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. 2025 Nov 12;10(46):55316–55324. doi: 10.1021/acsomega.5c04034

Efficient Isolation and Enrichment of SH2 Domain Protein Species from Plasma Exploiting Specific Peptide–Protein Interactions

Zhen-Cun Cai ‡, Long-Long Zhao †, Qing Chen †,§,*, Xue Hu †,§,*
PMCID: PMC12658793  PMID: 41322625

Abstract

Fiber SiO2 microspheres are modified with a phosphorylated peptide chain through a Schiff base reaction, and the product, pPeps@SiO2 microspheres, is obtained. The interaction between the phosphorylated peptide chain and the specific site of the SH2 domain offers the as-prepared microspheres with good capture performance toward SH2 domain proteins. Under the condition of pH 4, the capture efficiencies of pPeps@SiO2 microspheres for SH2-containing proteins SH2–SH2, SH2–SH3, and SH2–PTP are 91%, 61.3%, and 62.96%, respectively, which are much higher than those for proteins without an SH2 domain. The adsorbed SH2 proteins can be readily recovered by using 0.1 mol L–1 imidazole; therefore, a strategy for the isolation of SH2 domain proteins from complex matrices is proposed. SDS-PAGE results indicate that the isolation of the SH2–SH3 protein from plasma using pPeps@SiO2 microspheres is successfully achieved, and the isolation process is not affected by other high-abundance protein species in plasma. The concentration of SH2 domain protein in plasma increases from 12.4 pg mL–1 to 61.59 pg mL–1 after treating with pPeps@SiO2 microspheres, which well demonstrates the favorable separation and enrichment ability of pPeps@SiO2 microspheres. The isolation strategy based on protein domains not only provides the basis for in-depth investigation of SH2 domain protein functions but also provides new ideas for protein separation and purification.

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Introduction

Protein interactions are a complicated issue in proteomic research, and the protein domain is the starting point for studying protein interaction. The Src homology 2 (SH2) domain, a protein module comprising approximately 100 amino acids, was first identified in the oncogenic protein v-Src and subsequently found in hundreds of human protein species. All the known SH2 domains are conservative folded and include an antiparallel β-sheet in the middle and a sandwich-like structure of α-helices on each side. − The SH2 domain can specifically recognize phosphorylated tyrosine (pTyr) and 3–6 amino acid residues at the C-terminus of pTyr, − and it is an important part of the tyrosine kinase signal transduction pathway. The activation of cancer genes such as Src, Abl, Ras, and Raf, due to the enhancement of tyrosine kinase activity, is directly or indirectly mediated by the SH2 domain. The abnormality of the signaling pathway involving the SH2 domain is related to a variety of tumors and genetic diseases. In patients with chronic myelogenous leukemia, it was found that the SH2 domain with a self-inhibition function upstream of Abl is missing, which leads to increased kinase activity. The target molecules Shc and p62, recruited by the hyperphosphorylated SH2 domain, indirectly enhance the Ras pathway. Previous research has indicated that various growth factor receptors play significant roles in the initiation and progression of diverse tumor types. Grb2 is the main pathway of growth factor receptor-mediated signal transmission, so its SH2 domain target molecular mimics are expected to block the conduction of this signaling pathway. Abnormalities in the SH2 domain have also been found in some genetic diseases. Point mutations or deletion mutations in the SH2 domain of the SAP protein will cause X-linked lymphocytosis. A single-point mutation within the SH2 domain of Bruton’s tyrosine kinase has been implicated in the onset of X-linked agammaglobulinemia. Similarly, mutations in the kinase domain of ZAP-70 may result in severe combined immunodeficiency (SCID). Noonan syndrome patients have point mutations in the N-terminal gene encoding the SH2 domain of the SHP-2 molecule. STAT5b, expressed by patients with growth hormone insensitivity syndrome, contains only half of the SH2 domain and so on.

