A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE.

BACKGROUND: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. RESULTS: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 µM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 µg/mL) than classical Coomassie brilliant blue (14.11 µg/mL). SIGNIFICANCE: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Protein profile pattern analysis: A multifarious, in vitro diagnosis technique for universal screening.

Universal health care is attracting increased attention nowadays, because of the large increase in population all over the world, and a similar increase in life expectancy, leading to an increase in the incidence of non-communicable (various cancers, coronary diseases, neurological and old-age-related diseases) and communicable diseases/pandemics like SARS-COVID 19. This has led to an immediate need for a healthcare technology that should be cost-effective and accessible to all. A technology being considered as a possible one at present is liquid biopsy, which looks for markers in readily available samples like body fluids which can be accessed non- or minimally- invasive manner. Two approaches are being tried now towards this objective. The first involves the identification of suitable, specific markers for each condition, using established methods like various Mass Spectroscopy techniques (Surface-Enhanced Laser Desorption/Ionization Mass Spectroscopy (SELDI-MS), Matrix-Assisted Laser Desorption/Ionization (MALDI-MS), etc., immunoassays (Enzyme-Linked Immunoassay (ELISA), Proximity Extension Assays, etc.) and separation methods like 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE), Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Capillary Electrophoresis (CE), etc. In the second approach, no attempt is made the identification of specific markers; rather an efficient separation method like High-Performance Liquid Chromatography/ Ultra-High-Performance Liquid Chromatography (HPLC/UPLC) is used to separate the protein markers, and a profile of the protein pattern is recorded, which is analysed by Artificial Intelligence (AI)/Machine Learning (MI) methods to derive characteristic patterns and use them for identifying the disease condition. The present report gives a summary of the current status of these two approaches and compares the two in the use of their suitability for universal healthcare.

Two-Dimensional Polyacrylamide Gel Electrophoresis Coupled with Nanoliquid Chromatography-Tandem Mass Spectrometry-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in the MCF7 Breast Cancer Cell Line Transfected for Jumping Translocation Breakpoint Protein Overexpression.

The identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and understand the molecular mechanisms of BC initiation and progression. The jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors, including BC. Thus, the JTB protein could have the potential to be used as a biomarker in BC, but its neoplastic mechanisms still remain unknown or controversial. We previously analyzed the interacting partners of JTBhigh protein extracted from transfected MCF7 BC cell line using SDS-PAGE complemented with in-solution digestion, respectively. The previous results suggested the JTB contributed to the development of a more aggressive phenotype and behavior for the MCF7 BC cell line through synergistic upregulation of epithelial-mesenchymal transition (EMT), mitotic spindle, and fatty acid metabolism-related pathways. In this work, we aim to complement the previously reported JTB proteomics-based experiments by investigating differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB overexpression using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Statistically different gel spots were picked for protein digestion, followed by nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. We identified six DEPs related to the JTBhigh condition vs. control that emphasize a pro-tumorigenic (PT) role. Twenty-one proteins, which are known to be usually overexpressed in cancer cells, emphasize an anti-tumorigenic (AT) role when low expression occurs. According to our previous results, proteins that have a PT role are mainly involved in the activation of the EMT process. Interestingly, JTB overexpression has been correlated here with a plethora of significant upregulated and downregulated proteins that sustain JTB tumor suppressive functions. Our present and previous results sustain the necessity of the complementary use of different proteomics-based methods (SDS-PAGE, 2D-PAGE, and in-solution digestion) followed by tandem mass spectrometry to avoid their limitations, with each method leading to the delineation of specific clusters of DEPs that may be merged for a better understanding of molecular pathways and neoplastic mechanisms related to the JTB's role in BC initiation and progression.

Fiber Type Identification of Human Skeletal Muscle.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy. The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples. These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Hydrophobicity and molecular mass-based separation method for autoantibody discovery from mammalian total cellular proteins.

