Detection Of Post-translational Modifications Research Articles

Immunoaffinity Intact Top-Down Mass Spectrometry for Quantification of Neuron-Specific Enolase Gamma, a Low-Abundance Protein Biomarker.

Quantification of intact proteins in serum by liquid chromatography high-resolution mass spectrometry (HRMS) may be a useful alternative to bottom-up LC-MS or conventional ligand binding assays, due to reduced assay complexity and by providing additional information, such as isoform differentiation or detection of post-translational modifications. The 47.2 kDa lung cancer tumor marker neuron-specific enolase γ (NSEγ) was quantified in a clinically relevant concentration range of 6.25 to 100 ng/mL in NSE-depleted human serum using magnetic bead immunoprecipitation coupled to LC-high-resolution quadrupole-time-of-flight MS. The novelty of the described approach is in the combined setup of immunoaffinity extraction and the use of a full-length NSEγ calibrator and labeled NSEγ internal standard (IS) to reliably quantify the post-translationally acetylated form of this protein tumor marker in a top-down proteomics workflow. Isolation parameters and quantification using deconvolution and reconstructed extracted ion chromatograms were evaluated, and the development of a suitable liquid chromatography method was demonstrated. Various validation parameters were determined using both quantification methods, both showing acceptable performance. Additionally, deconvolution-based quantification enabled an accurate mass determination. The developed method was compared to a commercially available ECLIA and showed good correlation in sera of patients suspected of lung cancer. This assay may form the starting point for the development of a reference method for the standardization of immunoassays.

Exploring the Role of Unconventional Post-Translational Modifications in Cancer Diagnostics and Therapy.

Unconventional Post-Translational Modifications (PTMs) have gained increasing attention as crucial players in cancer development and progression. Understanding the role of unconventional PTMs in cancer has the potential to revolutionize cancer diagnosis, prognosis, and therapeutic interventions. These modifications, which include O-GlcNAcylation, glutathionylation, crotonylation, including hundreds of others, have been implicated in the dysregulation of critical cellular processes and signaling pathways in cancer cells. This review paper aims to provide a comprehensive analysis of unconventional PTMs in cancer as diagnostic markers and therapeutic targets. The paper includes reviewing the current knowledge on the functional significance of various conventional and unconventional PTMs in cancer biology. Furthermore, the paper highlights the advancements in analytical techniques, such as biochemical analyses, mass spectrometry and bioinformatic tools etc., that have enabled the detection and characterization of unconventional PTMs in cancer. These techniques have contributed to the identification of specific PTMs associated with cancer subtypes. The potential use of Unconventional PTMs as biomarkers will further help in better diagnosis and aid in discovering potent therapeutics. The knowledge about the role of Unconventional PTMs in a vast and rapidly expanding field will help in detection and targeted therapy of cancer.

On the Hunt for the Histone Code

Our genome is not made of naked DNA, but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don’s clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab’s effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don’s pioneering work in this area, many labs have been able to jump into the epigenetics field and “Hunt” down their own histone targets. A walkthrough of those early histone years in the Hunt Lab are described by three of us who were fortunate enough to be at the right place, at the right time.

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Abstract 1838: Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si

Abstract Protein sequencing has the potential of playing a crucial role in advancing cancer research by providing insights into the specific proteins involved in cancer pathways. New discoveries from protein sequencing advance our understanding of cancer and potential targeted therapies. Here, we demonstrate the power of next-generation protein sequencing™ using the insight-generating Platinum™ platform. Individual peptides are digested from proteins and immobilized on a semiconductor chip and probed by dye-labeled N-terminal amino acid (NAA) recognizers, followed by subsequent aminopeptidase cleavage to expose each NAA in the peptide for recognition. By recording and analyzing fluorescent intensity, lifetime, and binding kinetics of each NAA binding event, we can decipher single amino acid substitutions and modifications. To demonstrate the versatility of this methodology across proteins associated with cellular biology, we sequenced a series of interleukins: IL4, IL6, IL7, IL13, IL20, and IL34. Interleukins, a group of cytokines that play a vital role in cancer by modulating immune responses, promoting inflammation, and influencing tumor microenvironment, impacting tumor growth and the efficacy of treatments. We have successfully sequenced all the mentioned interleukins individually or within protein mixtures. Furthermore, we were able to sequence IL6 that was enriched through immunoprecipitation from human serum. Finally, we demonstrated the unique potential of Platinum in detecting post-translational modifications (PTMs) relevant to cancer, as exemplified by the successful identification of arginine citrullination in vimentin, an onco-protein that promotes cancer cell mobility and invasion into surround tissues. These results demonstrate the transformative potential of next-generation protein sequencing using Platinum™ in enabling the identification of proteins in complex mixtures and biofluids, as well as the detection of critical PTMs implicated in human pathophysiology, offering valuable insights into cancer research. Citation Format: Khanh Dinh Nguyen. Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1838.