Considering the extensive existence of the SH2 domain in different protein species and its role in regulating corresponding signal transduction pathways, the investigation on the SH2 domain’s ligand recognition characteristics and the corresponding target molecular mimics is quite favored by biologists and pharmacists. Cantley and colleagues demonstrated that the SH2 domain selectively recognizes phosphotyrosine-containing proteins or synthetic peptides based on specific amino acid sequences, while showing minimal affinity for their unphosphorylated counterparts. Fantl et al. reported that peptides containing the pTyr-Met/Val-X-Met sequence exhibit high affinity for phosphatidylinositol 3-kinase, while the replacement of the Met residue at the end of the sequence induces a significant reduction in affinity. Peptides containing the sequence Tyr-X-X-Leu/Ile (about 10 amino acids) can specifically recognize the SH2 domain contained in the Src family, and a bidentate-binding mode is formed between this amino acid sequence and tyrosine kinases with two SH2 domains. Several strategies have been previously developed for SH2 domain protein isolation using synthetic phosphorylated peptides. For instance, Testini et al. employed phosphopeptide-based affinity resins to investigate VEGFR2 signaling via SH2 interactions, while Höfener et al. utilized Inhibitor Affinity Purification (IAP) to probe SH2-domain interactions in proteomic workflows. , Building upon these foundational studies, we designed a novel platform by grafting phosphotyrosine-containing peptides onto fibrous SiO2 microspheres, which offer higher surface area and better accessibility compared to conventional supports. This design enables the selective enrichment of SH2-domain proteins through noncovalent interactions, forming the basis of our separation strategy.

Generally, materials with a pore structure are collectively referred to as porous materials. Based on pore size, pore structures are typically classified into three categories: macropores (>50 nm), mesopores (2–50 nm), and micropores (<2 nm). Studies have shown that the penetrating mesoporous and macroporous structures are beneficial for reducing diffusion resistance, providing a larger contact area, and offering more active sites. − Silica-based porous substances have gained widespread attention due to their excellent resistance to acidic and basic environments, ease of synthesis through various techniques, and abundant raw material sources. In particular, fibrous silica microspheres possess a broadly distributed mesoporous network, and their fiber-like architecture can also function as a macroporous framework, endowing them with a hierarchical porous structure. − Therefore, this work selects fibrous SiO2 microspheres as the carrier to carry the polypeptide chain to achieve the separation and purification of proteins.

The binding of the SH2 domain to tyrosine-phosphorylated proteins or peptide chains primarily depends on the amino acid sequence surrounding the phosphotyrosine residue, and the arrangement of amino acids around phosphorylated tyrosine is the key to achieving specific binding. Therefore, we delicately designed phosphorylated tyrosine-containing peptide chains with specific amino acid arrangements and immobilized them on porous fibrous SiO2 nanoparticles via Schiff base reactions to achieve the separation and enrichment of SH2 domain proteins from complex samples, i.e., human plasma.

Experimental Section

Materials and Reagents

See details in Supporting Information.

Instruments

See details in the Supporting Information.

Fabrication of pPeps@SiO2 Microspheres

The fibrous SiO2 microspheres were sequentially modified to obtain the final peptide-functionalized materials. First, SiO2–NH2 microspheres were prepared by reacting pristine fibrous SiO2 with (3-aminopropyl)­triethoxysilane (APTES), introducing surface amino groups. These amino-functionalized microspheres were then treated with 2.5% glutaraldehyde (GA) to obtain GA@SiO2 microspheres via Schiff base activation. Finally, phosphorylated peptides (pPep1) were covalently immobilized onto GA@SiO2 through the reaction with terminal amine groups, forming the final pPeps@SiO2 microspheres. These modified microspheres were thoroughly washed with PBS and stored at 4 °C prior to use. Detailed synthesis protocols for each modification step are provided in the Supporting Information.

Adsorption of Proteins by pPeps@SiO2 Microspheres

To evaluate the protein adsorption capacity of pPeps@SiO2 microspheres, a range of protein models, including SH2–SH2, SH2–SH3, SH2–PTP, PTP, HSA, IgG, and Trf, are employed. In the typical procedure, 1 mL of the protein solution is incubated with 2 mg of the microspheres at ambient temperature under vigorous shaking for 30 min to facilitate binding. Following incubation, the suspension is subjected to centrifugation at 6000 rpm for 5 min. The resulting supernatant is analyzed for unbound protein content via absorbance measurement at 595 nm, following the Bradford method. The quantity of adsorbed protein (Q) is subsequently determined using the corresponding formula:

Q=(C0−C1)×VM

where C 0 and C 1 represent the initial and residual protein concentrations (mg·L–1), V denotes the volume of the protein solution (L), and M refers to the mass of the adsorbent (g).