Serum autoantibody profiles are unique to individuals and reflect the level and history of autoimmunity and tumor immunity. The identification of autoantibody biomarkers is critical for the development of immune monitoring systems for immune-related disorders. Here, we present a practical method for large-scale autoantibody discovery using total cellular proteins from cultured mammalian cells. We found that nucleic acid-free and fully denatured water-soluble total cellular proteins from mammalian cells were superior, allowing precise separation by reversed-phase HPLC after preparing a large set of homogeneous total cellular proteins. After separating the proteins based on hydrophobicity, the fractionated samples were subjected to molecular mass analysis using conventional SDS-PAGE. The resulting two-dimensional gel electrophoresis was successfully employed for immune blotting and LC-MS/MS analysis. All procedures, including TRIzol-based total cellular protein extraction, solubilization of denatured proteins, reversed-phase HPLC separation, and SDS-PAGE, were highly reproducible and easily scalable. We propose this novel two-dimensional gel electrophoresis system as an alternative proteomics-based methodology suitable for large-scale autoantibody discovery.

A novel method for the component identification of human blood products: Mass spectrometric analysis of human fibrinogen digested after SDS-PAGE in-gel digestion.

Human fibrinogen, as a blood product of special origin, is relatively simple to prepare and purify. Therefore, completely isolating and removing the relevant impurity proteins is difficult. Further, which impurity protein components are present is not clear. In this study, human fibrinogen products from seven enterprises were collected from the market, and the presence of impurity proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the major 12 impurity proteins were identified and screened by in-gel enzymolysis mass spectrometry, and 7 major impurity proteins with different peptide coverage were identified by enzyme-linked immunosorbent assay, in agreement with the mass spectrometry results. The seven major impurity proteins included fibronectin, plasminogen, F-XIII, F-VIII, complement factor H, cystatin-A, and α-2-macroglobulin. The final test results were in the range of undetectable to 50.94 µg/mL, with correspondingly low levels of impurity proteins between different companies and a manageable risk. Moreover, we found that these impurity proteins existed in the form of polymers, which might also be an important cause of adverse reactions. This study established a protein identification technique applicable to fibrinogen products, which provided new ideas for studying the protein composition of blood products. In addition, it provided a new means of testing for companies to monitor the flow of proteomic fractions and improve the purification yield and product quality. It laid the foundation for reducing the risk of clinical adverse reactions.

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 PO 3 - ${\rm{PO}}_3^ - $ monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 PO 3 - $\;{\rm{PO}}_3^ - $ monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 PO 3 - ${\rm{PO}}_3^ - $ monomers), trimetaphosphate (trimetaP: 3 PO 3 - ${\rm{PO}}_3^ - $ ring), tripolyphosphate (tripolyP), pyrophosphate (PPi ), and inorganic orthophosphate (o-Pi ) were visualized after separation by an in situ hydrolytic degradation process to o-Pi that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. 31 P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PPi diffusivity by <10%. Linear regression between the Rf peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 PO 3 - ${\rm{PO}}_3^ - $ monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-Pi , but no PPi .

Determination of the biological origin of enzyme preparations using SDS-PAGE and peptide mass fingerprinting.

Enzymes are mainly extracted from the culture broth of microorganisms. Various commercially available enzyme preparations (EPs) are derived from different microorganisms, and the source of the EP should be the same as that mentioned in the manufacture's information. The development of analytical methods that can determine the origin of the final products is important for ensuring that the EPs are nontoxic, especially when used as food additives. In this study, various EPs were subjected to SDS-PAGE, and the main protein bands were excised. After in-gel digestion, the generated peptides were analysed using MALDI-TOF MS, and protein identification was performed by searching the set of peptide masses against protein databases. In total, 36 EPs including amylase, ß-galactosidase, cellulase, hemicellulase and protease were analysed, and the information about the enzyme sources was obtained for 30 EPs. Among these, the biological sources determined for 25 EPs were consistent with the manufacturer's information; for the remaining five, enzymes produced by closely-related species were shown as matching proteins due to high sequence similarity. Six enzymes derived from four microorganisms could not be identified because their protein sequences were not registered in the database. As these databases are expanded, this approach of using SDS-PAGE and peptide mass fingerprinting (PMF) can determine the biological origin of enzymes rapidly and contribute to ensuring the safety of EPs.

Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies.

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.

Monitoring the Kinase Activity of Heme-Based Oxygen Sensors and Its Dependence on O2 and Other Ligands Using Phos-Tag Electrophoresis.

The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [32P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).

Characterization of beta subunit variants of recombinant human chorionic gonadotrophin.

Human chorionic gonadotropin (hCG), an endogenous glycoprotein hormone, has been widely used for the treatment of infertility and corpus luteum defect in women. The biological specificity of hCG is essentially determined by its beta (ß-) subunit, whereas the alpha (α-) subunit is a common subunit shared among the gonadotropin family. In development of a therapeutic recombinant hCG, the purity analysis showed that the beta (ß-) subunit has two variants, ß1 and ß2. Structural characterization using a combination of analytical techniques has demonstrated that ß1-subunit is derived from non-glycosylation at Asn 13, whereas ß2-subunit is a normal species with complete N-glycosylation at both Asn 13 and Asn 30. In vivo Bioactivity evaluation of the r-hCG fractions with various ratios of ß1-and ß2-subunits showed that incomplete glycosylation at Asn 13 potentially reduced the biological activity of r-hCG to promote uterus growth. Although hCG has a long history of medicinal use, this is the first report to identify the structural difference of hCG ß-subunit variants, as well as to preliminary establish the structure-activity relationship of this variation. The obtained results also suggest the importance of variant characterization and necessary quality control of product variants during the development of recombinant protein therapeutics.

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk.

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography-tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.

Isolation and Characterization of Collagen and Collagen Peptides with Hyaluronidase Inhibition Activity Derived from the Skin of Marlin (Istiophoridae).

Type I and V collagens are the major components of fibrillogenic proteins in fish skin, and their hydrolysis products possess hyaluronidase inhibitory activity. In this study, for the first time, type I and V collagens were isolated from the skin of shortbill spearfish and striped marlin. Type I (2α1[I]α2[I]) and type V (α1[V]α3[V]α2[V]) collagens composed of distinct α-peptide chains with comparable structures were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometric chromatography. After enzymatic digestion, the collagen peptides were purified by using ultrafiltration (30 KDa) and high-performance liquid chromatography (RP-HPLC) to yield CPI-F3 and CPV-F4 fractions with strong hyaluronidase inhibition rates (42.17% and 30.09%, respectively). Based on the results of simulated gastrointestinal fluid, temperature, and pH stability assays, CPI-F3 and CPV-F4 exhibited stability in gastric fluid and showed no significant changes under the temperature range from 50 to 70 °C (p > 0.05). The results of this first research on the bioactivity of type V collagen peptides provide valuable information for the biomedical industry and show the potential for future bioactivity investigations of type V collagen and its peptides.

Effects of rice vinegar treatment on the antioxidant activities and protein structures of whole egg liquid before and after gastrointestinal digestion.

In this study, the effects of vinegar treatment on the antioxidant and structural properties of whole egg proteins were investigated. The results showed that the degrees of hydrolysis (DH) of vinegar-treated egg liquid (VE) and digested VE (DVE) increased after vinegar addition. A similar trend was also found for the antioxidant activity of DVE but not for that of VE. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that vinegar treatment increased the gastrointestinal hydrolysis of whole egg protein compared with that of digested egg liquid (DEL) protein, which was in agreement with the free amino acid content results. The Fourier transform infrared (FTIR) analysis results indicated that proteins from VE1:3 tended to form ß-sheet structures, whereas proteins from DVE1:3 accumulated α-helices and turns. Vinegar treatment has great potential as a nonthermal processing method for promoting gastrointestinal digestion and producing superior antioxidant peptides.

Improved Identification of Protease Cleavage Sites by In-gel Reductive Dimethylation.

