Culture substrate stiffness impacts human myoblast contractility-dependent proliferation and nuclear envelope wrinkling.

Understanding how biophysical and biochemical microenvironmental cues together influence the regenerative activities of muscle stem cells and their progeny is crucial in strategizing remedies for pathological dysregulation of these cues in aging and disease. In this study, we investigated the cell-level influences of extracellular matrix (ECM) ligands and culture substrate stiffness on primary human myoblast contractility and proliferation within 16 h of plating and found that tethered fibronectin led to stronger stiffness-dependent responses compared to laminin and collagen. A proteome-wide analysis further uncovered cell metabolism, cytoskeletal and nuclear component regulation distinctions between cells cultured on soft and stiff substrates. Interestingly, we found that softer substrates increased the incidence of myoblasts with a wrinkled nucleus, and that the extent of wrinkling could predict Ki67 (also known as MKI67) expression. Nuclear wrinkling and Ki67 expression could be controlled by pharmacological manipulation of cellular contractility, offering a potential cellular mechanism. These results provide new insights into the regulation of human myoblast stiffness-dependent contractility response by ECM ligands and highlight a link between myoblast contractility and proliferation.

All-in-One digital microfluidics pipeline for proteomic sample preparation and analysis.

Highly sensitive and reproducible analysis of samples containing low amounts of protein is restricted by sample loss and the introduction of contaminants during processing. Here, we report an All-in-One digital microfluidic (DMF) pipeline for proteomic sample reduction, alkylation, digestion, isotopic labeling and analysis. The system features end-to-end automation, with integrated thermal control for digestion, optimized droplet additives for sample manipulation and analysis, and an automated interface to liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Dimethyl labeling was integrated into the pipeline to allow for relative quantification of the trace samples at the nanogram level, and the new pipeline was applied to evaluating cancer cell lines and cancer tissue samples. Several known proteins (including HSP90AB1, HSPB1, LDHA, ENO1, PGK1, KRT18, and AKR1C2) and pathways were observed between model breast cancer cell lines related to hormone response, cell metabolism, and cell morphology. Furthermore, differentially quantified proteins (such as PGS2, UGDH, ASPN, LUM, COEA1, and PRELP) were found in comparisons of healthy and cancer breast tissues, suggesting potential utility of the All-in-One pipeline for the emerging application of proteomic cancer sub-typing. In sum, the All-in-One pipeline represents a powerful new tool for automated proteome processing and analysis, with the potential to be useful for evaluating mass-limited samples for a wide range of applications.

Comparison of Database Searching Programs for the Analysis of Single-Cell Proteomics Data.

Single-cell proteomics is emerging as an important subfield in the proteomics and mass spectrometry communities, with potential to reshape our understanding of cell development, cell differentiation, disease diagnosis, and the development of new therapies. Compared with significant advancements in the "hardware" that is used in single-cell proteomics, there has been little work comparing the effects of using different "software" packages to analyze single-cell proteomics datasets. To this end, seven popular proteomics programs were compared here, applying them to search three single-cell proteomics datasets generated by three different platforms. The results suggest that MSGF+, MSFragger, and Proteome Discoverer are generally more efficient in maximizing protein identifications, that MaxQuant is better suited for the identification of low-abundance proteins, that MSFragger is superior in elucidating peptide modifications, and that Mascot and X!Tandem are better for analyzing long peptides. Furthermore, an experiment with different loading amounts was carried out to investigate changes in identification results and to explore areas in which single-cell proteomics data analysis may be improved in the future. We propose that this comparative study may provide insight for experts and beginners alike operating in the emerging subfield of single-cell proteomics.

A mass-tagged MOF nanoprobe approach for ultra-sensitive protein quantification in tumor-educated platelets.

A mass-tagged metal-organic framework (MOF) nanoprobe approach was developed for ultra-sensitive quantification of platelet protein CD44 by integrating activable aptamer recognition and MOF nanoprobe signal amplification with mass spectrometric detection. This approach offered high sensitivity and quantitative capability for low abundant protein analysis in tumor-educated platelets (TEPs), exhibiting great potential in cancer diagnosis and management.