The pPeps@SiO2 microspheres with adsorbed proteins are then washed with deionized water and incubated with 1 mL of 0.1 mol·L–1 imidazole solution. After shaking for 30 min, the mixture is centrifuged at 6000 rpm for 5 min, and the supernatant is collected for subsequent SDS-PAGE analysis and quantitative assays.

Results and Discussion

Preparation and Characterization of pPeps@SiO2 Microspheres

The preparation of pPeps@SiO2 microspheres involves four steps, as illustrated in Scheme . pPeps@SiO2 microspheres are first synthesized according to a hydrothermal synthesis procedure and then modified with amino propyltriethoxysilane. The phosphorylated peptide pPep1 (sequence: Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu), containing a central phosphotyrosine residue, was designed based on canonical SH2-binding motifs, especially those recognized by Src-family kinase SH2 domains. Such sequences typically contain a central phosphotyrosine (pTyr) flanked by acidic or hydrophobic residues at the +1 to +3 positions, which are known to enhance specificity and affinity. To better contextualize the design of our phosphorylated peptide (pPep1), we compiled a comparison table (Table ) summarizing representative SH2-binding sequences previously reported in the literature. These canonical motifs generally feature a central phosphotyrosine flanked by residues that modulate domain-specific binding. In contrast, pPep1 was rationally designed to enhance both SH2-domain affinity and compatibility with immobilization onto fibrous SiO2 surfaces. The inclusion of multiple Glu and hydrophobic residues at the +1 to +5 positions provides enhanced electrostatic and hydrophobic interaction potential, distinguishing it from shorter or naturally occurring SH2-binding sequences. This peptide was covalently attached to the surface of fibrous SiO2 microspheres through a Schiff base reaction, which occurs between the aldehyde groups of glutaraldehyde and the amino groups of the peptides, resulting in the formation of pPeps@SiO2 microspheres. To confirm the phosphorylation-dependent binding specificity of the SH2 domain, a solution-phase binding assay was performed using the nonphosphorylated control peptide pPep2 (sequence: Glu-Pro-Gln-Tyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu). Given that SH2 domains are known to specifically recognize phosphotyrosine-containing motifs, the absence of detectable interactions with pPep2 served as a negative control, reinforcing the critical role of phosphorylation in mediating the observed binding. Accordingly, pPep2 was not immobilized on the microspheres and was exclusively employed in the solution-phase experiments.

1. Schematic Illustration of the Preparation Process for pPeps@SiO2 Microspheres.

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1. Comparison of Representative SH2 Domain-Binding Peptide Sequences.

Peptide Name Sequence (N→C)
pYVNV pTyr-Val-Asn-Val
Crk-SH2 canonical motif Glu-pTyr-Val-Asn
pYEEI pTyr-Glu-Glu-Ile
VEGFR2-SH2 binding Gln-pTyr-Leu-Asn
Inhibitor affinity probe Ac-pTyr-Ala-Pro-Leu
GpYLPQTV Gly-pTyr-Leu-Pro-Gln-Thr-Val
pYENV pTyr-Glu-Asn-Val
pPep1 (this work) Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu
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The SEM reveals that the fibrous SiO2 microspheres exhibit a spherical morphology composed of intertwined fibers, with an average diameter of approximately 500 nm, and display a typical mesoporous architecture (Figure A). Following modification with polypeptide chains, the mesoporous features of the microspheres are well preserved (Figure B). As shown in Figure D, the TEM image of the pPeps@SiO2 microspheres reveals an increase in particle size and a decrease in pore diameter compared to that of the unmodified fibrous SiO2 microspheres (Figure C). The EDX mapping images of pPeps@SiO2 microspheres reveal the successful formation of the fibrous SiO2 framework. C, N, and P are uniformly distributed across the surface, and the contents were 12%, 6%, and 23%, respectively (Figure ). These values represent the relative atomic percentages of each element detected on the microsphere surface, rather than the bulk composition. The relatively high phosphorus content arises from localized enrichment of phosphate groups introduced by the phosphotyrosine peptides. As EDX is surface-sensitive and detects elements within the top 1–2 μm of the sample, the observed phosphorus level reflects successful and dense peptide grafting rather than overall microsphere composition. , This shows that the polypeptide chain is successfully modified onto the surface of the fibrous SiO2 microspheres and is homogeneously distributed.

