Selectivity For Phosphopeptides Research Articles

Optimization of Enrichment Conditions on TiO2Chromatography Using Glycerol As an Additive Reagent for Effective Phosphoproteomic Analysis

Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.

Enrichment specificity of micro and nano‐sized titanium and zirconium dioxides particles in phosphopeptide mapping

Owning to their anion-exchange properties, titanium and zirconium dioxides are widely used in phosphopeptide enrichment and purification protocols. The physical and chemical characteristics of the particles can significantly influence the loading capacity, the capture efficiency and phosphopeptide specificity and thus the outcome of the analyses. Although there are a number of protocols and commercial kits available for phosphopeptide purification, little data are found in the literature on the choice of the enrichment media. Here, we studied the influence of particle size on the affinity capture of phosphopeptides by TiO2 and ZrO2. Bovine milk casein derived phosphopeptides were enriched by micro and nanoparticles using a single-tube in-solution protocol at different peptide-to-beads ratio ranging from 1 : 1 to 1 : 200. Unsupervised hierarchical cluster analysis based on the whole set of Matrix Assisted Laser Desorption/Ionization time-of-flight mass spectra of the phosphopeptide enriched samples revealed 62 clustered peptide peaks and shows that nanoparticles have considerably higher enrichment capacity than bulk microparticles. Moreover, ZrO2 particles have higher enrichment capacity than TiO2. The selectivity and specificity of the enrichment was studied by monitoring the ion abundances of monophosphorylated, multiphosphorylated and non-phosphorylated casein-derived peptide peaks at different peptide-to-beads ratios. Comparison of the resulting plots enabled the determination of the optimal peptide-to-beads ratios for the different beads studied and showed that nano-TiO2 have higher selectivity for phosphopeptides than nano-ZrO2 particles.

Two-in-One Strategy for Effective Enrichment of Phosphopeptides Using Magnetic Mesoporous γ-Fe2O3 Nanocrystal Clusters

Designed with a two-in-one strategy, the magnetic mesoporous γ-Fe(2)O(3) nanocrystal clusters (m-γ-Fe(2)O(3)) have been successfully prepared for integrating the functions of effective enrichment and quick separation of phosphopeptides into a single architecture. First, the mesoporous Fe(3)O(4) nanocrystal clusters (mFe(3)O(4)) were synthesized by solvothermal reaction and then were subjected to calcination in air to form m-γ-Fe(2)O(3). The obtained m-γ-Fe(2)O(3) have spherical morphology with uniform particle size of about 200 nm and mesoporous structure with the pore diameter of about 9.7 nm; the surface area is as large as 117.8 m(2)/g, and the pore volume is 0.34 cm(3)/g. The m-γ-Fe(2)O(3) possessed very high magnetic responsiveness (Ms = 78.8 emu/g, magnetic separation time from solution is less than 5 s) and were used for the selective enrichment of phosphopeptides for the first time. The experimental results demonstrated that the m-γ-Fe(2)O(3) possessed high selectivity for phosphopeptides at a low molar ratio of phosphopeptides/nonphosphopeptides (1:100), high sensitivity (the detection limit was at the fmol level), high enrichment recovery (as high as 89.4%), and excellent speed (the enrichment can be completed in 10 min). Moreover, this material is also quite effective for enrichment of phosphopeptides from the real sample (drinking milk), showing great potential in the practical application.

Highly selective recovery of phosphopeptides using trypsin-assisted digestion of precipitated lanthanide–phosphoprotein complexes

