which are found as docking sites for the Src homology 2 (SH2), phosphotyrosine binding (PTB), and 14-3-3 domains.2-4 The relatively small contact areas of many phosphorylationdependent peptide-protein interactions are reflected by low affinities and moderate specificities compared with the typical large surface interactions between structured proteins.5 Studies of intracellular signaling pathways by quantitative mass spectrometry in combination with phosphopeptide pull-downs have shown that individual phosphotyrosine sites can be responsible for specific protein-protein interactions.3,6 * To whom correspondence should be addressed. Tel. +49 30 94793221, Fax +49 30 94793222, E-mail: [email protected]. † Present address: Department of Biochemistry & Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark. 10.1021/pr1003054

 2010 American Chemical Society

TCR stimulation is accompanied by multiple tyrosine phosphorylation of kinases and adapter proteins (e.g., Fyn, SLP76, LAT, and ADAP).7,8 This leads to activation of integrins and to corresponding changes in the adhesive and migratory properties of the cell via nucleation of macromolecular signaling complexes.9 The adapter protein ADAP (adhesion and degranulation promoting adapter protein) is critical for the intracellular regulation of integrins10,11 and possesses a number of protein interaction motifs that mediate binding to proteins containing SH2, SH3, or EVH1 domains. Most of the Tyr phosphorylation sites in ADAP predicted to mediate proteinprotein interactions are located within the C-terminal region of the protein. It has been shown that phosphorylation of ADAP at tyrosine 625 provides a template for the binding of the Src kinase Fyn SH2 domain which also phosphorylates ADAP in vivo.8 Tyr 595 and 651 constitute binding sites for the adapter protein SLP76 which in turn interacts with guanine nucleotide exchange factor Vav1, adapter protein NCK, Tec kinase Itk, PLCγ1,and GRAP2, which are all essential components in T cell signaling and in the activation of integrins.7,12 However, since several of these T cell receptor proximal proteins contain SH2 domains themselves, there may be hierarchy or overlap in binding specificities with potential implications for the spatiotemporal organization of the inside-out-signaling complex. Journal of Proteome Research 2010, 9, 4113–4122 4113 Published on Web 06/23/2010

research articles Mass spectrometry (MS) has become a powerful tool for characterization of complex mixtures of proteins.13 Combined with tandem affinity purification (TAP) or single affinity purification protocols, capillary liquid chromatography (LC)MS approaches facilitate identification of specific proteinprotein interactions in a hypothesis-free manner.14-16 Affinity purification/MS has been successfully applied to study posttranslational modification (PTM)-dependent protein interactions which are mediated by protein domains. In such approaches, unmodified peptides and the corresponding modified peptides were immobilized and incubated with cell extracts.17-19 The bound proteins can be identified by mass spectrometry using an unbiased proteome screen which, for example, enables the identification of pyruvate kinase M2 as a novel phosphotyrosine binding protein18 or the determination of histone H3 methylation-dependent binding of transcription factors.19 Quantitative mass spectrometry using stable isotope labeling of amino acids in cell culture (SILAC)20 has been applied to the identification of specifically bound proteins (typically lowabundant signaling proteins) in the presence of a large number of background proteins. Using differentially labeled cell cultures with either “light” arginine and lysine or “heavy” arginine and lysine, nonspecifically bound proteins show MS signals of peptides with intensity ratios of about 1. In contrast, proteins with greater affinity to the modified peptide bait than to the unmodified analog have isotopically enriched mass signals. SILAC is a powerful method for relative quantification in peptide pull-down experiments. However, when metabolic labeling is impracticable (e.g., for whole organisms, primary cells), label-free approaches and isobaric tagging for relative and absolute quantification (iTRAQ)21 have been used for the relative quantification of protein abundances.22,23 Enzyme-catalyzed 18O-labeling involves the enzymatic incorporation of two 18O isotopes at the carboxyl group of peptides - formed during and after the tryptic digestion of proteins - resulting in a mass difference of 4 Da. This offers a flexible labeling technique24-26 which can be used to distinguish specific from nonspecific protein-protein interactions in affinity pull-down experiments at the low femtomole level.27 However, in comparison with the well established SILAC technique, the enzymatic 18O-labeling has experienced slower methodological development and the reported conclusions are sometimes inconclusive or even contradictory. A systematic study is lacking which compare the 18O-labeling approach with SILAC and evaluating it concerning robustness, sensitivity and dynamic range of quantified proteins in pull-down experiments. Here, we use the combination of pull-down assays and SILACbased quantitative mass spectrometry to identify protein interactions of site-specifically phosphorylated ADAP sequences. Peptide pull-down experiments were performed using three phosphotyrosine motifs – RPIEDDQEVY595DDVAE, DDDIY625DGIEE, and NDGEIY771DDIADG – and the corresponding nonphosphorylated sequences as baits. To examine whether enzymatic 18O-labeling can be used as a reliable substitute for the SILAC approach, we compared SILAC and labeling with 18O water for the unambiguous differentiation between phosphorylation-specific protein interaction and nonspecific binding to the peptide sequences.