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SEM images of fibrous SiO2 microspheres (A) and pPeps@SiO2 microspheres (B). TEM images of fibrous SiO2 microspheres (C) and pPeps@SiO2 microspheres (D).

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EDX mapping images of pPeps@SiO2 microspheres.

X-ray photoelectron spectroscopy (XPS) was utilized to investigate the chemical states of N and C elements in SiO2–NH2 microspheres, GA@SiO2 microspheres, and pPeps@SiO2 microspheres (Figure ). The N 1s spectrum shown in Figure A, compared with SiO2–NH2, shows that both GA@SiO2 microspheres and pPeps@SiO2 microspheres have OC–NH (401.3 eV) except −NH2 (399.6 eV), and GA@SiO2 microspheres also have −NH– (400.4 eV), which is attributed to peptide chain side chain functional groups. The C 1s spectra (Figure B) of SiO2–NH2 were fitted into C–C (284.6 eV) and −C–N (285.3 eV). Compared with SiO2–NH2, both GA@SiO2 microspheres and pPeps@SiO2 microspheres have OC–NH (285.9 eV) and C–O (286.4 eV). Compared with GA@SiO2, SiO2@pPeps shows a significant increase in the N 1s peak of OC–NH after modifying the polypeptide chain. This further proves that the polypeptide chain was effectively grafted onto the microsphere surface.

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XPS analysis of (A) high-resolution C 1s spectrum and (B) high-resolution N 1s spectrum of SiO2–NH2 microspheres, GA@SiO2 microspheres, and pPeps@SiO2 microspheres.

The BET analysis reveals that the specific surface areas and total pore volumes of fibrous SiO2, GA@SiO2, and pPeps@SiO2 microspheres are approximately 569.36 m2·g–1 and 2.111 cm3·g–1, 228.39 m2·g–1 and 1.365 cm3·g–1, and 148.65 m2·g–1 and 1.150 cm3·g–1, respectively. Nitrogen adsorption–desorption isotherms of pPeps@SiO2 microspheres exhibit a typical type IV pattern with an H3 hysteresis loop (Figure A), confirming that the mesoporous architecture is retained following functionalization with polypeptide chains. FT-IR spectra of fibrous SiO2 microspheres, GA@SiO2 microspheres, and pPeps@SiO2 microspheres are illustrated in Figure B. The GA@SiO2 and pPeps@SiO2 microspheres display characteristic absorption peaks at 2957 cm–1 and 2856 cm–1, corresponding to the asymmetric bending vibrations of −CH3 and −CH2 groups, respectively. The absorption peaks of pPeps@SiO2 microspheres at 1635 cm–1, 1460 cm–1, and 3437 cm–1 are attributed to the trans and cis structures of peptide bond stretching vibrations of −NH–. These above characteristic peaks suggest the functionalization of polypeptide chains on the final pPeps@SiO2 microspheres through polypeptide chain modification.

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Nitrogen adsorption–desorption isotherms of fibrous SiO2, GA@SiO2, and pPeps@SiO2 microspheres (A). FT-IR spectra of fibrous SiO2, GA@SiO2, and pPeps@SiO2 microspheres (B). XRD patterns of fibrous SiO2 and pPeps@SiO2 microspheres (C). TGA curves of fibrous SiO2, SiO2–NH2, GA@SiO2, and pPeps@SiO2 microspheres (D).

The broad diffraction peak observed at 2θ = 22.13° in the XRD patterns of both fibrous SiO2 microspheres and pPeps@SiO2 microspheres indicates that the structural integrity of the SiO2 framework remains unaffected by polypeptide chain modification (Figure C). TGA results indicate that fibrous SiO2, aminated SiO2 (SiO2–NH2), GA@SiO2, and pPeps@SiO2 microspheres all exhibit an initial weight loss around 100 °C, which is attributed to the release of physically adsorbed or crystalline water (Figure D). It can be seen from the curve that the organic layer on the surface of the fibrous SiO2 microspheres begins to decompose at about 200 °C. From the DTG curve of pPeps@SiO2 microspheres (Figure S1), it can be seen that the material has an endothermic peak at about 210 °C, which corresponds to the decomposition of the amino group on the surface of the microspheres, and there is an endothermic peak around 461 °C, which represents the decomposition peak of the modified polypeptide chain.