Selective enrichment tandem ß-elimination assisted strategy for N-phosphorylation analysis.

N-phosphorylation modifications are crucial for prokaryotic signal transduction and verified as intermediate for several metabolic enzymes, yet the landscape of N-phosphorylation remains an obstacle due to the lack of effective identification strategies. One of the difficulties derives from the labile phosphoramidate bond (P-N) under acidic conditions, making it easily hydrolyze during the routine analysis. Meanwhile, O-phosphopeptides influence the accurate identification of N-phosphorylation sites during mass spectrometry (MS) analysis. Herein, a selective enrichment tandem ß-elimination assisted identification (abbreviated as EnaBe) strategy was established to address the difficulties. Firstly, N-phosphoproteins could be captured within 10 min by SiO2@DpaZn microspheres under neutral conditions, which was benefited from rapid mass transfer. Secondly, the ß-elimination efficiency could reach over 92% within 3 h under the optimized condition, and MS signals of N-phosphopeptides were significantly enhanced by integrating the ß-elimination treatment. Finally, N-phosphoproteins from E. coli lysates were analyzed by EnaBe approach and 16 N-phosphoproteins with high confidence were identified. Furthermore, functional analysis showed that the proteins played vital roles in phosphorylation process and bacterial primary metabolic processes including glucose, purine and ADP metabolism. All above results demonstrated the superiority of the EnaBe strategy.

2,6-Dichloro-1,4-benzoquinone formation from chlorination of substituted aromatic antioxidants and its control by pre-ozonation in drinking water treatment plant.

Halobenzoquinones are frequently detected as disinfection by-products in drinking water. Among identified halobenzoquinones, 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) is particularly toxic and is frequently detected in drinking water. Synthetic aromatic antioxidants discharged to source water may increase the risk of 2,6-DCBQ formation, as many studies suggest that aromatic compounds are the most likely precursors to 2,6-DCBQ. Herein, we investigated the formation of 2,6-DCBQ from chlorination of three model aromatic antioxidants, including 3-tert-butyl-4-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT) and bis(4-tert-butylphenyl)amine (BBPA). Only BBPA produced 2,6-DCBQ under chlorination, while chlorination of BHA and BHT formed α, ß-unsaturated C4-dicarbonyl ring-opening products and phenolic compounds. Based on mass balance and intermediate transformation analysis, mechanisms for the formation of 2,6-DCBQ from BBPA chlorination involved hydrolysis, tert-butyl group cleavage, chlorine substitution, desamination and oxidation. Mitigating aromatic compounds will be an efficient method for 2,6-DCBQ control, such as pre-ozonation, because the intermediates involved in 2,6-DCBQ formation were aromatic compounds. Real water samples from two drinking water treatment plants (DWTPs), one with pre-ozonation (DWTP 2) and the other without pre-ozonation (DWTP1), were analyzed. The two DWTPs were built along the Yangtse river in Nanjing city. Raw water parameters from the two DWTPs, including dissolved organic carbon (DOC), UV absorbance at 254 nm (UV254) and NH3-N, indicated the water quality between these sources was similar. Pre-ozonation in DWTP 2 vanished 2,6-DCBQ in raw water. Concentrations of 2,6-DCBQ in finished water from DWTP 1 (5.69 ng/L) was higher than concentrations generated from DWTP 2 (1.31 ng/L). These results demonstrate that pre-ozonation, granular activated carbon (GAC) and quartz sand treatments at DWTP 2 remove more 2,6-DCBQ precursors than the conventional quartz sand and GAC treatments in DWTP 1. These results suggest the pre-ozonation, GAC and quartz sand treatments can help minimize concentrations of 2,6-DCBQ generated in DWTPs.

Zn(II)-DPA functionalized graphene oxide two-dimensional nanocomposites for N-phosphoproteins enrichment.

