Non-phosphorylated Peptides Research Articles

Phosphoproteomics: Searching for a needle in a haystack

Most of the cellular processes are regulated by reversible phosphorylation of proteins, which in turn plays a critical role in the regulation of gene expression, cell division, signal transduction, metabolism, differentiation, and apoptosis. Mass spectrometry of phosphopeptides obtained from tryptic protein digests has become a powerful tool for characterization of phosphoproteins involved in these processes. However, there is a general need to significantly enrich the phosphopeptide content to compensate their low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. This paper aims to give a comprehensive overview on the methods involved in recent phosphoproteomics. It presents a description of contemporary enrichment techniques with references to particular studies and compares different approaches to characterization of phosphoproteome by mass spectrometry.

Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

A simple method for on-target enrichment and subsequent separation and analysis of phosphorylated peptides is presented. The tryptic digest of a phosphorylated protein, in this case β-casein, is loaded onto a spot on a thin stripe made of mesoporous TiO(2) sintered onto a conductive glass surface. After washing with a salicylic buffer in order to remove the nonphosphorylated peptides, the stripe is placed in an elution chamber containing a phosphate solution. In a way analogous to thin layer chromatography (TLC), the phosphate solution acts as an eluent, clearly separating multi- and monophosphorylated peptides. By performing matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) along the stripe, the detection of all phosphorylated peptides present in the digest is facilitated, as they are isolated from each other. The method was also tested on commercial drinking milk, achieving successful separation between multi- and monophosphorylated peptides, as well as a detection limit in the femtomole range. As the enrichment, separation, and analysis take place in the same substrate, sample handling and risk of contamination and sample loss is minimized. The results obtained suggest that the method, once optimized, may successfully provide a complete phosphoproteome.

Integrin β3 Phosphorylation Dictates Its Complex with the Shc Phosphotyrosine-binding (PTB) Domain

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin β(3), Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated β(3)integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr(759), which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr(747) of the highly conserved NPXY motif of β(3). The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Hydroxyapatite as a concentrating probe for phosphoproteomic analyses

A novel method for the selective enrichment of casein phosphoproteins/phosphopeptides (CPP) from complex mixtures is reported herein. This method employs ceramic hydroxyapatite (HA) as a solid-phase adsorbent to efficiently capture phosphoproteins and CPP from complex media. Casein was chosen as the model phosphoprotein to test the protocol. CPP immobilized on HA microgranules formed a complex that was included in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI) matrix before desorbing directly from the well plate. Casein fractions with different levels of phosphorylation were desorbed based upon the specific concentration of trifluoroacetic acid (TFA) included in the MALDI matrix. The HA-bound casein enzymolysis was performed in situ with trypsin to remove non-phosphorylated peptides and isolate the immobilized CPP. The latter were recovered by centrifugation, dried, and co-crystallized with a 1% phosphoric acid (PA) solution in the matrix that was appropriate for detecting CPP in MALDI-MS spectra. This approach for the selection of casein/CPP resulted in the identification of 32 CPP by MALDI-time of flight (TOF). The analytical process involved two steps requiring ∼2 h, excluding the time required for the enzymatic reaction. The alkaline phosphatase (AP)-assisted de-phosphorylation of tryptic CPP allowed the phosphorylation level of peptides to be calculated concurrently with MALDI-TOF MS and liquid chromatography-electrospray ionization–mass spectrometry (LC–ESI–MS/MS). The effectiveness of the extraction procedure assayed on eggshell phosphoproteins resulted in the identification of 5 phosphoproteins and 14 derived phosphopeptides with a phosphoprotein global recovery of ∼70% at least.

EFFECT OF PHOSPHORYLATION ON THE RETENTION BEHAVIOR OF PEPTIDES IN ION PAIRING REVERSED-PHASE HPLC BASED ON A PREDICTION MODEL

A prediction model of the peptide retention time was developed on the basis of 358 non-phosphorylated peptides, which had various amino acid residues in the C-terminal from the casein pancreatic hydrolysates. This model was applied to predict the retention times (RT) of another 43 peptides on C4 reversed phase columns with trifluoroacetic acid (TFA) as the ion-pairing reagent, with a relatively high R2 value (0.969). Furthermore, the experimental RTs of 32 phosphopeptides were compared with the predictive RTs of their non-phosphorylated cognates. Mono- and poly-phosphopeptides seemed to elute after or before the non-phosphorylated predictive cognates, respectively. The positive charges of peptides were partly masked by the ion-pairing reagent TFA. Single and multiple phosphorylation might generally lead to an increase or reduction in the overall hydrophobicity of peptides, respectively.