Experimental Section SILAC Cell Culture. Jurkat T cells (clone E6-1) were cultured in RPMI1640 medium with 2 g/L NaHCO3 and 2 mM ac-Ala4114

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Lange et al. Gln (Biochrom AG, Berlin, Germany) without antibiotics in a humidified atmosphere with 5% CO2. Stable isotope labeling of amino acids in cell culture (SILAC) was performed as described.28 RPMI1640 medium deficient in arginine and lysine was used (SILAC quantification kit, Pierce). The “light” cell population was supplemented with 0.1 g/L L-Lys and 0.2 g/L L-Arg (Sigma, Deisenhofen, Germany) and the “heavy” cell population was supplemented with 10% dialyzed FBS, 0.1 g/L 13 C6-L-Lys, and 0.2 g/L 13C6 or 13C6,15N4-L-Arg (Cambridge Isotope Laboratories, Andover, MA). Cells were cultured in “heavy” or “light” media for 8 days in the presence of 10% dialyzed FBS (Biochrom). Labeling efficiencies were determined by mass spectrometry of tryptic digests of selected 1DE-bands and were found to be >95%. Preparation of Peptide Baits. Tyrosine-phosphorylated and the corresponding unmodified analogs of ADAP sequences were synthesized by standard solid-phase peptide synthesis (Fmoc chemistry) and purified by preparative HPLC as described earlier.29 After lyophilization, all peptides were shown to have >95% purity, according to HPLC with photometric detection at 220 nm, and gave the expected mass peaks by ESITOF-MS. To form the affinity matrix, peptides were immobilized on agarose beads (SulfoLink, Pierce) through the -SH group of the N-terminal cysteine. The coupling reaction was performed according to the manufacturer’s manual. The peptide loading of all matrices was determined by quantitative amino acid analysis. SILAC Peptide Pull-Down. “Heavy” and “light” labeled cells (∼2 × 107 each) were lysed in 2 × 100 µL of lysis buffer (10 mM Hepes (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 10 mM KCl, 0.5 mM EGTA) with 1% (v/v) NP-40, 1 mM Na3VO4, protease inhibitor cocktail (complete, EDTA free, Roche Applied Science, Mannheim, Germany) on ice for 30 min. The cell extracts were vortexed and centrifuged for 10 min at 8,000 × g at 4 °C. Peptide binding assays were performed using a reverse labeling strategy (crossover experiments). Hence, each form of matrix-bound peptide (phosphorylated and nonphosphorylated) was incubated with either labeled or unlabeled cell lysate resulting in two independent pull-down experiments. An equal amount of total protein (∼1.5 mg) was incubated with 20 µL agarose beads (∼15 nmol peptide) at 20 °C for 1 h. Beads were washed four times with lysis buffer to reduce nonspecific binders and bound proteins were eluted from the matrix with SDS sample buffer at 95 °C for 5 min. Labeled and unlabeled samples were combined and loaded onto Tris-glycine gradient gels (4-20%, Invitrogen) and PAGE was performed under standard conditions. Entire gel lanes were cut into 40 slices of equal size. Gel slices were washed with 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile and dried in a vacuum centrifuge. The gel pieces were reswollen in 10 µL of 50 mM ammonium bicarbonate containing 50 ng trypsin (sequencing grade modified, Promega). After 17 h incubation at 37 °C, the enzymatic reaction was terminated by addition of 10 µL of 0.5% (v/v) trifluoroacetic acid in acetonitrile, samples were sonicated for 2 min, and the separated liquid was taken to dryness under vacuum. Samples were reconstituted in 6 µL of 0.1% (v/v) TFA, 5% (v/v) acetonitrile in water. 18 O-Labeled Peptide Pull-Down. Jurkat T cells (∼4 × 107) were lysed in 200 µL lysis buffer as described in the SILAC section. The lysate was evenly divided into two sample tubes. To these samples, 20 µL of either phosphorylated or the corresponding nonphophorylated agarose-bound peptide was

