Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS.

Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone H3 N-terminal tail peptides (amino acids 1-50, 5-6 kDa) were separated by optimized weak cation exchange/hydrophilic interaction liquid chromatography (WCX/HILIC) and sequenced online by electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS). High mass accuracy and near complete sequence coverage allowed unambiguous mapping of the major histone marks and discrimination between isobaric and nearly isobaric PTMs such as trimethylation and acetylation. Hierarchical data analysis identified H3K27me2-H3K36me2 as the most frequently observed PTMs in H3. Modifications at H3 residues K27 and K36 often coexist with the abundant mark K23ac, and we identified two frequently occurring quadruplet marks 'K9me1K23acK27me2K36me2' and 'K9me3K23acK27me2K36me', which might indicate a role in crosstalk. Co-occurrence frequency analysis revealed also an interplay between methylations of K9, K27, and K36, suggesting interdependence between histone methylation marks. We hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells.

A probabilistic framework for peptide and protein quantification from data-dependent and data-independent LC-MS proteomics experiments.

A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods.

Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes.

Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.

Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36.

SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects of Suz12 deficiency on H3.2 and H3.3 from mouse ESCs. Using high mass accuracy MS combined with CID or electron transfer dissociation (ETD) tandem mass spectrometry, we identified a total of 81 unique modified peptides from H3.2 and H3.3 and assigned 46 modifications at 22 different positions, including distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides. The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone peptides (amino acids 27-40) showed the relative distribution of distinct coexisting histone marks. Our study revealed limitations of antibody-based Western blotting methods for detection of coexisting protein modifications and demonstrated the utility of quantitative tandem mass spectrometry for detailed analysis of the dynamics of coexisting post-translational modifications in proteins.

Proteomic analysis of GPI-anchored membrane proteins.

Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell-type specific surface molecules, candidate biomarkers and potential therapeutic targets.:

Effect of OATP1B1 (SLCO1B1) variant alleles on the pharmacokinetics of pitavastatin in healthy volunteers.

BACKGROUND: Pitavastatin is a potent, newly developed 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor for the treatment of hyperlipidemia. We characterized the effects of organic anion transporting polypeptide 1 B 1 (OATP 1 B 1) alleles *1a, *1b, and *15 on the pharmacokinetics of pitavastatin. METHODS: Twenty-four healthy Korean volunteers who had previously participated in a pharmacokinetic study of pitavastatin (single oral dose, 1--8 mg) were further investigated. Subjects were grouped according to OATP 1 B 1 genotype. Dose-normalized area under the plasma concentration-time curve (AUC) and peak plasma concentration (C(max)) values were analyzed, because different dosages were administered to subjects, whereas the pharmacokinetics showed linear characteristics. RESULTS: Dose-normalized pitavastatin AUCs for *1b/*1b (group 1), *1a/*1a or *1a/*1b (group 2), and *1a/*15 or *1b/*15 (group 3) were 38.8+/-13.3, 54.4 +/-12.4, and 68.1+/-6.3 ng.h.mL(-1).mg(-1) (mean+/-SD), respectively, with significant differences between all 3 groups (P=.008) and between subjects carrying and those not carrying the *15 allele (P = .004). Dose-normalized pitavastatin C(max) values were 13.2+/- 3.3, 18.2+/-5.7, and 29.4+/- 9.6 ng.mL(-1).mg(-1) in groups 1, 2, and 3, respectively, and also showed significant differences (P=.003) in a manner similar to that shown by AUC. No significant differences were found between the genotype groups in terms of dose-normalized AUC or C(max) values of pitavastatin lactone. CONCLUSION: OATP 1 B 1 variant haplotypes were found to have a significant effect on the pharmacokinetics of pitavastatin. These results suggest that the *15 allele is associated with decreased pitavastatin uptake from blood into hepatocytes and that OATP 1 B 1 genetic polymorphisms have no effect on the pharmacokinetics of pitavastatin lactone.

Effect of the UGT2B15 genotype on the pharmacokinetics, pharmacodynamics, and drug interactions of intravenous lorazepam in healthy volunteers.

