Publications by authors named "Shaolian Zhou"

27 Publications

  • Page 1 of 1

Toward Accurate and Robust Liquid Chromatography-Mass Spectrometry-Based Quantification of Antibody Biotherapeutics in Tissues.

Anal Chem 2020 11 6;92(22):15152-15161. Epub 2020 Nov 6.

The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, United States.

Liquid chromatography-mass spectrometry (LC-MS) affords a highly promising solution for absolute quantification of biotherapeutics/targets in tissues, which is critical for drug development. Nonetheless, accurate/robust tissue quantification remains challenging largely owing to the lack of optimal approaches to address the following fundamental prerequisites: (i) efficient removal of residual blood without losing tissue-associated biotherapeutics; (ii) an optimal method to exhaustively/quantitatively recover target proteins from tissues; and (iii) an appropriate strategy to prepare calibration/quality-control samples to ensure accurate tissue analysis. Here, we devised novel analytical procedures enabling extensive and systematic investigation of the above issues and thereby development of optimal strategies for accurate tissue analysis. Key discoveries include: first, using a novel procedure of sequential administration of nonlabeled and then stable-isotope-labeled monoclonal antibody (mAb); it was determined that perfusion with three blood volumes of heparinized saline is optimal, achieving efficient blood removal (95-99%) and low quantitative bias (0.5-13%); second, a reference sample set established by mass-balanced, exhaustive extraction, permitted accurate measurement of protein recovery from tissues of dosed animals; with this method, we found mAb biotherapeutics present in free-(49.3-75.4%) and bound-forms (24.6-50.7%) in tissues, even without a target; therefore, a denaturing detergent buffer is necessary for exhaustive extraction (recovery>90%); third, overnight-incubation of calibration samples after spiking mAb to tissue was found to improve quantitative accuracy, especially for nondenaturing buffer extraction. These investigations established the critical parameters and optimal protocols that can be universally applied to achieve accurate and robust quantification of biotherapeutics/targets in tissues. As a proof of concept, we conducted the first-ever extensive pharmacokinetics measurement of mAb in major tissues with a LC-MS-based method, where interesting features of mAb tissue disposition were observed.
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http://dx.doi.org/10.1021/acs.analchem.0c03620DOI Listing
November 2020

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

Bioanalysis 2019 Sep 30;11(18s):1-228. Epub 2019 Sep 30.

WuXi Apptec, Shanghai, China.

The 13 GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.
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http://dx.doi.org/10.4155/bio-2019-0207DOI Listing
September 2019

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Anal Chem 2019 03 15;91(5):3475-3483. Epub 2019 Feb 15.

The Department of Pharmaceutical Sciences , University at Buffalo, State University of New York , Buffalo , New York 14214 , United States.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption-desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI - 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87-95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.
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http://dx.doi.org/10.1021/acs.analchem.8b05046DOI Listing
March 2019

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

Bioanalysis 2018 Dec 29;10(23):1897-1917. Epub 2018 Nov 29.

F Hoffmann-La Roche, Basel, Switzerland.

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of , issues 22 and 24 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0285DOI Listing
December 2018

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics.

Anal Chem 2018 02 8;90(3):1870-1880. Epub 2018 Jan 8.

Department of Pharmaceutical Sciences, University at Buffalo, State University of New York , Buffalo, New York 14214, United States.

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.
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http://dx.doi.org/10.1021/acs.analchem.7b03949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960441PMC
February 2018

Practical considerations in enhancing LC-MS sensitivity for therapeutic protein bioanalysis.

Bioanalysis 2017 Sep;9(18):1353-1356

The Department of Pharmaceutical Sciences, School of Pharmacy & Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.

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http://dx.doi.org/10.4155/bio-2017-4982DOI Listing
September 2017

An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED.

Nat Chem Biol 2017 04 30;13(4):381-388. Epub 2017 Jan 30.