Binding Ability of Phosphotyrosine-Containing Peptides to SH2 Domain Protein

While biophysical techniques, such as isothermal titration calorimetry (ITC), fluorescence polarization (FP), or gel mobility shift assays, are widely used to characterize protein–ligand interactions with quantitative affinity data, the current study focuses on confirming the structural composition and stoichiometry of peptide–protein complexes. ESI-MS, though indirect in determining binding strength, provides a powerful and sensitive means to directly detect noncovalent complexes under mild conditions. In particular, it is well suited for verifying the successful formation of specific peptide–SH2 domain interactions, especially in combination with functional enrichment assays. Therefore, the use of ESI-MS in this context is both appropriate and effective for the intended analytical purpose. Figure shows the ESI mass spectra of the SH2 domain protein SH2–SH3 and the binding of SH2–SH3 to different ratios of specifically designed peptide chains. The molecular weight of SH2–SH3 protein was confirmed by electrospray ionization mass spectrometry (ESI-MS). As shown in Figure A, SH2–SH3 displayed a typical multiply charged pattern with characteristic m/z peaks at 820.02, 851.55, 885.56, 922.43, 962.49, 1006.15, 1054.08, 1106.76, and 1164.99. Among these, the peak at m/z = 1164.99 with a charge state of z = 19+ corresponds to a calculated molecular mass of 22,114.6 Da, which closely matches the theoretical molecular weight of SH2–SH3 (∼22.2 kDa). , This strongly supports the correct identification and integrity of the protein under native ESI-MS conditions. It can be seen from Figure B that a small amount of free SH2–SH3 exists when the ratio of SH2–SH3 to the phosphorylated tyrosine peptide chain is 1:1, but when the ratio increases to 1:10 (Figure C), the signal of free SH2–SH3 cannot be observed, indicating that SH2–SH3 has been completely combined with the peptide chain to form a noncovalent complex. To elucidate the mass shift upon peptide binding, we focus on the peak at m/z = 840.50 in Figure B, which corresponds to the [M + 28H]^28+ ion of the SH2–SH3–pPep1 complex. This peak reflects the addition of the peptide mass (approximately 1,554.6 Da) to the SH2–SH3 domain (22,114.6 Da), resulting in a complex with a total mass of 23,668.85 Da. The observed m/z shift from the unbound SH2–SH3 at m/z ≈ 814.13 to the complex at m/z = 840.50 confirms the binding of pPep1, as the mass difference is consistent with the peptide’s mass distributed over the 28 positive charges. , While for the nonphosphotyrosine peptide chain, even when the concentration is increased to 10 times that of SH2–SH3, the formation of complexes is still not observed (Figure D). These results strongly suggest the binding specificity of the SH2 domain protein toward the phosphorylated tyrosine peptide.

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Electrospray ionization mass spectra of SH2–SH3 (A), 1:1 SH2–SH3 protein:pPep1 molar ratio (B), 1:10 ratio with pPep1 (C), and 1:10 ratio with pPep2 (D).