ChipFilter: Microfluidic-Based Comprehensive Sample Preparation Methodology for Microbial Consortia.

The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.

Enrichment of histone tail methylated lysine residues via cavitand-decorated magnetic nanoparticles for ultra-sensitive proteomics.

Nearly every protein in the human body is modified with post-translational modifications (PTMs). PTMs affect proteins on many levels, including their function, interaction, half-life, and localization. Specifically, for histone proteins, PTMs such as lysine methylation and acetylation play essential roles in chromatin dynamic regulations. For this reason, methods to accurately detect and quantify PTMs are of paramount importance in cell biology, biochemistry, and disease biology. Most protein modifications are sub-stoichiometric, so, to be analyzed, they need methods of enrichment, which are mostly based on antibodies. Antibodies are produced using animals, resulting in high costs, ecological concerns, significant batch variations, and ethical implications. We propose using ferromagnetic nanoparticles functionalized with synthetic receptors, namely tetraphosphonate cavitands, as a tool for selective enrichment of methylated lysines present on histone tails. Before the enrichment step, histone proteins from calf thymus were digested to facilitate the recognition process and to obtain small peptides suitable for mass analyses. Cavitands were anchored on ferromagnetic nanoparticles to easily separate the PTM-peptides of interest from the rest of the proteolytic peptides. Our approach detects more modified peptides with higher signal intensity, rivaling commercial antibodies. This chemical strategy offers a cost-effective and efficient alternative for PTM detection, potentially advancing proteomic research.

Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER–ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection.

Integrated Mass Spectrometry‐based Pipeline for Characterization of Tau in Human Alzheimer’s Brain: Development and Comparison of Sample Preparation Methods

AbstractBackgroundTau protein aggregation into neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer’s disease (AD) and related tauopathies [1]. Though the mechanism of tau destabilization is not fully understood, tau protein exhibits several allele‐specific isoforms and post‐translational modifications (PTMs) specific to tau aggregates [2]. To explore in‐depth tau species in brain tissues of AD patients, we developed novel pipelines based on different tau fractions retrieved by buffer solubility, Filter Aided Sample Preparation (FASP) or immunoprecipitation, before high‐resolution mass spectrometry (HRMS) analysis.MethodAs a proof of concept, we used frozen brain samples from two AD patients (0.6‐1.2 g per replicate, middle frontal gyrus). The samples were divided and homogenized, ultracentrifuged at 180.000xg for 30min and solubilized in different buffers successively (2 replicates): (1) SarkMethodlow‐salt buffer, 10% Triton X‐100, sarkosyl buffer, and urea buffer; (2) RIPAMethodhigh‐salt buffer (twice), RIPA buffer and urea buffer; and, (3) GnHMethodguanidine hydrochloride and urea buffer. Then, we performed an antibody‐free approach by FASP or immunoprecipitation with a panel of commercial tau antibodies and analyzed on reversed‐phase capillary liquid chromatography, coupled with a qExactive HRMS.ResultHRMS demonstrated that the RIPA method performed better than the other methods for Tau analysis: coverage of 63% (full‐length, 2N4R) and 342 tau peptide‐spectrum matches (PSMs) (Sark: 49% and 183 PSMs; GnH: 41% and 169 PSMs). PTM‐modified tau species (phosphorylated, methylated, and acetylated) were primarily seen using the RIPA method (59 PSMs; Sark: 19 PSMs, GnH: 21 PSMs). Immunoprecipitation improved tau coverage and PTM detection compared to FASP.ConclusionThese findings highlight the best performance of the RIPA method followed by immunoprecipitation for the study of AD brain tau allele‐specific isoforms and PTMs using HRMS. Better characterization of Tau molecular profile in the brain of AD patients opens the gate for a better understanding of AD pathogenesis and represents an opportunity in the biomarker field.[1] Mroczko, B. et al. Int J Mol Sci 2019, 20, E4661, https://doi.org/10.3390/ijms20194661.[2] Boyarko, B.; Hook, V. Front. Neurosci. 2021, 15, 702788, https://doi.org/10.3389/fnins.2021.702788.

Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection.

Generating top-down tandem mass spectra (MS/MS) from complex mixtures of proteoforms benefits from improvements in fractionation, separation, fragmentation, and mass analysis. The algorithms to match MS/MS to sequences have undergone a parallel evolution, with both spectral alignment and match-counting approaches producing high-quality proteoform-spectrum matches (PrSMs). This study assesses state-of-the-art algorithms for top-down identification (ProSight PD, TopPIC, MSPathFinderT, and pTop) in their yield of PrSMs while controlling false discovery rate. We evaluated deconvolution engines (ThermoFisher Xtract, Bruker AutoMSn, Matrix Science Mascot Distiller, TopFD, and FLASHDeconv) in both ThermoFisher Orbitrap-class and Bruker maXis Q-TOF data (PXD033208) to produce consistent precursor charges and mass determinations. Finally, we sought post-translational modifications (PTMs) in proteoforms from bovine milk (PXD031744) and human ovarian tissue. Contemporary identification workflows produce excellent PrSM yields, although approximately half of all identified proteoforms from these four pipelines were specific to only one workflow. Deconvolution algorithms disagree on precursor masses and charges, contributing to identification variability. Detection of PTMs is inconsistent among algorithms. In bovine milk, 18% of PrSMs produced by pTop and TopMG were singly phosphorylated, but this percentage fell to 1% for one algorithm. Applying multiple search engines produces more comprehensive assessments of experiments. Top-down algorithms would benefit from greater interoperability.

Y-mtPTM: Yeast mitochondrial posttranslational modification database.

One powerful strategy of how to increase the complexity of cellular proteomes is through posttranslational modifications (PTMs) of proteins. Currently, there are ∼400 types of PTMs, the different combinations of which yield a large variety of protein isoforms with distinct biochemical properties. Although mitochondrial proteins undergoing PTMs were identified nearly 6 decades ago, studies on the roles and extent of PTMs on mitochondrial functions lagged behind the other cellular compartments. The application of mass spectrometry for the characterization of the mitochondrial proteome as well as for the detection of various PTMs resulted in the identification of thousands of amino acid positions that can be modified by different chemical groups. However, the data on mitochondrial PTMs are scattered in several data sets, and the available databases do not contain a complete list of modified residues. To integrate information on PTMs of the mitochondrial proteome of the yeast Saccharomyces cerevisiae, we built the yeast mitochondrial posttranslational modification (y-mtPTM) database (http://compbio.fmph.uniba.sk/y-mtptm/). It lists nearly 20,000 positions on mitochondrial proteins affected by ∼20 various PTMs, with phosphorylated, succinylated, acetylated, and ubiquitylated sites being the most abundant. A simple search of a protein of interest reveals the modified amino acid residues, their position within the primary sequence as well as on its 3D structure, and links to the source reference(s). The database will serve yeast mitochondrial researchers as a comprehensive platform to investigate the functional significance of the PTMs of mitochondrial proteins.

Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.