The basic idea of this study was to recover phosphopeptides after trypsin-assisted digestion of precipitated phosphoproteins using trivalent lanthanide ions. In the first step, phosphoproteins were extracted from the protein solution by precipitation with La(3+) and Ce(3+) ions, forming stable pellets. Additionally, the precipitated lanthanide-phosphoprotein complexes were suspended and directly digested on-pellet using trypsin. Non-phosphorylated peptides were released into the supernatants by enzymatic cleavage and phosphopeptides remained bound on the precipitated pellet. Further washing steps improved the removal of non-phosphorylated peptides. For the recovery of phosphopeptides the precipitated pellets were dissolved in 3.7% hydrochloric acid. The performance of this method was evaluated by several experiments using MALDI-TOF MS measurements and delivered the highest selectivity for phosphopeptides. This can be explained by the overwhelming preference of lanthanides for binding to oxygen-containing anions such as phosphates. The developed enrichment method was evaluated with several types of biological samples, including fresh milk and egg white. The uniqueness and the main advantages of the presented approach are the enrichment on the protein-level and the recovery of phosphopeptides on the peptide-level. This allows much easier handling, as the number of molecules on the peptide level is unavoidably higher, by complicating every enrichment strategy.

C60-fullerene bound silica for the preconcentration and the fractionation of multiphosphorylated peptides

Phosphorylation of proteins is an important cellular regulatory process. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of phosphorylated species. Mass spectrometry (MS) of phosphopeptides obtained from tryptic protein digests is the method-of-choice for characterization of phosphorylated proteins. However, determination of phosphopeptides by MS represents a major challenge, especially in the presence of unmodified peptides. Due to lower ionization efficiency of phosphopeptides, as well as the fact that the stoichiometry of phosphorylation is often present at low relative abundance, efficient enrichment of the phosphorylated peptides prior to MS analysis is therefore of high demand. In addition, successful identification of peptides with different phosphorylation grades still remains challenging.This work presents a new strategy for enrichment and subsequent selective elution of multi-, mono- and nonphosphorylated peptides based on their difference in pI by using pH gradient elution in presence of different concentration of acetonitrile prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis (MALDI-MS). The developed protocol was successfully applied for α-casein tryptic digest and bovine serum albumin digest spiked with 9 synthetic phosphopeptides. Further selectivity for phosphopeptides was demonstrated by fractionation of peptides from a milk digest.

Ti4+‐Immobilized Magnetic Composite Microspheres for Highly Selective Enrichment of Phosphopeptides

AbstractArchitectural design is essential to achieve ideal chemical and biological properties of nanomaterials. In this article, a novel route to fabricate high‐quality magnetic composite microspheres composed of a high‐magnetic‐response magnetic colloid nanocrystal cluster (MCNC) core, a poly(methylacrylic acid) (PMAA) interim layer, and a Ti4+‐immobilized poly(ethylene glycol methacrylate phosphate) (PEGMP) shell via two‐step distillation–precipitation polymerization is presented. The unique as‐synthesized MCNC@PMAA@PEGMP‐Ti4+ composite microsphere is investigated for its applicability for selective enrichment of phosphopeptides from complex biological samples. The experiment results demonstrate that, by taking advantage of the pure phosphate–Ti4+ interface and high Ti4+ loading amount, the MCNC@PMAA@PEGMP‐Ti4+ composite microsphere possesses remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/nonphosphopeptides (1:500). The extreme sensitivity, excellent recovery of phosphopeptides, and high magnetic susceptibility are also proven. These outstanding features demonstrate that the MCNC@PMAA@PEGMP‐Ti4+ composite microspheres have great benefit for the pretreatment before mass spectrometric analysis of phosphopeptides. Furthermore, the performance of the approach in selective enrichment of phosphopeptides from drinking milk and human serum gives powerful evidence for its high selectivity and effectiveness in identifying the low‐abundant phosphopeptides from complicated biological samples.

Tailor-Made Magnetic Fe3O4@mTiO2 Microspheres with a Tunable Mesoporous Anatase Shell for Highly Selective and Effective Enrichment of Phosphopeptides

