ENL reads histone ß-hydroxybutyrylation to modulate gene transcription.

Histone modifications are typically recognized by chromatin-binding protein modules (referred to as 'readers') to mediate fundamental processes such as transcription. Lysine ß-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap should be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark, and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation led to the suppressed expression of genes such MYC that drive cell proliferation. Together, our work offered a chemoproteomics approach and identified ENL as a novel histone ß-hydroxybutyrylation effector that regulates gene transcription, providing new insight into the regulation mechanism and function of histone Kbhb.

Elucidating the binding partners of histone post-translational modifications (hPTMs) is key to understanding epigenetic regulatory pathways. Lysine ß-hydroxybutyrylation (Kbhb) is a novel hPTM that couples metabolism to transcription. However, the effectors reading this mark are poorly understood as the Kbhb-mediated protein­protein interactions are weak and transient. Here, we presented a quantitative chemical proteomics approach using multivalent photoaffinity probes to robustly capture interactors of this mark. Thus, we identified ENL as a novel binder of Kbhb of histone H3 lysine 9 (H3K9bhb). Biochemical studies and CUT&Tag analysis further revealed that ENL recognizes H3K9bhb and co-localizes with it on gene promoters to modulate transcription and tumorigenesis. This study highlights ENL as a histone Kbhb reader for the regulation of transcription.

TmcA functions as a lysine 2-hydroxyisobutyryltransferase to regulate transcription.

Protein lysine 2-hydroxyisobutyrylation (Khib) has recently been shown to play a critical role in the regulation of cellular processes. However, the mechanism and functional consequence of Khib in prokaryotes remain unclear. Here we report that TmcA, an RNA acetyltransferase, functions as a lysine 2-hydroxyisobutyryltransferase in the regulation of transcription. We show that TmcA can effectively catalyze Khib both in vitro and intracellularly, and that R502 is a key site for the Khib catalytic activity of TmcA. Using quantitative proteomics, we identified 467 endogenous candidates targeted by TmcA for Khib in Escherichia coli. Interestingly, we demonstrate that TmcA can specifically modulate the DNA-binding activity of H-NS, a nucleoid-associated protein, by catalysis of Khib at K121. Furthermore, this TmcA-targeted Khib regulates transcription of acid-resistance genes and enhances E. coli survival under acid stress. Our study reveals transcription regulation mediated by TmcA-catalyzed Khib for bacterial acid resistance.

Systematic Proteome and Lysine Succinylome Analysis Reveals Enhanced Cell Migration by Hyposuccinylation in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and posttranslational modifications (PTMs) leading to the pathogenesis of ESCC remain unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome, and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and PTM pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

Deciphering the Interactome of Histone Marks in Living Cells via Genetic Code Expansion Combined with Proximity Labeling.

Deciphering the endogenous interactors of histone post-translational modifications (hPTMs, also called histone marks) is essential to understand the mechanisms of epigenetic regulation. However, most of the analytical methods to determine hPTM interactomes are in vitro settings, lacking interrogating native chromatin. Although lysine crotonylation (Kcr) has recently been considered an important hPTM for the regulation of gene transcription, the interactors of Kcr still remain to be explored. Herein, we present a general approach relying upon a genetic code expansion system, APEX2 (engineered peroxidase)-mediated proximity labeling, and quantitative proteomics to profile interactomes of the selected hPTMs in living cells. We genetically fused APEX2 to the recombinant histone H3 with a crotonyl lysine inserted site specifically to generate APEX2-H3K9cr that incorporated into native chromatin. Upon activation, APEX2 triggered in vivo biotin labeling of H3K9cr interactors that can then be enriched with streptavidin beads and identified by mass spectrometry. Proteomic analysis further revealed the endogenous interactomes of H3K9cr and confirmed the reliability of the method. Moreover, DPF2 was identified as a candidate interactor, and the binding interaction of DPF2 to H3K9c was further characterized and verified. This study provides a novel strategy for the identification of hPTM interactomes in living cells, and we envision that this is key to elucidating epigenetic regulatory pathways.