Novartis Institutes for BioMedical Research, Shanghai, China.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.
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http://dx.doi.org/10.1038/nchembio.2304DOI Listing
April 2017

Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS.

J Proteome Res 2017 02 11;16(2):780-787. Epub 2017 Jan 11.

Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.

Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using N-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.
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http://dx.doi.org/10.1021/acs.jproteome.6b00788DOI Listing
February 2017

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 2 - Hybrid LBA/LCMS and input from regulatory agencies).

Bioanalysis 2016 Dec;8(23):2457-2474

Roche Pharma Research and Early Development, Roche Innovation Center, Basel, Switzerland.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10 WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
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http://dx.doi.org/10.4155/bio-2016-4988DOI Listing
December 2016

Quantitative Profiling of Combinational K27/K36 Modifications on Histone H3 Variants in Mouse Organs.

J Proteome Res 2016 Mar 5;15(3):1070-9. Epub 2016 Feb 5.

China Novartis Institutes for BioMedical Research Co. Ltd. , Building 8, Lane 898 Halei Road, Shanghai 201203, China.

The coexisting post-translational modifications (PTMs) on histone H3 N-terminal tails were known to crosstalk between each other, indicating their interdependency in the epigenetic regulation pathways. H3K36 methylation, an important activating mark, was recently reported to antagonize with PRC2-mediated H3K27 methylation with possible crosstalk mechanism during transcription regulation process. On the basis of our previous studies, we further integrated RP/HILIC liquid chromatography with MRM mass spectrometry to quantify histone PTMs from various mouse organs, especially the combinatorial K27/K36 marks for all three major histone H3 variants. Despite their subtle difference in physicochemical properties, we successfully obtained decent separation and high detection sensitivity for both histone H3.3 specific peptides and histone H3.1/3.2 specific peptides. In addition, the overall abundance of H3.3 can be quantified simultaneously. We applied this method to investigate the pattern of the combinatorial K27/K36 marks for all three major histone H3 variants across five mouse organs. Intriguing distribution differences were observed not only between different H3 variants but also between different organs. Our data shed the new insights into histone codes functions in epigenetic regulation during cell differentiation and developmental process.
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http://dx.doi.org/10.1021/acs.jproteome.5b01164DOI Listing
March 2016

SETDB1 modulates PRC2 activity at developmental genes independently of H3K9 trimethylation in mouse ES cells.

Genome Res 2015 Sep 9;25(9):1325-35. Epub 2015 Jul 9.

China Novartis Institutes for BioMedical Research, Shanghai 201203, China;

SETDB1, a histone methyltransferase responsible for methylation of histone H3 lysine 9 (H3K9), is involved in maintenance of embryonic stem (ES) cells and early embryonic development of the mouse. However, how SETDB1 regulates gene expression during development is largely unknown. Here, we characterized genome-wide SETDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SETDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SETDB1 solo peaks, particularly those near neural development-related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins JARID2 and MTF2. Genetic deletion of Setdb1 reduced EZH2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SETDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SETDB1 methyltransferase activity. The currently identified reciprocal action between SETDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation.
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http://dx.doi.org/10.1101/gr.177576.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561491PMC
September 2015

Absolute quantification of histone PTM marks by MRM-based LC-MS/MS.

Anal Chem 2014 Oct 8;86(19):9679-86. Epub 2014 Sep 8.

China Novartis Institutes for BioMedical Research Co. Ltd. , Building 8, Lane 898 Halei Road, Shanghai 201203, China.

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells.
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http://dx.doi.org/10.1021/ac502333aDOI Listing
October 2014

A liquid chromatography/mass spectrometry-based generic detection method for biochemical assay and hit discovery of histone methyltransferases.

Anal Biochem 2013 Dec 7;443(2):214-21. Epub 2013 Sep 7.

Novartis Institutes for BioMedical Research, Cambridge, MA, 02139, USA. Electronic address:

Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.
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http://dx.doi.org/10.1016/j.ab.2013.08.029DOI Listing
December 2013

NSD2 is recruited through its PHD domain to oncogenic gene loci to drive multiple myeloma.