Selective enrichment of phosphoproteins or phosphopeptides from complex mixtures is essential for MS-based phosphoproteomics, but still remains a challenge. In this article, we described an unprecedented approach to synthesize magnetic mesoporous Fe(3)O(4)@mTiO(2) microspheres with a well-defined core/shell structure, a pure and highly crystalline TiO(2) layer, high specific surface area (167.1 m(2)/g), large pore volume (0.45 cm(3)/g), appropriate and tunable pore size (8.6-16.4 nm), and high magnetic susceptibility. We investigated the applicability of Fe(3)O(4)@mTiO(2) microspheres in a study of the selective enrichment of phosphopeptides. The experiment results demonstrated that the Fe(3)O(4)@mTiO(2) possessed remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), large enrichment capacity (as high as 225 mg/g, over 10 times as that of the Fe(3)O(4)@TiO(2) microspheres), extreme sensitivity (the detection limit was at the fmol level), excellent speed (the enrichment can be completed in less than 5 min), and high recovery of phosphopeptides (as high as 93%). In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Fe(3)O(4)@mTiO(2) composite microspheres high benefit for mass spectrometric analysis of phosphopeptides.

Src homology 2 domain proteomimetics: developing phosphopeptide selective receptors

SH2 domains are the paradigm of phosphotyrosine protein recognition modules and are involved in mediating numerous cancer-promoting protein–protein complexes. In an effort to develop protein-selective receptors for inhibition of protein complexation events, we herein report the de novo rational and computationally aided design of SH2 domain proteomimetics. Lead mimetics exhibit target phosphopeptide selectivity (Ka ≈ 107 M−1) and potently disrupt SH2 domain:phosphopeptide interactions.

Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides

Phosphorylation, one of the most important post-translational modifications of protein, plays a crucial role in a large number of biological processes. Large-scale identification of protein phosphorylation by mass spectrometry is still a challenging task because of the low abundance of phosphopeptides and sub-stoichiometry of phosphorylation. In this work, a novel strategy based on the specific affinity of zirconium arsenate to the phosphate group has been developed for the effective enrichment of phosphopeptides. Zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe(3)O(4)@SiO(2)) were prepared by covalent immobilization of zirconium arsenate on Fe(3)O(4)@SiO(2) magnetic nanoparticles under mild conditions, and characterized by transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometer (VSM). The prepared ZrAs-Fe(3)O(4)@SiO(2) was applied for the selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA). Our results demonstrated that the ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles possess higher selectivity for phosphopeptides and better capture capability towards multiply-phosphorylated peptides than commercial zirconium dioxide (ZrO(2)), which has been widely employed for the enrichment of phosphopeptides. In addition, endogenous phosphopeptides from human serum can be effectively captured by ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles. It is the first report, to the best of our knowledge, in which the zirconium arsenate-modified magnetic nanoparticles were successfully applied to the enrichment of phosphopeptides, which offers the potential application of this new material in phosphoproteomics study.

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004 [1]; Larsen et al., 2005 [2]; Kweon and Hakansson, 2006 [3]; Cantin et al., 2007 [4]; Collins et al., 2007 [5]). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO 2-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007 [6]). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO 2 and ZrO 2 columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.

Improved method of phosphopeptides enrichment using biphasic phosphate‐binding tag/C18 tip for versatile analysis of phosphorylation dynamics

A number of factors including low stoichiometry of phosphorylation, ion suppression, and reduced peptide backbone fragmentation interfere with precise identification of proteins in phosphoproteomic analysis by MS. Therefore, enrichment of phosphopeptides is an important process for subsequent mass spectrometric analysis. Here, we have developed a simple and efficient method for phosphopeptides enrichment, which employs a biphasic phosphate-binding tag (Phos-tag)/C18 tip consisting of overlaid Phos-tag on the C18 resin in a pipet tip. The improvement in selectivity for phosphopeptides was achieved by using a 40% ACN solution under the phosphopeptides binding conditions. We also assessed the adequacy of Phos-tag/C18 tip for quantitative phosphoproteomic analysis using the iTRAQ technology. After protein digestion and subsequent iTRAQ labeling, interfering substances including excess iTRAQ reagent were directly removed by Phos-tag/C18 tip in a single step. Applying this method, phosphoproteomic analysis of HeLa cells stimulated with tumor necrosis factor -alpha was rapidly and successfully achieved.

Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano‐powders

The potential of an organic monolith with incorporated titanium dioxide (TiO(2)) and zirconium dioxide (ZrO(2)) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.

Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution

Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO 2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO 2 using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.

Sequence, Structure and Energetic Determinants of Phosphopeptide Selectivity of SH2 Domains

Here, we present an approach for the prediction of binding preferences of members of a large protein family for which structural information for a number of family members bound to a substrate is available. The approach involves a number of steps. First, an accurate multiple alignment of sequences of all members of a protein family is constructed on the basis of a multiple structural superposition of family members with known structure. Second, the methods of continuum electrostatics are used to characterize the energetic contribution of each residue in a protein to the binding of its substrate. Residues that make a significant contribution are mapped onto the protein sequence and are used to define a “binding site signature” for the complex being considered. Third, sequences whose structures have not been determined are checked to see if they have binding-site signatures similar to one of the known complexes. Predictions of binding affinity to a given substrate are based on similarities in binding-site signature. An important component of the approach is the introduction of a context-specific substitution matrix suitable for comparison of binding-site residues. The methods are applied to the prediction of phosphopeptide selectivity of SH2 domains. To this end, the energetic roles of all protein residues in 17 different complexes of SH2 domains with their cognate targets are analyzed. The total number of residues that make significant contributions to binding is found to vary from nine to 19 in different complexes. These energetically important residues are found to contribute to binding through a variety of mechanisms, involving both electrostatic and hydrophobic interactions. Binding-site signatures are found to involve residues in different positions in SH2 sequences, some of them as far as 9 Å away from a bound peptide. Surprisingly, similarities in the signatures of different domains do not correlate with whole-domain sequence identities unless the latter is greater than 50%. An extensive comparison with the optimal binding motifs determined by peptide library experiments, as well as other experimental data indicate that the similarity in binding preferences of different SH2 domains can be deduced on the basis of their binding-site signatures. The analysis provides a rationale for the empirically derived classification of SH2 domains described by Songyang & Cantley, in that proteins in the same group are found to have similar residues at positions important for binding. Confident predictions of binding preference can be made for about 85% of SH2 domain sequences found in SWISSPROT. The approach described in this work is quite general and can, in principle, be used to analyze binding preferences of members of large protein families for which structural information for a number of family members is available. It also offers a strategy for predicting cross-reactivity of compounds designed to bind to a particular target, for example in structure-based drug design.

Phosphoproteome Analysis of Capacitated Human Sperm: EVIDENCE OF TYROSINE PHOSPHORYLATION OF A KINASE-ANCHORING PROTEIN 3 AND VALOSIN-CONTAINING PROTEIN/p97 DURING CAPACITATION

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

A Single Point Mutation Switches the Specificity of Group III Src Homology (SH) 2 Domains to That of Group I SH2 Domains

Src homology 2 (SH2) domains recognize phosphotyrosine-containing sequences, and thereby mediate the association of specific signaling proteins in response to tyrosine phosphorylation (Pawson, T., and Schlessinger, J. (1993) Curr. Biol. 3, 434-442). We have shown that specific binding of SH2 domains to tyrosine-phosphorylated sites is determined by sequences adjacent to the phosphotyrosine. Based on the phosphopeptide specificity and crystal structures, SH2 domains were classified into four different groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778). The beta D5 residues of SH2 domains were predicted to be critical in distinguishing these groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778; Eck, M. J., Shoelson, S. E., and Harrison, S. C. (1993) Nature 362, 87-91). We report here that replacing the aliphatic residues at the beta D5 positions of two Group III SH2 domains (phosphoinositide 3-kinase N-terminal SH2 domain and phospholipase C-gamma C-terminal SH2 domain) with Tyr (as found in Group I SH2 domains) results in a switch in phosphopeptide selectivity, consistent with the specificities of Group I SH2 domains. These results establish the importance of the beta D5 residue in determining specificities of SH2 domains.

SH2 domains recognize specific phosphopeptide sequences

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-lle/Pro while another group (SH2 domains of p85, phosphollpase C-γ, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, F́yn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.