Serum phosphopeptide profiling for colorectal cancer diagnosis using liquid chromatography-mass spectrometry.

RATIONALE: The identification and evaluation of novel biomarkers are essential to clinical diagnosis and prognosis of colorectal cancer (CRC). Serum phosphopeptides have been recognized as a potential signature pool for cancers; therefore, we aim to profile the expression of serum phosphopeptides and to evaluate their feasibility in CRC diagnosis. METHODS: We conducted the characterization and absolute quantification of endogenous phosphopeptides in sera using liquid chromatography-mass spectrometry analysis in combination with enrichment of phosphopeptides by ZrAs-Fe3 O4 @SiO2 nanoparticles and use of deuterium-labeled standards. Differentially expressed analysis of four phosphopeptides was performed, generating a two-phosphopeptide-based biomarker, LF3-4 , by logistic regression analysis, where LF3-4 is equal to (5.85 - 5.13 × [F3] - 3.57 × [F4]), and [F3] and [F4] are the concentration of phosphopeptides DpSGEGDFLAEGGGVR and ADpSGEGDFLAEGGGVR in sera, respectively. RESULTS: The LF3-4 values showed significant difference in CRC cases compared with controls, and yielded a specificity of 100%, leading to correct classification of 56 (93%) out of 60 CRC patients, including 12 (92.3%) of 13 CRC cases in stage I. Double-blind validation showed that 97.5% of CRC cases were discriminated accurately. CONCLUSIONS: The LF3-4 value was firstly verified to be a potential biomarker for CRC diagnosis, and may expand our view in underlying mechanisms for CRC.

An Integrated Approach for Combinatorial Readout of Dual Histone Modifications by Epigenetic Tandem Domains.

Histone post-translational modifications (HPTMs) serve as signal platforms for recruitment of binding proteins (readers) to regulate gene expression. Accumulated evidence suggests that the intensive distribution of HPTMs may result in crosstalk, which increases or inhibits the recruitment of reader proteins, further altering the functional outcome of HPTMs. Therefore, the comprehensive identification of multiple interactions between combinatorial HPTMs and reading domains is essential to understand the chromatin-templated processes. However, it is still a big challenge to profile these complicated interactions due to various limitations including rather weak, transient and multiple interactions between HPTMs and readers, the high dynamic property of HPTMs as well as the low abundance of reader proteins. Here we developed an integrated approach to profile the complicated interactions between combinatorial HPTMs and dual domains. Based on a combinatorial HPTM peptide library (trimethylation of histone H3 lysine 4 and its neighboring PTMs) and five affinity tag proteins containing tandem-domain probes, histone interactions can be profiled by pull-down assay combined with mass spectrometry analysis. The interactions were further verified by isothermal titration calorimetry and proximity ligation assay, as well as molecular docking. By use of combinatorial HPTMs, we demonstrated that this integrated approach can be successfully utilized for the characterization of multiple interactions between reading domains and combinatorial HPTMs including novel HPTMs with low stoichiometry. Thus, a novel chemical proteomics tool for profiling of multiple PTM-mediated protein-protein interactions was successfully developed and can be adapted for broad biomedical applications.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

RATIONALE: Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. OBJECTIVE: The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. METHOD AND RESULTS: In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. CONCLUSIONS: YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2.

Systematic Identification of Lysine 2-hydroxyisobutyrylated Proteins in Proteus mirabilis.