OBJECTIVE: Our objective was to investigate the effect of the uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B15 genetic polymorphism on the pharmacokinetics and pharmacodynamics of lorazepam in basal, inhibited, and induced metabolic states in healthy normal volunteers. METHODS: Twenty-four healthy subjects were enrolled and grouped into UGT2B15*1/*1 or UGT2B15*2/*2 genotype groups. The pharmacokinetic and pharmacodynamic profiles of intravenous lorazepam were characterized before and after inhibition with 600 mg valproate once daily for 4 days and after induction with rifampin (INN, rifampicin) pretreatment (600 mg once daily for 10 days), with a washout period of 10 days between. The plasma concentrations of lorazepam and lorazepam glucuronide were analyzed before and at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, and 48 hours after lorazepam administration by liquid chromatography-tandem mass spectrometry. Visual analog scale assessments and psychomotor coordination tests were administered before and up to 12 hours after drug administration. RESULTS: The UGT2B15*2/*2 group showed 0.58-fold (95% confidence interval, 0.43-0.72; P < .0001) lower systemic clearance during the basal state and 1.37-fold (95% confidence interval, 1.05-1.88; P = .037) higher area under the visual analog scale-time curve during the induced state compared with the UGT2B15*1/*1 group. The mean systemic clearance of lorazepam decreased by 20% in the inhibited state and increased by 140% in the induced state. During the inhibited or induced state, absolute values of clearance were consistently lower in the *2/*2 group, but the percent changes from baseline did not differ significantly by genotype. CONCLUSIONS: Our results suggest that the UGT2B15*2 polymorphism is a major determinant of interindividual variability with respect to the pharmacokinetics and pharmacodynamics of lorazepam.

A variant 2677A allele of the MDR1 gene affects fexofenadine disposition.

BACKGROUND AND OBJECTIVES: There have been considerable disagreements regarding the influence of MDR1 (ABCB1) polymorphisms on the disposition of P-glycoprotein (P-gp) substrates. We speculated that the unknown function of the A allele of exon 21 G2677T/A (Ala893Ser/Thr) provides one of the reasons for the contradictory results. This study was performed to clarify the effects of major MDR1 gene polymorphisms, including a variant A allele in exon 21, on fexofenadine pharmacokinetics. METHODS: We investigated the occurrence of 3 high-frequency single-nucleotide polymorphisms (SNPs) in exons 12 (C1236T), 21 (G2677T/A), and 26 (C3435T) of the MDR1 gene in 232 healthy Koreans, using a polymerase chain reaction-restriction fragment length polymorphism method, and performed haplotype analysis on these 3 SNPs. A single oral dose of 180 mg fexofenadine hydrochloride was administered to 33 healthy Korean male volunteers, who were divided into 6 groups based on the MDR1 genotype for the G2677T/A polymorphism in exon 21 and the C3435T polymorphism in exon 26. RESULTS: A strong linkage disequilibrium was observed among the 3 SNPs. The frequencies of the 4 major haplotypes, 1236C-2677A-3435C, C-G-C, T-G-C, and T-T-T, were 16.4%, 18.6%, 21.6%, and 32.2%, respectively. Fexofenadine disposition varied considerably among the groups. In the 2677AA/3435CC genotype group (n=3), the values of area under the concentration-time curve from time 0 to 24 hours [AUC(0-24)] were significantly lower (P=.014) than those of the other 5 genotype groups (GG/CC, GT/CT, TT/TT, GA/CC, and TA/CT). As compared with the 2677GG/3435CC subjects, the AUC(0-24) values were 17% lower in the 2677AA/3435CC subjects and 47% higher in the 2677TT/3435TT subjects (GG/CC versus AA/CC versus TT/TT, 4017 +/- 1137 ng . h/mL versus 3315 +/- 958 ng . h/mL versus 5934 +/- 2,064 ng . h/mL; P=.018). By stratification for genotypes at position 3435, homozygous 3435TT subjects were found to have significantly higher AUC(0-24) (P=.024) and maximum plasma concentration (P=.040) values than CC subjects [AUC(0-24), 5934 +/- 2064 ng . h/mL versus 3998 +/- 1241 ng . h/mL; maximum plasma concentration, 958 +/- 408 ng/mL versus 673 +/- 242 ng/mL]. CONCLUSIONS: The plasma concentrations of fexofenadine after a single oral administration were lower in 2677AA/3435CC subjects than in subjects with the other 5 genotype combinations of the SNPs of G2677T/A and C3435T. These findings confirm the importance of analyzing MDR1 haplotypes and provide a plausible explanation for the conflicting results regarding the effect of MDR1 polymorphisms on the disposition of P-gp substrates.