Protein-Capturing Behaviors by pPeps@SiO2 Microspheres

The capturing behaviors of SH2–SH2, SH2–SH3, SH2–PTP, PTP, HSA, IgG, and Trf onto pPeps@SiO2 microspheres were examined across a range of pH conditions. As illustrated in Figure A, SH2 domain-containing proteins exhibit significantly higher adsorption compared to non-SH2 proteins under pH 4.0 conditions. The adsorption efficiencies for SH2–SH2, SH2–SH3, and SH2–PTP at pH 4.0 are 91%, 61.3%, and 62.96%, respectively. While at the same pH conditions, the adsorption of non-SH2 proteins is rather poor, and adsorption efficiencies for PTP, HSA, IgG, and Trf are 19.67%, 19.14%, 15.53%, and 12.18%, respectively. The adsorption difference between SH2 domain-containing proteins and non-SH2 proteins can be attributed to multivalent interactions between the SH2 motifs and the peptide chains anchored on the SiO2 microsphere surface. It is proposed that electrostatic interactions may occur between Arg205 (βD1) in the SH2 domain and both the phosphorylated tyrosine residue and the second Glu residue near the phosphorylated tyrosine in the peptide chain of pPeps@SiO2 microspheres. Additionally, potential hydrogen bonding might be formed between Lys200 (βD3) and/or Tyr202 (βD5) of the β-4 strand in the SH2 domain and the first Glu residue near the phosphorylated tyrosine. At the same time, the Ile in the peptide chain is likely to bind to the hydrophobic pocket of the SH2 domain through van der Waals forces, which possibly consist of the methyl groups of Tyr202 (βD5), Thr215 (EF1), and the side chains of Ile214 (βE4) and Leu237 (BG4). These multiple interactions thus contribute to the favorable adsorption of SH2 domain proteins on pPeps@SiO2 microspheres.

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Adsorption selectivity and surface charge properties of the pPeps@SiO2 microspheres. Capture efficiencies of different standard proteins on pPeps@SiO2 microspheres at various pH values (A), Zeta potential measurements of pPeps@SiO2 microspheres under the same pH conditions (B). Experimental conditions: protein solution, 100 mg·L–1, 1.0 mL; pPeps@SiO2 microspheres, 2 mg; adsorption time, 30 min.

The capture efficiency of the material for SH2–SH3 at pH 7 is significantly higher than that of SH2–SH2 and SH2–PTP. This is because the phosphorylated tyrosine peptide chain mainly binds to specific sites in the β-sheet in the SH2 domain. At pH 7, the fibrous SiO2 microspheres display a net negative surface charge due to the deprotonation of silanol and carboxyl groups. The SH2 fusion proteins used in this study have theoretical isoelectric points between 6.5 and 7.2, indicating that at pH 7 they are close to neutral or mildly positively charged. More importantly, positively charged residues near the phosphotyrosine-binding site of SH2 domains enable favorable electrostatic interactions with the negatively charged microsphere surface, enhancing binding specificity. It can be seen from the circular dichroism spectrum that SH2–SH3 presents a configuration dominated by β-sheets (Figure B), which is structurally compatible with the canonical β-sheet-based phosphotyrosine-binding groove of SH2 domains. In contrast, SH2–SH2 and SH2–PTP (Figure A and C) exhibit mixed secondary structures rather than clear α-helix dominance, suggesting partial disruption or shielding of the β-sheet interface involved in peptide recognition. This structural variation may arise from domain–domain packing (in SH2–SH2) or interference by adjacent functional domains (e.g., the PTP domain in SH2–PTP), which could reduce the binding site accessibility. These conformational effects likely weaken the interaction between the peptide and the SH2 domain, thus reducing the capture efficiency. The sudden increase in the capture efficiency of IgG at pH 7 is due to the fact that IgG is positively charged when the pH of the solution is 7, and the pPeps@SiO2 microspheres are negatively charged at this time (Figure B). There is an electrostatic force between the pPeps@SiO2 microspheres and IgG.

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CD spectra of SH2–SH2 (A), SH2–SH3 (B), and SH2–PTP (C) at different pH buffers.

Capturing of SH2 Domain Protein from Human Plasma by pPeps@SiO2 Microspheres

As the as-prepared pPeps@SiO2 microspheres exhibit superior adsorption performance toward SH2 domain proteins over non-SH2 proteins, their potential for selectively capturing SH2-containing proteins from human plasma was further explored. In order to highlight the capturing ability of pPeps@SiO2 microspheres, 1 mL of a plasma sample spiked with 100 μg mL–1 SH2–SH3 protein was subjected to the adsorption procedure and then recovered with an imidazole solution (0.1 mol L–1).