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Table 1. ADAP Sequences with Crucial Phosphorylation Site Used for Peptide Pull-down Experiments

a

identifier

AA-position

AA-sequencesa

phosphorylation site

ADAP-595 ADAP-625 ADAP-771

586-600 621-630 766-777

C-RPIEDDQEVY(p)DDVAE C-DDDIY(p)DGIEE C-NDGEIY(p)DDIADG

595 625 771

N-terminal cysteine was added for covalent binding to agarose beads.

added. After incubation at 20 °C for 1 h, the beads were washed four times with lysis buffer and bound proteins were eluted from the matrix with SDS sample buffer at 95 °C for 5 min. Samples eluted from the phosphorylated or nonphosphorylated peptide baits were separated by SDS-PAGE (Tris-glycine gradient gel, 4-20%, Invitrogen) side-by-side. Whole gel lanes corresponding to eluates from the phosphorylated and nonphosphorylated ADAP peptides were cut into 40 slices in parallel fashion. Protein digestion and in-gel 16O/18O-labeling was performed as described.30 In brief, the gel pieces were incubated with 50 ng trypsin (sequencing grade modified, Promega) in 10 µL of 50 mM ammonium bicarbonate. The tryptic digestions were performed in the presence of H218O (Campro Scientific GmbH, 97% 18O) and H216O for phosphorylated and nonphosphorylated ADAP peptide pull-downs, respectively. Pull-down experiments were performed as duplicates in a crossover manner. To prevent oxygen back-exchange after mixing the samples, 10 µL of 0.5% TFA in acetonitrile was added and the separated supernatant was dried under vacuum. Samples were reconstituted in 3 µL of 0.1% (v/v) TFA, 5% (v/v) acetonitrile in water and paired gel slices (16O and 18O samples of adjoining slices) were combined immediately before nano-LC-mass spectrometry analysis. Liquid Chromatography-Tandem Mass Spectrometry. LC-MS/MS analyses were performed on a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) equipped with an Eksigent 2D nanoflow LC system (Axel Semrau GmbH). The LC system was coupled to the mass spectrometer via a nanoelectrospray source (Proxeon) with a 10 µm i.d. PicoTip ESI emitter (New Objective). Six µL of the sample was injected and concentrated on a trap column (PepMap C18, 5 µm, 100 Å, 5 mm × 300 µm i.d., Dionex) equilibrated with 0.1% TFA, 2% acetonitrile in water. After switching the trap column inline, LC separations were performed on a capillary column (Atlantis dC18, 3 µm, 100 Å, 150 mm × 75 µm i.d., Waters) at an eluent flow rate of 250 nL/min using a linear gradient of 0-40% B in 50 min. Mobile phase A was 0.1% formic acid (v/v) in water; mobile phase B was 0.1% formic acid in acetonitrile. Mass spectra were acquired in a data-dependent mode with one MS survey scan (with a resolution of 60 000) in the Orbitrap and MS/MS scans of the five most intense precursor ions in the LTQ. The MS survey range was m/z 350-1500. The dynamic exclusion time (for precursor ions) was set to 120 s and automatic gain control was set to 3 × 106 and 20,000 for Orbitrap-MS and LTQ-MS/MS scans, respectively. Data Processing and Quantification. For SILAC experiments, identification and quantification of proteins were carried out with version 1.0.12.31 of the MaxQuant software package as described.31 In brief, generated peak lists (msm files) were submitted to a MASCOT search engine (version 2.2, Matrix Science Ltd., London) and searched against an IPI human protein database (version 3.52). The mass tolerance of precursor and sequence ions was set to 7 ppm and 0.35 Da, respectively. Methionine oxidation and the acrylamide modification of cysteine were used as variable modifications. False discovery