Cancer Res 2013 Oct 26;73(20):6277-88. Epub 2013 Aug 26.

Authors' Affiliations: Novartis Institutes for BioMedical Research (China), Shanghai, P.R. China; Genomics Institute of the Novartis Research Foundation, San Diego, California; and Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.

Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1000DOI Listing
October 2013

Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS.

Anal Chem 2013 Feb 6;85(4):2253-9. Epub 2013 Feb 6.

China Novartis Institutes for BioMedical Research Co. Ltd., Building 8, lane 898 Halei road, Shanghai 201203, China.

Histones participate in epigenetic regulation via a variety of dynamic posttranslational modifications (PTMs) on them. Mass spectrometry (MS) has become a powerful tool to investigate histone PTMs. With the bottom-up mass spectrometry approach, chemical derivatization of histones with propionic anhydride or deuterated acetic anhydride followed by trypsin digestion was widely used to block the hydrophilic lysine residues and generate compatible peptides for LC-MS analysis. However, certain severe side reactions (such as acylation on tyrosine or serine) caused by acid anhydrides will lead to a number of analytical issues such as reducing results accuracy and impairing the reproducibility and sensitivity of MS analysis. As an alternative approach, we report a novel derivatization method that utilizes N-hydroxysuccinimide ester to specifically and efficiently derivatize both free and monomethylated amine groups in histones. A competitive inhibiting strategy was implemented in our method to effectively prevent the side reactions. We demonstrated that our method can achieve excellent specificity and efficiency for histones derivatization in a reproducible manner. Using this derivatization method, we succeeded to quantitatively profile the histone PTMs in KMS11 cell line with selective knock out of translocated NSD2 allele (TKO) and the original parental KMS11 cell lines (PAR) (NSD2, a histone methyltransferase that catalyzes the histone H3 K36 methylation), which revealed a significant crosstalk between H3 protein K27 methylation and adjacent K36 methylation.
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http://dx.doi.org/10.1021/ac303171hDOI Listing
February 2013

Detecting S-adenosyl-L-methionine-induced conformational change of a histone methyltransferase using a homogeneous time-resolved fluorescence-based binding assay.

Anal Biochem 2012 Apr 27;423(1):171-7. Epub 2012 Jan 27.

China Novartis Institute for Biomedical Research, Pudong New Area, Shanghai 201203, China.

A homogeneous time-resolved fluorescence (HTRF)-based binding assay has been established to measure the binding of the histone methyltransferase (HMT) G9a to its inhibitor CJP702 (a biotin analog of the known peptide-pocket inhibitor, BIX-01294). This assay was used to characterize G9a inhibitors. As expected, the peptide-pocket inhibitors decreased the G9a-CJP702 binding signal in a concentration-dependent manner. In contrast, the S-adenosyl-L-methionine (SAM)-pocket compounds, SAM and sinefungin, significantly increased the G9a-CJP702 binding signal, whereas S-adenosyl-L-homocysteine (SAH) showed minimal effect. Enzyme kinetic studies showed that CJP702 is an uncompetitive inhibitor (vs. SAM) that has a strong preference for the E:SAM form of the enzyme. Other data presented suggest that the SAM/sinefungin-induced increase in the HTRF signal is secondary to an increased E:SAM or E:sinefungin concentration. Thus, the G9a-CJP702 binding assay not only can be used to characterize the peptide-pocket inhibitors but also can detect the subtle conformational differences induced by the binding of different SAM-pocket compounds. To our knowledge, this is the first demonstration of using an uncompetitive inhibitor as a probe to monitor the conformational change induced by compound binding with an HTRF assay.
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http://dx.doi.org/10.1016/j.ab.2012.01.019DOI Listing
April 2012

Structure of human SMYD2 protein reveals the basis of p53 tumor suppressor methylation.

J Biol Chem 2011 Nov 31;286(44):38725-38737. Epub 2011 Aug 31.