Protein N-phosphorylation is increasingly recognized as a critical post-translational modification in central metabolism and cell signaling. However, other functions of N-phosphorylation in organism are largely unexplored which is attributed to lack high efficient enrichment methods. To address this, Bis(zinc(II)-dipicolylamine) functionalized graphene oxide nanocomposites (Zn(II)-DPA-GO) was designed via loading bis(zinc(II)-dipicolylamine) (Zn(II)-DPA) on graphene oxide surface by π-π stacking, with the advantages of large immobilization amount of Zn(II)-DPA, layered structure, excellent solubility and low non-special adsorption. The material was firstly verified excellent enrichment ability for standard proteins and E.coli lysates. By integrating the enrichment method into proteomics workflow, 36 N-phosphoproteins functioned in metabolic process, biological regulation, localization, cellular processes were identified from Bacillus subtilis. Furthermore, motif analysis showed that leucine and isoleucine were enriched near the sites, providing potential clues for the discovery of new sites. The method provides an alternative for the discovery of N-phosphoproteins with significant biological functions.

Mass spectrometry-based chemical mapping and profiling toward molecular understanding of diseases in precision medicine.

Precision medicine has been strongly promoted in recent years. It is used in clinical management for classifying diseases at the molecular level and for selecting the most appropriate drugs or treatments to maximize efficacy and minimize adverse effects. In precision medicine, an in-depth molecular understanding of diseases is of great importance. Therefore, in the last few years, much attention has been given to translating data generated at the molecular level into clinically relevant information. However, current developments in this field lack orderly implementation. For example, high-quality chemical research is not well integrated into clinical practice, especially in the early phase, leading to a lack of understanding in the clinic of the chemistry underlying diseases. In recent years, mass spectrometry (MS) has enabled significant innovations and advances in chemical research. As reported, this technique has shown promise in chemical mapping and profiling for answering "what", "where", "how many" and "whose" chemicals underlie the clinical phenotypes, which are assessed by biochemical profiling, MS imaging, molecular targeting and probing, biomarker grading disease classification, etc. These features can potentially enhance the precision of disease diagnosis, monitoring and treatment and thus further transform medicine. For instance, comprehensive MS-based biochemical profiling of ovarian tumors was performed, and the results revealed a number of molecular insights into the pathways and processes that drive ovarian cancer biology and the ways that these pathways are altered in correspondence with clinical phenotypes. Another study demonstrated that quantitative biomarker mapping can be predictive of responses to immunotherapy and of survival in the supposedly homogeneous group of breast cancer patients, allowing for stratification of patients. In this context, our article attempts to provide an overview of MS-based chemical mapping and profiling, and a perspective on their clinical utility to improve the molecular understanding of diseases for advancing precision medicine.

Antibody-free enrichment method for proteome-wide analysis of endogenous SUMOylation sites.

SUMOylation is a reversible post-translational modification that plays crucial roles in numerous cellular processes. Although anti-SUMO antibodies have been applied to analyze exogenous and endogenous SUMOylation, such immunoprecipitation enrichment strategy is applicable only for the enrichment of one specific SUMO type in mammalian cells, unable to map the global landscape of all endogenous SUMOylation simultaneously. To address this issue, we proposed an antibody-free strategy to enrich and profile endogenous SUMO1/2/3-modified peptides simultaneously. Upon trypsin digestion, the SUMO1- and SUMO2/3-modified peptides contained SUMO remnants with 7 and 9 acidic amino acids respectively, which carried more negative charges at high pH and could interact with strong anion exchange (SAX) materials more strongly than non-SUMOylated peptides, thus enabling the specific enrichment of endogenous SUMOylated peptides. Followed by the secondary digestion with Asp-N/Glu-C to generate smaller SUMOylated peptides with proper length for MS identification, off-line high-pH C18 pre-fractionation and low pH nanoRPLC-ESI-MS/MS analysis, 177 SUMO1-modified sites and 74 SUMO2/3-modified sites were unbiasedly identified in HeLa cell lysate. To the best of our knowledge, this was the first antibody-free strategy to comprehensively profile various endogenous SUMOylation sites, demonstrating the great potential in the comprehensive analysis of endogenous SUMOylation across various species and organs, which might further facilitate the understanding of SUMO's function in physiology and pathology.