Roles of Electrostatics and Conformation in Protein-Crystal Interactions

In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65–80 (pSHDHMDDDDDDDDDGD) and 220–235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC50 = 1.93 µg/ml) and OPN220-235 (IC50 = 1.48 µg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC50 = 2.97 µg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.

A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis

For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.

Inhibitor screening of protein kinases using MALDI-TOF MS combined with separation and enrichment of phosphopeptides by TiO2 nanoparticle deposited capillary column

A MALDI-TOF mass spectrometric method for rapid screening of protein tyrosine kinase (PTK) inhibitors has been developed. To circumvent the ion suppression of phosphorylated substrate peptides caused by the presence of high abundant non-phosphorylated peptides in the enzymatic reaction mixtures, a separation and enrichment process of the phosphorylated peptides from complex mixtures was carried out by using an in-house fabricated TiO(2) nanoparticle-coated capillary column prior to the MS analysis. With a synthetic phosphopeptide (DAIpYAAPFAKKK), of which the sequence is similar to that of the substrate (EAIYAAPFAKKK) of the Abelson tyrosine kinase (Abl), as the internal standard, the signal ratio of the phosphorylated substrate to the standard detected by MALDI-TOF MS is linearly correlated with the molar ratio of the two phosphopeptides over the range of 0.3 to 3 with r(2) = 0.99. We validated the MS method by determining the IC(50) value of imatinib, an Abl inhibitor for clinical treatment of chronic myelogenous leukaemia (CML). The obtained IC(50) value (234 nM) is consistent with that determined by ELISA (291 nM). Then, six analogues of imatinib synthesized in our laboratory were screened using the method, giving rise to inhibitory potential results which are in good agreement with the docking analysis data. The developed method is sensitive, operationally simple, does not require isotope-labelling and is cost/time effective, providing an alterative method for rapid screening of PTK inhibitors as therapeutic agents for tumours.

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO(2)-MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO(2)-MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO(2)-MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO(2)-MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO(2)-MOAC showed higher specificity than immobilized gallium (Ga(3+)), immobilized iron (Fe(3+)), or zirconium dioxide (ZrO(2)) affinity chromatography for phosphopeptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides.

Concurrent Quantification of Proteome and Phosphoproteome to Reveal System-wide Association of Protein Phosphorylation and Gene Expression

Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression. Concurrent quantification of phosphopeptides and non-phosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and gene expression.

Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-γ1

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-gamma1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.

A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3-hydroxypicolinic acid (3-HPA) and alpha-cyano-4-hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3-HPA and CCA were found to be hot matrices, and 3-HPA not as good as CCA and 2,5-dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3-HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive-ion and negative-ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (-80 Da) and phosphoric acid (-98 Da) from the phosphorylated-residue-containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for 'sweet' spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in-solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass-to-charge values and LIFT TOF-TOF spectra.

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI‐TOF/TOF MS

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high-sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti-phosphotyrosine antibody 4G10 was coupled covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non-phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro-phosphorylated recombinant beta-intracellular region of the granulocyte-macrophage colony-stimulating factor receptor, which was subsequently analysed by MALDI-TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.

A Phosphorylation Cascade Controls the Degradation of Active SREBP1

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

Inhibition of binding of phospholipase C gamma 1 SH2 domains to phosphorylated epidermal growth factor receptor by phosphorylated peptides.

A series of tyrosine-containing peptides 1-12: [formula: see text] (six pairs with and without the tyrosine phosphorylated) has been synthesized. The peptides were derived from tyrosine autophosphorylation sites in the epidermal growth factor receptor (EGFR): Tyr 992, 1068, 1148 and 1173. Peptide 1, derived from the Tyr 992 site, inhibited binding of a 35S-labelled fusion protein containing both of the SH2 domains from PLC gamma 1 to the phosphorylated EGFR with an IC50 of 8 microM. All of the phosphorylated peptides except 11 (1, 3, 5, 7 and 9) inhibited this binding to some degree (20-55%) at 10 microM. The nonphosphorylated peptides were inactive in this assay. The nonphosphorylated peptides 2, 4, 6, 8, 10 and 12 were obtained by standard solid-phase synthetic methodologies using both Boc/benzyl and Fmoc/tert-butyl strategies. The phosphorylated peptides 1, 3, 5, 7, 9 and 11 were similarly obtained using a Fmoc/tert-butyl strategy incorporating unprotected N alpha-Fmoc-Tyr, followed by phosphitylation and oxidation of the tyrosine in the resin-bound peptide. In addition, Asp-Ala-Asp-Glu-Phe992(4-CH2PO3H2)-Leu-Ile-Pro-Gln-Gln-Gly-O H (15), an analog of 1 incorporating an enzymatically stable phosphotyrosine mimic, 4-phosphonomethyl-L-phenylalanine, was synthesized and found to be inactive.