SDS-PAGE analysis was carried out following Laemmli’s protocol, employing a conventional discontinuous buffer system. As shown in Figure , multiple protein bands are observed in the untreated human plasma sample (Lane 2), corresponding to a wide molecular weight range of 6.5–200 kDa. These bands are primarily attributed to IgG (150 kDa), transferrin (Trf, 80 kDa), human serum albumin (HSA, 66.4 kDa), IgG heavy chain (50.0 kDa), IgG light chain (25.0 kDa), and SH2–SH3 (22.11 kDa), as well as other low-abundance proteins. Lane 3 displays the result of the supernatant treated with pPeps@SiO2 microspheres. It can be seen that after treatment with pPeps@SiO2 microspheres, there is almost no change in the bands of high-abundance plasma proteins HSA, IgG, and Trf in the plasma, while the bands of SH2–SH3 are significantly lighter. Lane 4 is the recovered solution, in which only one band of SH2–SH3 appears, suggesting that pPeps@SiO2 microspheres can efficiently capture SH2 domain proteins from plasma and eliminate the interference of high-abundance proteins.

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SDS-PAGE analysis of the SH2 domain protein enrichment from plasma using pPeps@SiO2 microspheres. Lane 1: protein molecular weight markers (kDa); Lane 2: 100-fold diluted human plasma spiked with SH2–SH3; Lane 3: supernatant after treatment with pPeps@SiO2 microspheres; Lane 4: protein eluate recovered after desorption.

In order to ascertain the efficiency of pPeps@SiO2 microspheres in capturing SH2 domain proteins from plasma, the contents of SH2 domain proteins in raw plasma and plasma after capturing are quantified using a Human Csk/Src molecule C-terminal kinase (C-src) enzyme-linked immunoassay kit. As shown in Figure , the original concentration of SH2 domain protein detected in plasma is determined to be 12.4 pg mL–1, and the final concentration of SH2 domain protein in the recovered solution is determined to be 61.59 pg mL–1, indicating a total recovery of 61.59 pg of SH2 domain proteins. This corresponds to a 5.1-fold enrichment, further demonstrating the microspheres’ selective capture capability from complex biological samples.

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Quantitative enrichment of SH2 domain proteins from human plasma by pPeps@SiO2 microspheres. The results confirm the effective capture and recovery of target SH2 proteins from complex biological samples, highlighting the practical applicability of the designed material.

Conclusions

The SH2 domain plays a crucial role in signal transduction pathways mediated by tyrosine kinases. The abnormality of the signal transduction pathway involving the SH2 domain is related to a variety of tumors and genetic diseases. A novel phosphorylated peptide chain-functionalized pPeps@SiO2 microsphere has been prepared. Attributed to its structural features and specific interactions between phosphorylated peptide chains and SH2 domains, the pPeps@SiO2 microspheres exhibit excellent adsorption selectivity and high binding capacity for SH2 domain-containing proteins. The pPeps@SiO2 microsphere can eliminate the interference of high-peak proteins and effectively separate and purify proteins containing the SH2 domain from human plasma, providing effective help for further research on the relationship between SH2 domain proteins and cancer.

Supplementary Material

ao5c04034_si_001.pdf (294.8KB, pdf)

Acknowledgments

This work was supported by the PhD Start-up Foundation of Liaoning Province (2022-BS-339), the Natural Science Foundation Project of Liaoning Province (No. 2024-MS-222), and the Science and Technology Foundation of Shenyang Medical College (20229049), the Key R&D Project of the Liaoning Provincial Department of Science and Technology ( 2025JH2/102800051), the Future Industry Frontier Technology Project of the Liaoning Provincial Department of Science and Technology ( 2025080219-JH2/1013).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04034.

  • Materials and reagents and Instruments. The fabrication of pPeps@SiO2 microspheres. ESI-MS/MS of SH2 domain–peptide Complexes. Capture of SH2–SH3 proteins from plasma using pPeps@SiO2 microspheres. Isolation and detection of SH2 domain proteins from human plasma. DTG curves of pPeps@SiO2 microspheres. The SDS-PAGE assay results of expressed protein. The codon optimized SH–SH2 sequence for E. coli expression. The codon optimized SH2–SH3 sequence for E. coli expression. The codon optimized SH2–PTP sequence for E. coli expression. The codon optimized PTP sequence for E. coli expression (PDF)

ETHICS: All human whole blood experiments were conducted in accordance with applicable laws and institutional guidelines, with approval from the ethics committee of Shenyang Medical College.

The authors declare no competing financial interest.

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