Simultaneous and quantitative monitoring transcription factors in human embryonic stem cell differentiation using mass spectrometry-based targeted proteomics.

Human embryonic stem cells (hESCs) can be self-propagated indefinitely in culture while holding the capacity to generate almost all cell types. Although this powerful differentiation ability of hESCs has become a potential source of cell replacement therapies, application of stem cells in clinical practice relies heavily on the exquisite control of their developmental fate. In general, an essential first step in differentiation is to exit the pluripotent state, which is precariously balanced and depends on a variety of factors, mainly centering on the core transcriptional mechanism. To date, much evidence has indicated that transcription factors such as Sox2, Oct4, and Nanog control the self-renewal and pluripotency of hESCs. Their expression displays a restricted spatial-temporal pattern and their small changes in level can significantly affect directed differentiation and the cell type derived. So far, few assays have been developed to monitor this process. Herein, we provided a mass spectrometry (MS)-based approach for simultaneous and quantitative monitoring of these transcription factors, in an attempt to provide insight into their contributions in hESC differentiation.

Quantitative proteomics of epigenetic histone modifications in MCF-7 cells under estradiol stimulation.

Estrogen exposure has already been considered to be associated with tumorigenesis and breast cancer progression. To study the epigenetic regulation mechanism in MCF-7 cells under estrogen exposure, which normally results in cell proliferation and malignancy, a stable isotope labeling of amino acid (SILAC) based quantitative proteomics strategy was used to analyse histone post-translational modifications (PTMs) and protein differential expressions. In total, we have unambiguously identified 49 histone variants and quantified 42 of them, in which two differentially expressed proteins were found to be associated with breast cancers. Through the quantitative analysis of 470 histone peptides with a combination of different PTM types, including methylation (mono-, di-, and tri-), acetylation and phosphorylation, 150 of them were found to be differentially expressed. Through the biological analysis of the quantification results of both histone PTMs and proteins in MCF-7 cells, we found that (1) the histone variants H10 and H2AV have an effect on the adjustment of the nucleosome or chromatin structure and activate target genes; (2) after estrogen receptor (ER) activation by estrogen, the recruitment of histone acetyltransferase KAT7 might affect the acetylation at the N terminal of H4 (K5, K8 and K12) and also result in cross-talk between different acetylation sites; (3) different expression of histone deacetylase HDAC2 and its nucleo-cytoplasmic transportation process is important in the regulation of histone acetylation in MCF-7 cells under estrogen exposure.

Bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell microspheres for the analysis of N-phosphoproteome.

Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell silica microspheres (SiO2@DpaZn) are tailored for rapid and effective N-phosphopeptides enrichment. Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions.

Dual-Probe Approach for Mass Spectrometric Quantification of MUC1-Specific Terminal Gal/GalNAc In Situ.

Protein glycosylation is a prevalent post-translational modification that mediates a variety of cellular processes. For membrane proteins, glycosylation at their terminal motif is usually more functional. Among the various glycosylation types found in membrane proteins, O-glycosylation is the most common and is closely correlated with a variety of cancer types, including breast cancer. Slightly aberrant expression of certain O-glycans can significantly affect cancer progression, especially at the cancer-related membrane protein level. To collect biological information on protein-specific glycosylation and further explore clinical applications, quantitative detection of glycosylation is essential. However, few assays have been reported for the in situ detection of protein-specific glycosylation to date. Herein, we developed a dual-probe approach for mass spectrometric quantification of protein-specific glycosylation using the terminal galactose/N-acetylgalactosamine (Gal/GalNAc) of MUC1 as a model. The dual-probe (i.e., protein probe and glycan probe) system was first designed and built. The protein probe contained an aptamer for MUC1 protein recognition and a capture DNA sequence. Correspondingly, the glycan probe had a DNA sequence complementary to that of the capture DNA, a substrate peptide containing a reporter peptide, and a tryptic cleavage site, and could be covalently linked with the terminal Gal/GalNAc. Exonuclease III enabled recycling of the hybridization-dehybridization process in a restricted space. Finally, the reporter peptide was tryptically released and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass response of the reporter peptide represented the amount of MUC1-specific terminal Gal/GalNAc. This dual-probe approach was applied for in situ detection of MUC1-specific terminal Gal/GalNAc in three human breast cancer cell lines and 32 pairs of matched breast cancer tissue samples. The relationship between MUC1-specific terminal Gal/GalNAc expression and breast cancer diagnosis/prognosis was also assessed.