Abstract 5301: Detection of arginine posttranslational modifications by single-molecule protein sequencing on the Quantum-Si platform

Abstract Aberrant post-translational modifications (PTMs) on arginine residues, such as methylation and citrullination, are closely linked to oncogenic processes. Detection of these PTMs is challenging with current technology and can be hampered by low abundance and mass differences that are difficult to resolve by mass spectrometry. To overcome these challenges, we developed methods for PTM detection using Quantum-Si's Platinum single-molecule protein sequencing platform. Proteins are sequenced on Quantum-Si's Platinum instrument using our library prep protocol. Briefly, proteins are digested into peptide fragments, conjugated to linkers, then immobilized at the bottom of nanoscale reaction chambers on a semiconductor chip, resulting in exposed N-termini for sequencing. During sequencing, the immobilized peptides are exposed to a solution of dye-labeled N-terminal amino acid (NAA) recognizers that reversibly bind to their cognate NAAs with distinct kinetic properties. Aminopeptidases sequentially remove NAAs to expose subsequent amino acids for recognition. Fluorescence lifetime, intensity, and kinetic data are collected in real time and analyzed to determine primary sequence and PTM content. The data output consists of distinct pulsing regions called recognition segments (RSs). Each RS corresponds to a period of time between aminopeptidase cleavage events, during which an NAA recognizer binds on/off to its exposed target NAA. Chemical modification to a NAA or to a nearby downstream amino acid can modulate recognizer affinity, resulting in a characteristic change in the average pulse duration (PD) during an RS. To demonstrate the detection and differentiation of arginine PTMs, we applied our platform to distinguish between asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) and between citrulline and native arginine residues. Sequencing traces revealed that arginine and ADMA bind the arginine recognizer (PS621) with similar PD, whereas SDMA exhibited no binding. These results indicate that SDMA, in contrast to ADMA, has reduced binding affinity with PS621, providing a clear kinetic difference between these isomeric arginine PTMs. Moreover, citrulline and arginine side chains also exhibited distinguishable kinetic signatures. Citrullination eliminated the N-terminal arginine recognized by PS621 and resulted in a large increase in the median PD of preceding amino acid residues. Taken together, single-molecule protein sequencing offers an alternative approach to detection of arginine PTMs that is not based on m/z, but rather on the kinetic signature of binding between recognizers and N-terminal amino acids. The ability to directly detect arginine PTMs offers potential for biomedical research. We envisage applications using Quantum-Si's platform to key areas of cancer research including biomarker development and drug discovery. Citation Format: Kenneth Skinner, David Kamber, Brian Reed. Detection of arginine posttranslational modifications by single-molecule protein sequencing on the Quantum-Si platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5301.

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis.

As the common and significant chemical modifications, post-translational modifications (PTMs) play a key role in the functional proteome. Affected by the signal interference, low concentration, and insufficient ionization efficiency of impurities, the direct detection of PTMs by mass spectrometry (MS) still faces many challenges. Therefore, sample preparation and enrichment are an indispensable link before MS analysis of PTMs in proteomics. The rapid development of functionalized materials with diverse morphologies and compositions provides an avenue for sample preparation and enrichment for PTMs analysis. In this review, we summarize recent advances in the application of novel functionalized materials in sample preparation for phosphoproteomes and glycoproteomes analysis. In addition, this review specifically discusses the design and preparation of functionalized materials based on different enrichment mechanisms, and proposes research directions and potential challenges for proteomic PTMs research.

Discovery of Targets for Cancer Immunoprevention.

Antibodies against autologous tumor-associated antigens have been demonstrated as being useful biomarkers for early cancer diagnosis and prognosis. They have several advantages such as long half-life (7-30days depending on subtiter of Ig), inherent stability in patients' blood due to not being subjected to proteolysis, well-studied biochemical properties, and their easy detections via secondary antibodies or antigens. Moreover, they can be easily screened in the serum using a noninvasive approach. Consequently, many technical approaches have been developed to study autoantibodies. We used serological proteome analysis (SERPA) for analyzing antibodies in pancreatic cancer patients' sera, and the technique will be discussed in detail. SERPA has several advantages over other approaches currently used such as SEREX (serological analysis of tumor antigens by recombinant cDNA expression cloning) and phage display. SEREX involves the construction of a lambda phage cDNA library from tumor samples to infect bacteria. While library construction is a quite laborious and time-consuming procedure in SEREX, detection of posttranslational modifications that could be fundamental for antibody recognition is a major limitation of both SEREX and phage display techniques. SERPA avoids the time-consuming construction of cDNA libraries. In addition, since it does not rely on bacterial expression of antigens, antigens will have their usual posttranslational modifications preventing false-positive or -negative results in autoantibody profiling.