rates were 20 GRB2-related adapter protein 2 >20 >20 Cytoplasmic protein NCK1 >20 >20 Cytoplasmic protein NCK2 >20 >20 Phosphatidylinositol 3-kinase regulatory subunit alpha 10.5 >20 1-Phosphatidylinositol-4,5-bisphosphate phosphodiesterase >20 gamma-1 >20 Lymphocyte cytosolic protein 2 12.7 >20 Crk-like protein n.b.

FER

P16591

Proto-oncogene tyrosine-protein kinase FER

RASA1

P20936

Ras GTPase-activating protein 1

SH21A

O60880

WD40A

Q5T6F0

6 4 22 7 44 63 24 30 24 38 107 123 101 106 -

SH2 domain-containing protein 1A

6.7 16.8 11.0 >20 n.i.

6 6 11 25 -

WD repeat-containing protein 40A

n.i.

-

>20 >20 10.5 17.6 >20 >20 >20 >20 >20 >20 >20 >20 13.4 18.0 12.3 >20 8.2 >20 n.i.

3 11 12 15 20 40 16 18 44 67 69 85 25 52 11 10 17 29 -

8.9 10.8 5.1 7.0

4 8 2 8

>20 >20 17.2 >20 >20 >20 >20 >20 14.4 8.3 >20 >20 >20 >20 >20 >20 n.q.

6 9 7 10 22 19 8 14 16 7 54 25 43 29 7 10 -

n.q.

-

n.i.

-

n.i.

-

a Ratios of two independent pull-down experiments with enrichment factors >5 are listed. n.i., protein not identified; n.q., no quantification possible; n.b., no phospho-specific binding (ratio 6) but was not found in ADAP771 pull-downs. The novel potential binding partner RASA1 was exclusively recruited by ADAP-595. Surprisingly, using the SILAC approach, we could not find a phosphorylation-dependent interaction with the protein tyrosine kinase Fyn, even though this has been reported to phosphorylate ADAP in vivo 4118

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and has been identified previously as a predominant interaction partner of ADAP.8 However, we could show that ADAP-625 binds with high affinity and specificity to recombinant FynSH2 domain (data not shown). We suspect that the effective concentration of soluble and interaction-competent Fyn is low in our lysates. It is well-known that the protein is lipid modified

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Figure 5. Result of pull-down experiments based on the 18O labeling approach. A, plots of the number of quantified peptides (MASCOT distiller) versus protein ratios of the ADAP-595, ADAP-625, and ADAP-771 pull-downs. Most of the identified proteins bind irrespective of the Tyr-phosphorylation (isotope ratios of about 1). Two independent pull-down experiments were performed. Every dot represents one protein (• regular pull-down, [ crossover pull-down). B, Venn diagram of quantified proteins from replicate experiments.

