•Quantitatively detect p53-MDM2 interaction using LC-MS/MS-based targeted proteomics•The assay circumvents the trade-off between sensitivity and specificity.•The assay provides an accurate and dynamic value of protein-protein interaction.•Observe differential p53-MDM2 interaction between normal and breast cancer casesIn breast cancer, p53 could be functionally compromised by interaction with several proteins. Among those proteins, MDM2 serves as a pivotal negative regulator and counteracts p53 activation. Thus, the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction (PPI), provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far. In this study, a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation (Co-IP) for the quantification of p53-MDM2 complex. A p53 antibody with the epitope residing at 156–214 residues achieved the greatest IP efficiency. 321KPLDGEYFTLQIR333 (p53) and 327ENWLPEDK334 (MDM2) were selected as surrogate peptides in the targeted analysis. Stable isotope-labeled synthetic peptides were used as internal standards. An LOQ (limit of quantification) of 2 ng/mL was obtained. Then, the assay was applied to quantitatively detect total p53, total MDM2 and p53-MDM2 in breast cells and tissue samples. Western blotting was performed for a comparison. Finally, a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored.Biological significanceProteins do not function as single entities but rather as a team player that has to communicate. Protein-protein interaction (PPI), normally by means of non-covalent contact among binary or large protein complex, is essential for many cellular processes including cancer progression. Thus, the ability to quantitatively and accurately monitor the changes in level of PPI with disease state can enable an improved understanding of PPI, provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI so far. The major issue of current available approaches is the trade-off between sensitivity and specificity. Thus, techniques with the ability to quantify PPIs with both high sensitivity (low false-negative rate) and high specificity (low false-positive rate) are eagerly desired. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has shown its potential to study biomolecules because of its high sensitivity, high selectivity and wide dynamic range. In this study, we made an effort to develop a LC-MS/MS-based targeted proteomics assay for the quantitative detection of p53-MDM2 interaction in breast cells and tissue samples.

Parkinson’s disease (PD) is a neurodegenerative disorder with an incidence second only to Alzheimer’s disease. The main pathological feature of PD is the death of dopaminergic neurons in the substantia nigra pars compacta. Nicotinic receptor agonists are neuroprotective in several PD models and there is considerable evidence that α7 nicotinic acetylcholine receptors (α7-nAChRs) are important therapeutic targets for neurodegenerative diseases. However, the involvement of α7-nAChRs and underlying signaling mechanisms in PD pathogenesis are unclear. The objective of the present study was to explore the potential functions of α7-nAChRs in PD pathology, and to determine whether these effects are exerted via Wnt/β-catenin signaling in a mouse PD model. In the in vivo study, α7-nAChR knockout (α7-KO) reversed the beneficial effects of nicotine on motor deficits, dopaminergic neuron loss, astrocyte and microglia activation, and reduced striatal dopamine release induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Injury to SH-SY5Y cells by 1-methyl-4-phenylpyridinium treatment was also ameliorated by nicotine, and this effect was abolished by methyllycaconitine (MLA), a selective α7-nAChR antagonist, or by siRNA-mediated α7-nAChR knockdown. Furthermore, nicotine increased expression levels of Wnt/β-catenin signaling proteins in the PD mouse model or in the SH-SY5Y cells treated by 1-methyl-4-phenylpyridinium, and these effects were also reversed by MLA or α7-siRNA treatment in vivo or in vitro. These results suggest that endogenous α7-nAChR mechanisms play a crucial role in a mouse PD model via regulation of Wnt/β-catenin signaling.Download high-res image (65KB)Download full-size image

Circular RNAs (circRNAs) are recently identified as widespread and diverse endogenous noncoding RNAs that may harbor vital functions in human and animals. However, the role of circRNAs in the process of tumorigenesis and development of colorectal cancer (CRC) remains vague. Here we characterized the circRNA expression profile from three paired CRC cancerous and adjacent normal tissues by human circRNA array, and identified 136 significantly overexpressed circRNAs and 243 downregulated circRNAs in CRC cancerous tissues (>2-fold changes). We further validated one circRNA generated from Exon 5–11 of BANP gene, termed circ-BANP. In addition, RT-PCR result showed that circ-BANP was overexpressed in 35 CRC cancerous tissues. Knockdown of circ-BANP with siRNA significantly attenuate the proliferation of CRC cells. In summary, our findings demonstrated that dysregulated circ-BANP appears to have an important role in CRC cells and could serve as a prognostic and therapeutic marker for CRC.