A critical step in the functional characterization of proteases is the identification of physiologically relevant substrates, which often starts with a collection of candidate proteins. To test these candidates and identify specific processing sites, in vitro cleavage assays are typically used, followed by polyacrylamide gel electrophoresis (SDS-PAGE) to separate and visualize the cleavage products. For the identification of cleavage sites, the sequences at the N- or C-terminal ends of the cleavage products need to be identified, which is the most challenging step in this procedure. Here, we describe a method for the reliable identification of the N-termini of polypeptides after separation by SDS-PAGE. The procedure relies on in-gel labeling of the N-terminal-free amino group by reductive dimethylation, followed by tryptic digestion and analysis of resulting peptides by mass spectrometry. N-terminal peptides are readily identified by the 28 Da mass dimethyl tag linked to their first amino acid.

Cloning, Expression, and Purification of the Human Synthetic Survivin Protein in Escherichia coli Using Response Surface Methodology (RSM).

Survivin is one of the novel members of the apoptosis inhibitor protein family in humans. The main activity of the Survivin protein is to suppress caspases activity resulting in negative regulation of apoptosis. Survivin protein can be a potential target for the treatment of cancers between cancerous and normal cells. In the present research, the synthetic Survivin gene with PelB secretion signal peptide was cloned into a prokaryotic expression vector pET21a. The recombinant plasmid pET21a-PelB-Surv was expressed in Escherichia coli (E.coli) BL21, and the relative molecular mass of expressed protein was calculated 34,000 g/mol, approximately. The recombinant protein was purified through chromatography column and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Response surface methodology (RSM) was used to design 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 10 h for post-induction period, and 3.40768 for cell density (OD600). These findings resulted in 4.14-fold increases in the Survivin production rate of optimum expression conditions (93.6363 mg/ml).

Quantification of the presence of enzymes in gelatin zymography using the Gini index.

Gel zymography quantifies the activity of certain enzymes in tumor processes. These enzymes are widely used in medical diagnosis. In order to analyze them, experts classify the zymography spots into various classes according to their tonalities. This classification is done by visual analysis, which is what makes it a subjective process. This work proposes a methodology to carry out this classifications with a process that involves an unsupervised learning algorithm in the images, denoted as the GI algorithm. With the experiments shown in this paper, this methodology could constitute a tool that bioinformatics scientists can trust to perform the desired classification since it is a quantitative indicator to order the enzymatic activity of the spots in a zymography.

Identification of two SPRY isoforms SPRY1 and SPRY3 by atomic force microscopy at the single-molecule level.

Growing reports indicate that Sprouty (SPRY) isoforms act as inhibitors or promoters in various types of cancers. And the occurrence of different cancers may be related to the abnormal expression of one of the SPRY isoforms. The identification of SPRY isoforms thus plays a particularly important role in determining which isoform's aberrant expression inhibits or promotes cancer. But their own properties, such as similarities in the structure and molecular weight, make their identification particularly difficult. In this article, we propose a novel method to identify SPRY isoforms using atomic force microscopy (AFM) by observing differential binding of different SPRY isoforms to bovine serum albumin (BSA), which can be used to distinguish SPRY isoforms at the single-molecule level. Specific binding of SPRY1 and BSA was observed by AFM. The reduction in the number of monomeric protein molecules caused by the partial depletion of these two proteins during binding is also consistent with the weakening of the monomeric protein bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). At the same time, the arrangement of the two proteins in a tightly bound complex was also observed. However, the SPRY3 isoform did not interact with BSA to cause aggregation, and the diameter and height of the two proteins did not change significantly compared to those before the reaction. In this way, with the participation of BSA, the two isoforms, SPRY1 and SPRY3, can be identified and separated using atomic force microscopy. In addition, the experimental result that the formation of the SPRY1-BSA complex can selectively reduce the concentration of SPRY1 isoforms in the environment will also contribute to future research on anticancer drugs influenced by SPRY1.