Ionic liquid-assisted protein extraction method for plant phosphoproteome analysis.

Protein phosphorylation is one of the most important post-translational modifications (PTM) and plays critical roles in maintaining many biological processes of plant species, such as being a significant signal related to resistance to tobacco mosaic virus (TMV) infection in tobacco. Compared to other organisms, in-depth profiling of plant phosphoproteome remains challenging due to the harsh extraction environment of plant proteins and low abundance of plant phosphorylation, generally requiring large amount of plant materials. Herein, we developed an integrated strategy for efficient sample preparation of amounts of plant tissues, by integrating ionic liquid (IL)-assisted protein extraction, in-solution digestion, precipitation-assisted IL removal, as well as immobilized metal ion affinity chromatography (IMAC) enrichment of phosphopeptides together. In this strategy, to improve the efficiency of protein extraction and enzymatic digestion, IL of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was used as the solubilizer due to its excellent solubilizing ability and enzyme compatibility demonstrated in our previous work. Briefly, the extraction capability of C12Im-Cl for protein amount from tobacco leaves was improved 1.9-fold compared to the commonly used urea-assisted method. Notably, to avoid its interference with subsequent LC-MS analysis, the IL was easily removed from the peptide solution by our proposed ion substitution-mediated C12Im + precipitation strategy with high efficiency. By handling 10 mg of starting protein materials of tobacco leaves, 14,441 unique phosphopeptides, assigned to 5153 unique phosphoproteins were confidently identified. To the best of our knowledge, this was the most comprehensive phosphorylation dataset for tobacco so far. All the results demonstrated our strategy was of great potential to promote the large-scale analysis of plant phosphoproteome.

Quantitative site- and structure-specific N-glycoproteomics characterization of differential N-glycosylation in MCF-7/ADR cancer stem cells.

BACKGROUND: Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. METHODS: In this study, we report our comparative and quantitative site- and structure-specific N-glycoproteomics study of MCF-7/ADR cancer stem cells (CSCs) vs. MCF-7/ADR cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptide level. RESULTS: 4016 intact N-glycopeptides were identified with spectrum-level FDR ≤ 1%. With the criteria of ≥ 1.5 fold change and p value < 0.05, 247 intact N-glycopeptides were found differentially expressed in MCF-7/ADR CSCs as putative markers. Raw data are available via ProteomeXchange with identifier PXD013836. CONCLUSIONS: Quantitative site- and structure-specific N-glycoproteomics characterization may help illustrate the cell stemness property.

[Advancesin enrichment and detection methods for N-phosphorylated proteins].

Protein phosphorylation plays an important role in various life processes, such as signaling, metabolism, and growth. In addition to the well-known O-phosphorylation, which occurs on serine, threonine and tyrosine side chains, N-phosphorylation, occurring on histidine, arginine and lysine side chains, has attracted much attention. However, due to the chemical instability of N-phosphorylation, the studies on it are still in the nascent stage. In this review, enrichment and detection methods for protein N-phosphorylation are reviewed, and future research in this field is prospected.

Combination of continuous digestion by peptidase and spectral similarity comparisons for peptide sequencing.

Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS2 spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase). Then, based on the characteristics of peptide ladders, complete sequencing was realized by comparing MS2 spectral similarity of the generated peptide ladders. The complete amino acid sequences of bivalirudin, adrenocorticotropic hormone, and oxytocin were determined with high accuracy. This approach is beneficial to the quality control of drug peptides as well as the identification of novel bioactive peptides.