The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarkers. However, one of prerequisites for the application of miRNAs in clinical practice is to accurately profile their expression. Currently available assays normally require pre-enrichment, amplification, and labeling steps, and most of them are semiquantitative. In this study, we converted the signal of target miR-21 into reporter peptide by a DNA-peptide probe and the reporter peptide was ultimately quantified using LC-MS/MS-based targeted proteomics. Specifically, substrate peptide GDKAVLGVDPFR containing reporter peptide AVLGVDPFR and tryptic cleavage site (lysine at position 3) was first designed, followed by the conjugation with DNA sequence that was complementary to miR-21. The newly formed DNA-peptide probe was then hybridized with miR-21, which was biotinylated and attached to streptavidin agarose in advance. After trypsin digestion, the reporter peptide was released and monitored by a targeted proteomics assay. The obtained limit of quantification (LOQ) was 1 pM, and the detection dynamic range spanned ∼5 orders of magnitude. Using this assay, the developed quasi-targeted proteomics approach was applied to determine miR-21 level in breast cells and tissue samples. Finally, qRT-PCR was also performed for a comparison. This report grafted the strategy of targeted proteomics into miRNA quantification.

Protein arginine methylation is one of the common post-translational modifications in cellular processes. To date, two isomeric forms of dimethylated arginine have been identified: asymmetric NG,NG-dimethylarginine (aDMA), and symmetric NG,N′G-dimethylarginine (sDMA). Evidence indicated that these isomers can coexist and have different or even opposite functions, with aDMA and sDMA forms of arginine 2 on histone H3 (i.e., H3R2me2a and H3R2me2s) being an example. Thus, specific detection and quantification of each isomeric form is important. Current methods are capable of predicting and detecting thousands of methylarginine sites in proteins, whereas differentiation and stoichiometric measurement of dimethylated protein isomers are still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS)-based targeted proteomics has emerged as a promising technique for site-specific quantification of protein methylation using enzymatic peptides as surrogates of target proteins. However, it should be pointed out that a routine targeted proteomics strategy cannot easily distinguish sDMA- and aDMA-containing surrogate peptides due to their common nature. The estimated amount should be considered as the sum of both arginine dimethylated isomers. In this study, compositional analysis based on a linear algebra algorithm as an add-on to targeted proteomics was employed to quantify H3R2me2a and H3R2me2s (i.e., surrogate peptides of AR(me2a)TK(me1/2)QT and AR(me2s)TK(me1/2)QT). To achieve this simultaneous quantification, a targeted proteomics assay was developed and validated for each isomer first. With the slope and intercept of their calibration curves for each multiple reaction monitoring (MRM) transition, linear algebraic equations were derived. Using a series of mock mixtures consisting of isomers in varying concentrations, the reliability of the method was confirmed. Finally, the H3R2 dimethylation status was analyzed in normal MCF-10A cells, parental drug-sensitive MCF-7/WT cancer cells, and drug-resistant MCF-7/ADR cancer cells. Dimethylated H3R2 was also monitored in MCF-7/WT cells with the treatment of doxorubicin (DOX) for confirmation.

Soluble transferrin receptor (sTfR) in serum has been suggested as a marker for breast cancer diagnosis, monitoring and treatment. However, sTfR levels in some situations could be far below the limit of quantification (LOQ) of most assays. Thus, an efficient sample pretreatment strategy is required. In this study, molecularly imprinted polymers (MIPs) were developed and coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based targeted proteomics for sTfR measurement. The key to this effort was that the same surrogate peptide of sTfR (VEYHFLSPYVSPK, VK13) was employed in both the enrichment by MIPs and the quantification by targeted proteomics. Specifically, three peptide templates with different lengths were evaluated for the synthesis of MIPs, and the imprinting conditions were optimized. The characteristics of MIPs, including the adsorption capacity, adsorption kinetics, and binding selectivity, were also investigated. As a result, a ∼12-fold enhancement of sensitivity was achieved using MIPs. An LOQ of 200 ng·mL–1 was obtained. The intra- and interday precision were <10.7 and 7.8%, respectively. The accuracy was 7.5% at the lower limit of quantification (LLOQ) and <8.4% for the other QC levels. After validation, the assay was applied to determine the sTfR levels in breast cancer patients (n = 20) and healthy volunteers (n = 20) using the standard addition method. The corresponding levels of sTfR were 1.59 ± 0.36 μg·mL–1 (range: 0.96–2.34 μg·mL–1) in the volunteers and 1.82 ± 0.42 μg·mL–1 (range: 0.95–2.47 μg·mL–1) in the patients. This study is among the first to combine MIPs and LC-MS/MS targeted proteomics for protein quantification at the peptide level.