Synthesis of diazo functionalized solid supports and their application towards the enrichment of phosphorylated peptides

We describe herein the synthesis and application of a range of diazo-functionalized materials as platforms for the solid phase enrichment of phosphorylated peptides from protein digests. Diazo-functionalized solid phase resins are employed to bind covalently and specifically with the phosphate moiety of a peptide substrate, allowing for non-phosphorylated peptides to be efficiently removed by filtration. The technique described herein is compatible with enrichment of phospho-serine, threonine and tyrosine residues and was used to successfully enrich phosphorylated substrates from beta-casein and ovalbumin digests in low-picomolar quantities.

Characterization of Phosphorylated Peptides Using Traveling Wave-Based and Drift Cell Ion Mobility Mass Spectrometry

Phosphorylation is one the most studied and important post translational modifications. Nano electrospray mass spectrometry coupled with traveling wave (T-Wave)-based ion mobility has been used to filter for phosphorylated peptides in tryptic protein digests. T-Wave parameters have been optimized to maximize the separation between phosphorylated and non-phosphorylated peptides. A method to calibrate the T-Wave device, to provide estimates of collision cross sections, is presented, and these estimates are in excellent agreement with values obtained on drift cell instrumentation. Phosphorylated peptides have smaller cross sections which enables their separation from non-phosphorylated peptides of the same m/z. Post-mobility fragmentation is used to obtain the primary sequence for peptides of interest. This approach is shown to have potential as an additional screen for phosphorylated peptides, where up to 40% of observed peptides can be eliminated from the study.

Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry

Most current technologies for the enrichment of phosphopeptides rely on a tandem combination of different chromatography modes. Here, a fully automatic two-dimensional liquid chromatography mass spectrometry method was developed for global phosphopeptide identification. The peptide mixtures were loaded on a strong anion exchange (SAX) column under basic pH conditions and eluted with a continuous gradient to pH 2.0. This SAX system could be coupled online with reversed-phase liquid chromatography mass spectrometry (RP-LC-MS/MS). For peptide digests from a standard protein mixture spiked with synthesized phosphopeptides, most of the nonphosphorylated peptides were eluted in more basic pH than phosphopeptides, and the phosphopeptides were focused to acidic pH ranges and gradually eluted according to the phosphorylated states of peptides. Compared with the pH step elution method, the continuous gradient method displayed better repeatability and less peptide cross-overlap between fractions. This system provided a robust and fully automatic approach to large-scale phosphoproteomic profiling. For protein tryptic digests from HeLa cells, 1833 nonredundant phosphorylation sites were identified based on this two-phase separation. Compared with the method combining cation exchange and titanium dioxide, this anion-exchange based system preferred to identify more acidic and multiphosphorylated peptides. It also covered a more complete series of phosphorylation states of peptides.

The Phosphotyrosine Interactome of the Insulin Receptor Family and Its Substrates IRS-1 and IRS-2

The insulin signaling pathway is critical in regulating glucose levels and is associated with diabetes, obesity, and longevity. A tyrosine phosphorylation cascade creates docking sites for protein interactions, initiating subsequent propagation of the signal throughout the cell. The phosphotyrosine interactome of this medically important pathway has not yet been studied comprehensively. We therefore applied quantitative interaction proteomics to exhaustively profile all potential phosphotyrosine-dependent interaction sites in its key players. We targeted and compared insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) as central distributors of the insulin signal, the insulin receptor, the insulin-like growth factor 1 receptor, and the insulin receptor-related receptor. Using the stable isotope labeling by amino acids in cell culture (SILAC) approach with phosphorylated versus non-phosphorylated bait peptides, we found phosphorylation-specific interaction partners for 52 out of 109 investigated sites. In addition, doubly and triply phosphorylated motifs provided insight into the combinatorial effects of phosphorylation events in close proximity to each other. Our results retrieve known interactions and substantially broaden the spectrum of potential interaction partners of IRS-1 and IRS-2. A large number of common interactors rationalize their extensive functional redundancy. However, several proteins involved in signaling and metabolism interact differentially with IRS-1 and IRS-2 and thus provide leads into their different physiological roles. Differences in interactions at the receptor level are reflected in multisite recruitment of SHP2 by the insulin-like growth factor 1 receptor and limited but exclusive interactions with the IRR. In common with other recent reports, our data furthermore hint at non-SH2 or phosphotyrosine-binding domain-mediated phosphotyrosine binding.