Lysine 2-hydroxyisobutyrylation (Khib) is a novel post-translational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of Khib in Proteus mirabilis (P. mirabilis). By combining affinity enrichment with two-dimensional liquid chromatography and high-resolution mass spectrometry, 4735 2-hydroxyisobutyrylation sites were identified on 1051 proteins in P. mirabilis. These proteins bearing modifications were further characterized in abundance, distribution and functions. The interaction networks and domain architectures of these proteins with high confidence were revealed using bioinformatic tools. Our data demonstrate that many 2-hydroxyisobutyrylated proteins are involved in metabolic pathways, such as purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. The extensive distribution of Khib also indicates that the modification may play important influence to bacterial metabolism. The speculation is further supported by the observation that carbon sources can influence the occurrence of Khib Furthermore, we demonstrate that 2-hydroxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that Khib may change the binding formation of ENO and its substrate 2-phospho-d-glycerate (2PG) and cause the substrate far from the active sites of enzyme. We hope this first comprehensive analysis of nonhistone Khib in prokaryotes is valuable for further functional investigation of this modification.

Combinatorial Peptide Ligand Library-Based Photoaffinity Probe for the Identification of Phosphotyrosine-Binding Domain Proteins.

Phosphotyrosine (pY) serves as a docking site for the recognition proteins containing pY-binding (pYB) modules, such as the SH2 domain, to mediate cell signal transduction. Thus, it is vital to profile these binding proteins for understanding of signal regulation. However, identification of pYB proteins remains a significant challenge due to their low abundance and typically weak and transient interactions with pY sites. Herein, we designed and prepared a pY-peptide photoaffinity probe for the robust and specific enrichment and identification of its binding proteins. Using SHC1-pY317 as a paradigm, we showed that the developed probe enables to capture target protein with high selectivity and remarkable specificity even in a complex context. Notably, we expanded the strategy to a combinatorial pY-peptide-based photoaffinity probe by using combinatorial peptide ligand library (CPLL) technique and identified 24 SH2 domain proteins, which presents a deeper profiling of pYB proteins than previous reports using affinity probes. Moreover, the method can be used to mine putative pYB proteins and confirmed PKN2 as a selective binder to pY, expanding the repertoire of known domain proteins. Our approach provides a general strategy for rapid and robust interrogating pYB proteins and will promote the understanding of the signal transduction mechanism.

An Integrated Approach Based on a DNA Self-Assembly Technique for Characterization of Crosstalk among Combinatorial Histone Modifications.

Combinatorial histone post-translational modifications (HPTMs) form a complex epigenetic code that can be decoded by specific binding proteins, termed as readers. Their specific interplays have been thought to determine gene expression and downstream biological functions. However, it is still a big challenge to analyze such interactions due to various limitations including rather weak, transient, and complicated interactions between HPTMs and readers, the high dynamic property of HPTMs, and the low abundance of reader proteins. Here we sought to take advantage of DNA-templated and photo-cross-linking techniques to design a group of combinatorial histone PTM peptide probes for the identification of multivalent interactions among histone PTMs and readers. By use of trimethylation on histone H3K4 (H3K4me3) and phosphorylation on H3T3, we demonstrated that this approach can be successfully utilized for identification of the PTM crosstalk on the same histone. By use of H3K4me3 and acetylation on H4K16, we showed the potential application of the probe in the multivalent interactions among PTMs on different histones. Thus, this new chemical proteomics tool combined with mass spectrometry holds a promising potential in profiling of the readers of combinatorial HPTMs and characterization of crosstalk among multiple PTMs on histones and can be adapted for broad biomedical applications.

An Efficient Approach for Selective Enrichment of Histone Modification Readers Using Self-Assembled Multivalent Photoaffinity Peptide Probes.