Figure 6. Plots of isotope ratios (log2 fold) determined by 18O labeling versus the corresponding SILAC data. Phosphorylation-dependent binders are significant outliers in both the SILAC and 18O-pull-downs.

at its N-terminus,34,35 thereby driving its compartmentalization into distinct membrane fractions that are resistant to standard detergent solubilization protocols.36

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Table 3. List of Potential Phospho-Specific Binding Partners of Different ADAP Peptide Sequences Based on Enrichment Factors Determined with 18O Labelinga ADAP-595 UniProtKB/Swiss-Prot protein accession no. CRK

P46108

GRAP2

O75791

NCK1

P16333

NCK2

O43639

PIK3R1

P27986

PLCG1

P19174

SLP76

Q13094

CRKL

P46109

ADAP-625

ADAP-771

# peptides # peptides # peptides ratio (quant) ratio (quant) ratio (quant)

description

>20 10.8 GRB2-related adapter protein 2 >20 16.2 Cytoplasmic protein NCK1 >20 >20 Cytoplasmic protein NCK2 >20 >20 Phosphatidylinositol 3-kinase regulatory subunit alpha >20 16.2 1-Phosphatidylinositol-4,5-bisphosphate phosphodiesterase >20 gamma-1 19.6 Lymphocyte cytosolic protein 2 >20 16.6 Crk-like protein n.q.

3 6 4 3 14 15 5 10 4 5 23 34 9 13 2 2 4 5 -

Proto-oncogene C-crk

FER

P16591

Proto-oncogene tyrosine-protein kinase FER

RASA1

P20936

Ras GTPase-activating protein

FYN

A0JNB0

Proto-oncogene tyrosine-protein kinase Fyn

9.2 6.7 15.0 >20 n.i.

LCK

P06239

Proto-oncogene tyrosine-protein kinase LCK

n.b.

-

P85B

O00459

Phosphatidylinositol 3-kinase regulatory subunit beta

n.i.

-

UBS3B

Q8TF42

Ubiquitin associated and SH3 domain-containing protein B n.i.

-

>20 >20 >20 >20 11.4 >20 10.9 >20 >20 >20 >20 >20 >20 >20 5.6 >20 >20 >20 n.q.

2 2 5 7 8 5 2 9 11 12 14 23 8 6 3 5 7 8 -

11.3 9.0 12.8 17.4 >20 >20 15.4 9.2

4 4 7 16 2 3 7 10

>20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 n.q.

3 9 5 13 8 12 6 9 10 12 16 22 10 16 7 12 -

n.q.

-

n.b.

-

n.b.

-

n.q.

-

n.i.

-

a Ratios of two independent pull-down experiments with enrichment factors >5 are listed. n.i., protein not identified; n.q., no quantification possible; n.b., no phospho-specific binding (ratio 20 throughout while peptides of background proteins, such as NADH show 18O/16O ratios of about 1. All pull-downs resulted in the identification and quantification of more than 500 proteins, with most of them being identified in both the phosphorylated and the corresponding nonphosphorylated peptide pull-down experiments (18O/16O ratios of about 1). A graphical representation of protein ratios versus the amount of quantified peptides shows a wider distribution for proteins which were quantified on the basis of only a few peptides (Figure 5). Furthermore, the number of quantified proteins varies from pull-down to pull-down. More than 1100 proteins were quantified consistently in the ADAP-771 pull-downs, whereas the ADAP-595 pull-downs revealed between 600 and 700 proteins. We suspect that, irrespective of the labeling approach, these variances, together with the experiment-toexperiment deviations within the replicate pull-downs, may result from minor differences in the efficiency of rinsing of nonspecific binding proteins, despite repeated washing steps. However, notably fewer proteins could be quantified by the 18 O labeling approach than with SILAC pull-downs. This can probably be explained by the different quantification algorithms used for SILAC and 18O. MaxQuant provides more accurate quantification of SILAC-labeled peptide pairs31 than does the Mascot algorithm for 16O/18O peptides. In addition, natural isotopes can overlap with the 18O-signals, leading to reduced detection sensitivity for the heavy-labeled species, thus reducing the probability of particularly low abundant proteins being quantified. However, even though the overall count of quantified proteins is reduced, the 18O approach consistently revealed all