Novartis Institutes for BioMedical Research, Building 8, Lane 898, Halei Road, Pudong, Shanghai 201203, China. Electronic address:

SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel β7 and β8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.
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http://dx.doi.org/10.1074/jbc.M111.262410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207477PMC
November 2011

Histone methylation by PRC2 is inhibited by active chromatin marks.

Mol Cell 2011 May;42(3):330-41

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.
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http://dx.doi.org/10.1016/j.molcel.2011.03.025DOI Listing
May 2011

The use of chemical derivatization to enhance liquid chromatography/tandem mass spectrometric determination of 1-hydroxypyrene, a biomarker for polycyclic aromatic hydrocarbons in human urine.

Rapid Commun Mass Spectrom 2005 ;19(22):3331-8

Covance Laboratories Inc., 3301 Kinsman Boulevard, Madison, Wisconsin, USA.

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.
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http://dx.doi.org/10.1002/rcm.2196DOI Listing
March 2006

Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry--experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity.

J Chromatogr B Analyt Technol Biomed Life Sci 2005 Oct;825(2):223-32

Department of Bioanalytical Chemistry, Covance Laboratories Inc., 3301 Kinsman Blvd. Madison, WI 53704, USA.

In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.
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http://dx.doi.org/10.1016/j.jchromb.2005.01.012DOI Listing
October 2005

High-throughput biological sample analysis using on-line turbulent flow extraction combined with monolithic column liquid chromatography/tandem mass spectrometry.

Rapid Commun Mass Spectrom 2005 ;19(15):2144-50

DMPK-Bioanalytical Chemistry Department, Covance Laboratories Inc., Madison, WI 53704, USA.

A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, which combines on-line sample extraction through turbulent flow chromatography with a monolithic column separation, has been developed for direct injection analysis of drugs and metabolites in human plasma samples. By coupling a monolithic column into the system as the analytical column, the method enables running 'dual-column' extraction and chromatography at higher flow rates, thus significantly reducing the time required for the transfer and mixing of extracted fraction onto the separation column as well as the time for gradient separation. A strategy of assessing and reducing the matrix suppression effect on the on-line extraction LC/MS/MS has also been discussed. Experiments for evaluating the resolution, peak shape, sensitivity, speed, and matrix effect were conducted with dextromethorphan and its metabolite dextrorphan as model compounds in human plasma matrix. It was demonstrated that the total run time for this assay with a baseline separation of two analytes is less than 1.5 min.
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http://dx.doi.org/10.1002/rcm.2037DOI Listing
August 2005

Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco-specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations.

Rapid Commun Mass Spectrom 2004 ;18(21):2549-57

Covance Bioanalytical Chemistry, Madison, WI 53704, USA.

A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.
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http://dx.doi.org/10.1002/rcm.1656DOI Listing
November 2004

Tandem mass spectrometry for structure assignments of wye nucleosides from transfer RNA.

Nucleosides Nucleotides Nucleic Acids 2004 ;23(1-2):41-50

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA.

The tricyclic wye nucleoside family of eight known members constitutes one of the most complex and interesting series of posttranscriptionally modified nucleosides in transfer RNA. The principal reaction paths represented in collision-induced dissociation mass spectra of wye bases and their analogs have been studied in order to determine those structural features that can be readily established by mass spectrometry. The main routes of fragmentation are determined by the presence vs. absence of an amino acid side chain at C-7 (1H-imidazo[1,2-a]purine nomenclature). The common methionine-related side chain is cleaved at two points, providing a ready means of establishing the presence and net level of side chain modification. For those molecules without a side chain, the initial reaction steps are characteristically controlled by the presence vs. absence of methyl at N-4, allowing determination of the methylation status of that site. In the latter case initial opening of the central (pyrimidine) ring, in analogy to the dissociation behavior of guanine, causes loss of identity of C-6/C-7 so that placement of a single methyl at either site is not possible. Subsequent complex reaction paths follow, which include loss of CO and sequential loss of two molecules of HCN.
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http://dx.doi.org/10.1081/ncn-120027816DOI Listing
October 2004

Structures of two new "minimalist" modified nucleosides from archaeal tRNA.