Histone post-translational modifications (HPTMs) provide signaling platforms to recruit proteins or protein complexes (e.g., transcription factors, the so-called "readers" of the histone code), changing DNA accessibility in the regulation of gene expression. Thus, it is an essential task to identify HPTM readers for understanding of epigenetic regulation. Herein we designed and prepared a novel HPTM probe based on self-assembled multivalent photo-cross-linking technique for selective enrichment and identification of HPTM readers. By use of trimethylation of histone H3 lysine 4, we showcased that the functionalized HPTM probe was able to capture its reader with high enrichment efficiency and remarkable specificity even in a complex environment. Notably, this approach was readily applicable for exploring crosstalk among multiple HPTMs. Combining the probes with a mass spectrometry-based proteomic approach, our approach reached a fairly high coverage of known H3K4me3 readers. We further demonstrated that the HPTM probes can enrich a new type of HPTM readers and uncovered several novel putative binders of crotonylation of histone H3 lysine 9, expanding the repertoire of readers for this epigenetic mark. More broadly, our work provides a general strategy for rapid and robust interrogating HPTM readers and will be of great importance to elucidate epigenetic mechanism in regulating gene activity.

DNA-Templated Aptamer Probe for Identification of Target Proteins.

Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

Maleic Anhydride Labeling-Based Approach for Quantitative Proteomics and Successive Derivatization of Peptides.

Chemical derivatization is a simple approach for stable-isotope covalent labeling of proteins in quantitative proteomics. Herein we describe the development of a novel maleyl-labeling-based approach for protein quantification. Under optimized conditions, maleic anhydride can serve as a highly efficient reagent to label the amino groups of tryptic peptides. Furthermore, "click chemistry" was successfully applied to obtain the second modification of maleylated peptides via thiol-Michael addition reaction. Accurate quantification was further achieved via the first or/and second step stable-isotope labeling in this study. Our data thus demonstrate that the maleyl-labeling-based method is simple, accurate, and reliable for quantitative proteomics. The developed method not only enables an enhanced sequence coverage of proteins by improving the identification of small and hydrophilic peptides, but also enables a controllable, successive, second derivatization of labeled peptides or proteins, and therefore holds a very promising potential for in-depth analysis of protein structures and dynamics.

The Unexpected and Exceptionally Facile Chemical Modification of the Phenolic Hydroxyl Group of Tyrosine by Polyhalogenated Quinones under Physiological Conditions.

The phenolic hydroxyl group of tyrosine residue plays a crucial role in the structure and function of many proteins. However, little study has been reported about its modification by chemical agents under physiological conditions. In this study, we found, unexpectedly, that the phenolic hydroxyl group of tyrosine can be rapidly and efficiently modified by tetrafluoro-1,4-benzoquinone and other polyhalogenated quinones, which are the major genotoxic and carcinogenic quinoid metabolites of polyhalogenated aromatic compounds. The modification was found to be mainly due to the formation of a variety of fluoroquinone-O-tyrosine conjugates and their hydroxylated derivatives via nucleophilic substitution pathway. Analogous modifications were observed for tyrosine-containing peptides. Further studies showed that the blockade of the reactive phenolic hydroxyl group of tyrosine in the substrate peptide, even by very low concentration of tetrafluoro-1,4-benzoquinone, can prevent the kinase catalyzed tyrosine phosphorylation. This is the first report showing the exceptionally facile chemical modification of the phenolic hydroxyl group of tyrosine by polyhalogenated quinones under normal physiological conditions, which may have potential biological and toxicological implications.

Identification of hydroxylation at aromatic amino acid residues in yeast kinase using mass spectrometry with affinity enrichment.

RATIONALE: Protein kinases represent the key elements in phosphorylation-based signal transmission. Recent studies suggest that hydroxylation may mediate activities of protein kinases. This paper aims to examine the hydroxylation in protein kinases for improving our understanding of the protein modification. METHODS: We combined affinity-based protein purification with MS analysis for identification of novel hydroxylation at aromatic amino acid residues in yeast kinases. RESULTS: We identified 17 hydroxylation at aromatic amino acid residues (10 at Phe, 1 at Tyr and 6 at Trp) using MS analysis. We further characterized the localization and studied the potential significance of these modifications. CONCLUSIONS: This is a new report on the identification of hydroxylation at aromatic amino acid residues in yeast kinases. This study expands the catalog of hydroxylation in kinases and suggests the potential function of hydroxylation. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a DNA-Templated Peptide Probe for Photoaffinity Labeling and Enrichment of the Histone Modification Reader Proteins.