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relevant phospho-specific binding partners which were found by the SILAC strategy. Plotting the protein ratios determined by 18O labeling versus the corresponding SILAC data shows adequate correlation between the two quantification methods (Figure 6). Despite the slightly wider distribution of nonspecific proteins found for 18O pull-downs, known ADAP interaction partners such as SLP76 could be identified by both SILAC and 18 O labeling. Table 3 lists proteins which were identified as potential phosphorylation-dependent binders by 18O labeling. The data revealed 9 and 8 relevant interaction partners for ADAP-595 and ADAP-771, respectively, and this is exactly the same results as found with SILAC. The 18O results confirm the recently discovered direct interaction between phosphorylated ADAP and NCK1 and provide further evidence for site-specific recruitment of RASA1 activating protein by Tyr-595 phosphorylation. For ADAP-625, 9 proteins were consistently identified as phospho-specific binders by 18O and SILAC. However, 4 and 2 additional SH2 domain-containing proteins were found exclusively by the 18O and SILAC approach, respectively. Interestingly, protein tyrosine kinases Fyn could not be found in SILAC experiments, even though it has been reported to be an interaction partner of ADAP via Tyr-625 phosphorylation;8 this was clearly identified by the use of two independent 18O pull-downs, with mean enrichment factors of 11.3 and 9.0 (see discussion above).

Conclusion We used synthetic peptides covalently coupled to agarose beads in combination with quantitative mass spectrometry to study phosphorylation-mediated interactions of the T cell adapter protein ADAP. The identification of specifically bound signaling molecules in the presence of an excess of nonspecific protein background requires high accuracy mass spectrometry with a maximum of MS/MS capacity, sensitivity, and dynamic range. Peptide pull-downs with Tyr-595, Tyr-625, or Tyr-771 of ADAP resulted in the identification of more than 600 proteins for each phosphopeptide. However, only a limited number of phospho-dependent binding proteins of Tyr-phosphorylated ADAP with abundance ratios (phosphopeptide vs nonphosphorylated peptide) in the range of 20 to 150 were consistently identified. In addition to previously known proteins, for example, SLP76, we identified the recently described interaction partner NCK adapter protein 1 and the novel interaction with Ras GTPase activating protein, which both belong to the larger TCR proximal signaling complex. Presumably, the individual SH2-domain containing proteins interact in a certain hierarchy with the individual ADAP phospho-Tyr sites, depending on affinity constants, relative abundance and compartmentation. Novel interactions need therefore be verified in the physiological context however, the fact that most of the potential binding partners of ADAP are reported to play a role in T cell receptor proximal signaling events39 indicates a sequence-specific contribution of tyrosine phosphorylation motifs to the formation of ADAP protein complexes. While pull-down experiments in combination with SILAC have been frequently reported, this study provides a thorough evaluation of the potency and reliability of the enzymatic 18O labeling for quantitative mass spectrometry in peptide-protein and protein-protein interaction studies. Even though the number of identified and quantified proteins is higher in pulldowns from SILAC labeled cells, our results demonstrate that the in-gel 18O labeling provides a limit-of-detection in the lowfemtomole sensitivity, which allows the identification of specif-

ically binding proteins in pull-down experiments. All of the potential binding partners of ADAP peptides which were identified by the SILAC approach were also found using the 18 O method with similar ratios. The enzymatic 18O method can be broadly used for isotope labeling, does not require any chemical modification or removal of excessive reagents, and provides the advantage that identical basic material, such as protein extracts from all kind of cells or subcellular structures, can be used for comparative studies.

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