Heterogeneous sensing of post-translational modification enzymes by integrating the advantage of homogeneous analysis

Heterogeneous analysis has great application prospects in the detection of post-translational modification (PTM) enzymes with the advantages of signal enhancement, less sample demand, and high sensitivity and selectivity. Nevertheless, once the substrate was fixed on a solid interface, the steric hindrance might limit the approaching of catalytic center to the substrate, thus reducing the efficiency of PTM. Herein, we suggested that the avidin-modified interface could be used to develop heterogeneous sensing platforms with biotin-labeled substrates as the probes, in which the enzymatic PTM was performed in solution and the heterogeneous assay was conducted on a solid surface. The sensing strategy integrates the advantages but overcomes the defects of both homogeneous and heterogeneous assays. Protein kinase A (PKA) and histone acetyltransferase (HAT) were determined as the examples by using sequence-specific peptide substrates. The signal changes were monitored by HRP-based colorimetric assay and antibody-amplified surface plasmon resonance (SPR). The methods were used for analysis of cell lysates and evaluation of inhibition efficiency with satisfactory results. The strategy can be used for the detection of a variety of biological enzymes and provide a new idea for the design of various heterogeneous biosensors. Thus, this work should be of great significance to the popularization and practical application of biosensors.

Enhanced identification of Tau acetylation and phosphorylation with an engineered aerolysin nanopore.

Posttranslational modifications (PTMs) affect protein function/dysfunction, playing important roles in the occurrence and development of tauopathies including Alzheimer's disease. PTM detection is significant and still challenging due to the requirements of high sensitivity to identify the subtle structural differences between modifications. Herein, in terms of the unique geometry of the aerolysin (AeL) nanopore, we elaborately engineered a T232K AeL nanopore to detect the acetylation and phosphorylation of Tau segment (Pep). By replacing neutral threonine (T) with positively charged lysine (K) at the 232 sites, the T232K and K238 rings of this engineered T232K AeL nanopore corporately work together to enhance electrostatic trapping of the acetylated and phosphorylated Tau peptides. Translocation speed of the monophosphorylated Pep-P was decelerated by up to 46 folds compared to the wild-type (WT) AeL nanopore. The prolonged residences within the T232K AeL nanopore enabled to simultaneously identify the monoacetylated Pep-Ac, monophosphorylated Pep-P, di-modified Pep-P-Ac and non-modified Pep. The tremendous potential is demonstrated for PTM sensing by manipulating non-covalent interactions between nanopores and single analytes.

Probing multiple enzymatic methylation events in real time with NMR spectroscopy

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

A novel proteomics approach to epigenetic profiling of circulating nucleosomes

Alteration of epigenetic modifications plays an important role in human cancer. Notably, the dysregulation of histone post-translational modifications (PTMs) has been associated with several cancers including colorectal cancer (CRC). However, the signature of histone PTMs on circulating nucleosomes is still not well described. We have developed a fast and robust enrichment method to isolate circulating nucleosomes from plasma for further downstream proteomic analysis. This method enabled us to quantify the global alterations of histone PTMs from 9 CRC patients and 9 healthy donors. Among 54 histone proteoforms identified and quantified in plasma samples, 13 histone PTMs were distinctive in CRC. Notably, methylation of histone H3K9 and H3K27, acetylation of histone H3 and citrullination of histone H2A1R3 were upregulated in plasma of CRC patients. A comparative analysis of paired samples identified 3 common histone PTMs in plasma and tumor tissue including the methylation and acetylation state of lysine 27 of histone H3. Moreover, we highlight for the first time that histone H2A1R3 citrulline is a modification upregulated in CRC patients. This new method presented herein allows the detection and quantification of histone variants and histone PTMs from circulating nucleosomes in plasma samples and could be used for biomarker discovery of cancer.

Detection of Post-translational Modifications on MYC.

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.