Bioorg Chem 2004 Apr;32(2):82-91

Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112-5820, USA.

The wyeosine (or wye) family of tricyclic ribonucleosides from archaeal and eukaryal tRNA(Phe) constitutes one of the most complex and interesting series of posttranscriptional RNA modifications, and has been the object of numerous studies of their chemical and biological synthesis and distribution. We report the structures of two minimally elaborated wye derivatives from archaea, raising the known number of wye nucleosides to eight: 3,4-dihydro-6-methyl-3-beta-d-ribofuranosyl-9H-imidazo[1,2-a]purine-9-one (symbol imG-14), and 3,4-dihydro-6,7-dimethyl-3-beta-d-ribofuranosyl-9H-imidazo[1,2-a]purine-9-one (symbol imG2). Structures were determined primarily by mass spectrometry, and confirmed by comparison of physicochemical properties with those of chemically synthesized nucleosides. The nucleosides contain no amino acid side chains at C-7 (1H-imidazo[1,2-a]purine nomenclature) and are the only wye derivatives not methylated at N-4. These features suggest a minimal role for wye methyl groups and side chains in maintenance of anticodon stem-loop structures, and support the concept that archaeal tRNA nucleoside modification motifs are generally simpler than those of their counterparts in eukarya and bacteria.
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http://dx.doi.org/10.1016/j.bioorg.2003.09.005DOI Listing
April 2004

Profiling pH changes in the electrospray plume.

Anal Chem 2002 Oct;74(19):4885-8

Department of Chemistry, University of Tennessee, Knoxville 37996-1600, USA.

Laser-induced fluorescence spectrometry is used to profile pH changes as droplets evaporate in an electrospray plume by measuring emission spectra of 2(or 4)-[10-(dimethylamino)-3-oxo-3H-benzo[c]xanthene-7-yl]-benzenedicarboxylic acid (carboxy SNARF-1), a pH-sensitive fluorescent dye. The observed pH changes depend on initial droplet pH and polarity. In some instances, small or negligible changes of pH are observed, consistent with expected buffering. The pH of initially acidic droplets decreases along the spray axis in both positive and negative ion modes, to an extent larger than expected from solvent evaporation. This phenomenon may be a manifestation of droplet cooling, droplet subdivision, or heterogeneous charge distribution within the spray plume or within individual ES droplets.
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http://dx.doi.org/10.1021/ac025960dDOI Listing
October 2002

Qualitative assessment of monomer ratios in putative ionic terpolymer samples by electrospray ionization mass spectrometry with collision-induced dissociation.

J Am Soc Mass Spectrom 2002 Aug;13(8):921-7

Department of Chemistry, University of Tennessee, Knoxville 37996-1600, USA.

Collision-induced dissociation in the source of an electrospray (ES) mass spectrometer was employed to characterize putative samples of the ionic terpolymer poly(styrene sulfonate-co-acrylic acid-co-2-acrylamido-2-methyl-1-propanesulfonic acid). Qualitative and semi-quantitative information about the monomer content was quickly obtainable from ES spectra, and indicated that some samples contained little or none of one or two expected comonomers. For two representative samples, confirmatory nuclear magnetic resonance (NMR) data were acquired. The NMR experiments required sample clean-up (to remove additives) and long acquisition times (up to 720 min) for 13C NMR. Cleanup also improved the ES results, providing better agreement with the NMR data. However, qualitative and semi-quantitative information was obtainable by ES (but not by NMR) without the cleanup step. Full quantitation of monomer ratios would require suitable standards, but even without such standards the ES measurements provide a rapid (<1 min) means for differentiating these samples (e.g., for process or quality control).
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http://dx.doi.org/10.1016/S1044-0305(02)00411-7DOI Listing
August 2002