Site-Specific Quantification of Persulfidome by Combining an Isotope-Coded Affinity Tag with Strong Cation-Exchange-Based Fractionation.

Protein persulfidation is one of the most important oxidative translational modifications and plays vital roles in various important biological processes. However, the proteome-wide identification of persulfidation sites is a great challenge because of the difficulties in accurately differentiating persulfide groups with disulfide and thiol groups in proteins as well as the extremely low abundance of persulfidated peptides. By current approaches, the persulfidated peptides were often identified by the cleavage of their persulfide groups by reductants prior to MS analysis; therefore, it would bring about a false positive identification and was unable to identify persulfidation sites accurately for a single peptide with multiple cysteine residues. In this study, a novel strategy for the site-specific quantification of persulfidome (SSQPer) was developed. By this strategy, the persulfidated proteins were first labeled with cleavable isotope-coded affinity tag (c-ICAT) reagents. After digestion, the labeled persulfidated peptides were selectively enriched with streptavidin beads and fractionated by strong cation exchange chromatography, followed by LC-MS/MS identification. To evaluate the performance of SSQPer, the persulfidated BSA digests with 20 persulfidation sites identified were used to spike HeLa cell digests with mass ratios of 1:100 and 1:1000, and 16 and 13 persulfidated sites were respectively identified. We applied SSQPer to the site-specific quantification of persulfidome in the epithelial-mesenchymal transition (EMT) process, and 226 endogenous persulfidation sites were identified, of which 74.3% were newly discovered. All of these results demonstrated that the SSQPer strategy would provide a promising tool to profile the site-specific persulfidome and pave the way for future investigation to expand our knowledge of persulfidation.

[A novel method for the selective enrichment of persulfidated peptides based on iodoacetic acid functionalized dendrimer].

Protein persulfidation is an important oxidative translational modification which plays vital roles in many important processes including cellular senescence, endoplasmic reticulum stress, vasorelaxation, and apoptosis. The proteome-wide analysis of persulfidation is of great importance; therefore, this study combines filter-aided sample preparation with an iodoacetic acid functionalized polyamidoamine dendrimer to enrich persulfidated peptides (denoted as filter-aided dendrimer enrichment strategy, FADE). To evaluate the performance of this strategy, the synthetic persulfidated standard peptide was spiked into bovine serum albumin (BSA) digests at a mass ratio of 1:100, and was successfully identified by FADE. Moreover, in combination with stable isotope labelling by amino acids in cell culture technology, the FADE strategy was applied to enrich persulfidated peptides from NaHS-stimulated SHSY5Y cells over a concentration gradient, resulting in the identification of 163 persulfidated peptides. Bioinformatic analysis indicated that persulfidation might play important roles in the central nervous system.

Aptamer functionalized magnetic graphene oxide nanocomposites for highly selective capture of histones.

The binding coverage of aptamer was an important restricted factor for aptamer-based affinity enrichment strategy for capturing target molecules. Herein, we designed and prepared aptamer functionalized graphene oxide based nanocomposites (GO/NH2 -NTA/Fe3 O4 /PEI/Au), and the coverage density of aptamer was high to 33.1 nmol/mg. The high aptamer coverage density was contributed to the large surface area of graphene oxide. The successive modification of Nα,Nα-Bis(carboxymethyl)-L-lysine, magnetic nanoparticles, polyethylenimine, and Au nanoparticles ensured the histone purification with fast speed and high purity. Histones could be captured rapidly and specifically from nucleoproteins by our aptamer based purification strategy, while traditional acid-extraction could not specifically enrich histones. Compared with traditional acid-extraction method, rapid and efficient discovery of histones and their post-translational modifications, such as several kinds of methylation at H3.1K9 and H3.1K27, were achieved confidently. It demonstrated that our aptamer functionalized magnetic graphene oxide nanocomposites have a great potential for histone analysis.