Histone post-translational modifications (HPTMs) provide signal platforms to recruit proteins or protein complexes to regulate gene expression. Therefore, the identification of these recruited partners (readers) is essential to understand the underlying regulatory mechanisms. However, it is still a major challenge to profile these partners because their interactions with HPTMs are rather weak and highly dynamic. Herein we report the development of a HPTM dual probe based on DNA-templated technology and a photo-crosslinking method for the identification of HPTM readers. By using the trimethylation of histone H3 lysine 4, we demonstrated that this HPTM dual probe can be successfully utilized for labeling and enrichment of HPTM readers, as well as for the discovery of potential HPTM partners. This study describes the development of a new chemical proteomics tool for profiling HPTM readers and can be adapted for broad biomedical applications.

A proximity labeling-based orthogonal trap strategy identifies HDAC8 promotes cell motility by modulating cortactin acetylation.

It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates in vivo. Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac. By leveraging this approach, we identified numerous of putative acetylated proteins targeted by HDAC8, and further confirmed CTTN as a bona fide substrate in vivo. Next, we demonstrated that HDAC8 facilitates cell motility via deacetylation of CTTN at lysine 144 that attenuates its interaction with F-actin, expanding the underlying regulatory mechanisms of HDAC8. We developed a general strategy to profile the transient enzyme-substrate interactions mediated by PTMs, providing a powerful tool for identifying the spatiotemporal PTM-network regulated by enzymes in living cells.

HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription.

Lysine lactylation (Kla) links metabolism and gene regulation and plays a key role in multiple biological processes. However, the regulatory mechanism and functional consequence of Kla remain to be explored. Here, we report that HBO1 functions as a lysine lactyltransferase to regulate transcription. We show that HBO1 catalyzes the addition of Kla in vitro and intracellularly, and E508 is a key site for the lactyltransferase activity of HBO1. Quantitative proteomic analysis further reveals 95 endogenous Kla sites targeted by HBO1, with the majority located on histones. Using site-specific antibodies, we find that HBO1 may preferentially catalyze histone H3K9la and scaffold proteins including JADE1 and BRPF2 can promote the enzymatic activity for histone Kla. Notably, CUT&Tag assays demonstrate that HBO1 is required for histone H3K9la on transcription start sites (TSSs). Besides, the regulated Kla can promote key signaling pathways and tumorigenesis, which is further supported by evaluating the malignant behaviors of HBO1- knockout (KO) tumor cells, as well as the level of histone H3K9la in clinical tissues. Our study reveals HBO1 serves as a lactyltransferase to mediate a histone Kla-dependent gene transcription.

Hypoxia promotes histone H3K9 lactylation to enhance LAMC2 transcription in esophageal squamous cell carcinoma.

Hypoxia promotes tumorigenesis and lactate accumulation in esophageal squamous cell carcinoma (ESCC). Lactate can induce histone lysine lactylation (Kla, a recently identified histone marks) to regulate transcription. However, the functional consequence of histone Kla under hypoxia in ESCC remains to be explored. Here, we reveal that hypoxia facilitates histone H3K9la to enhance LAMC2 transcription for proliferation of ESCC. We found that global level of Kla was elevated under hypoxia, and thus identified the landscape of histone Kla in ESCC by quantitative proteomics. Furthermore, we show a significant increase of H3K9la level induced by hypoxia. Next, MNase ChIP-seq and RNA-seq analysis suggest that H3K9la is enriched at the promoter of cell junction genes. Finally, we demonstrate that the histone H3K9la facilitates the expression of LAMC2 for ESCC invasion by in vivo and in vitro experiments. Briefly, our study reveals a vital role of histone Kla triggered by hypoxia in cancer.

Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by titania coated magnetic mesoporous silica microspheres for screening of protein kinase inhibitors.

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO(2)/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r(2) being 0.99. The IC(50) values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.