The extended use of doxorubicin (DOX) could be limited because of the emergence of drug resistance associated with its treatment. To reverse the drug resistance, two thiol-modified peptide sequences HAIYPRHGGC and THRPPMWSPVWPGGC were, respectively, conjugated to DOXO-EMCH, forming a maleimide bridge in this study (i.e., T10-DOX and T15-DOX). The structures and properties of peptide–DOX conjugates were characterized using 1H NMR, 13C NMR, mass spectrometry, and high-performance liquid chromatography. Their stability was also evaluated. By using MCF-7/ADR cells as an in vitro model system and nude mice bearing MCF-7/ADR xenografts as an in vivo model, the ability of these novel peptide–DOX conjugates to reverse drug resistance was accessed as compared with free DOX. As a result, the IC50 values for T10-DOX and T15-DOX significantly decreased (31.6 ± 1.6 μM and 27.2 ± 0.8 μM), whereas the percentage of apoptotic cell population increased (35.4% and 39.3%). The in vivo extent of inhibition was more evident in the mice groups treated with peptide–DOX conjugates (59.6 ± 8.99% and 46.4 ± 6.63%), which had DOX primarily accumulated in tumor. These conjugates also showed a longer half-life in plasma and cleared much more slowly from the body. Furthermore, T10-DOX may be more effective than T15-DOX with a higher efficacy and a lower side effect. Most importantly, evidence was provided to support the enhanced intracellular drug accumulation and the induction of lysosomal pathway of apoptosis underlying the drug resistance. As an endosomal/lysosomal marker, cathepsin D permealized the destabilized organelle membrane and was detected in the cytoplasm, leading to the activation of the effector caspase-3 in cell apoptosis. This report is among the first to demonstrate that peptide–DOX-like conjugates promote apoptosis through the initiation of the lysosomal pathway.

•LC/MS/MS-based targeted proteomics was used to study HSP27 in P-gp mediated MDR.•Simultaneous quantification of HSP27 and P-gp was performed in a time course.•Stoichiometry of HSP27 phosphorylation at Ser15 and Ser82 was monitored.•The targeted proteomics assay was compared with conventional analytical methods.One missing puzzle piece to study heat shock protein 27 (HSP27) in P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) was the amount of HSP27 and the extent of its phosphorylation in the biological context. Liquid chromatography–tandem mass spectrometry (LC/MS/MS)-based targeted proteomics allows researchers to monitor associated proteins and their modification simultaneously and quantitatively. In this study, a targeted proteomics assay was first developed and validated for the quantification of HSP27 and its phosphorylated forms. Using this assay, the level of HSP27 was determined in non-tumoral cells MCF-10A, parental drug-sensitive cancer cells MCF-7/WT and drug-resistant cancer cells MCF-7/ADR. A decrease of HSP27 expression was observed in P-gp overexpressed MCF-7/ADR cells. A quantitative time-course analysis of both HSP27 and P-gp in doxorubicin (DOX)-treated MCF-7/WT cells also implied that HSP27 may participate in the P-gp modulation. Furthermore, stoichiometry of site-specific HSP27 phosphorylation indicated that DOX treatment rapidly induced the HSP27 phosphorylation at Ser82. Moreover, conventional analytical methods were also performed for a comparison.Biological significanceLC/MS/MS-based targeted proteomics turns out to be a promising quantification approach for the study of proteins in the preclinical and clinical environment. Unfortunately, rare studies applied this technology to detect multiple associated proteins or protein modification in one experiment. This study demonstrated the potential of LC/MS/MS-based targeted proteomics to understand the cell events in a more accurate context of biological system. By the quantitative time-course analysis of HSP27 and its phosphorylated forms at sites of Ser15 and Ser82, the possible role of HSP27 in P-gp mediated MDR was suggested. Further development of targeted proteomics in future may provide more insight into signal transduction pathways upon perturbation of a protein network or changes to a panel of proposed biomarkers in a given disease state.

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC–MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

The extended use of doxorubicin (DOX) could be limited due to the emergence of drug resistance and cardiotoxicity associated with its treatment. Conjugates of DOX with transferrin (DOX–TRF) can effectively alleviate these side effects, thereby leading to a better treatment. The effectiveness of DOX–TRF could result from the enhancement of transferrin receptor (TfR)-mediated transportation. However, detailed TfR-mediated DOX delivery has not been fully elucidated thus far, which may rely on the quantitative subcellular study of DOX distribution and metabolism. In this study, an immunoisolation assay was developed to isolate the organelles with high purity, yield and integrity. Using this immunoisolation assay together with liquid chromatography–tandem mass spectrometry (LC/MS/MS), the subcellular distribution profiles of DOX and its main metabolite doxorubicinol (DOXol) in human breast cancer cells MCF-7/WT and MCF-7/ADR were determined and compared after the treatment of DOX and DOX–TRF. As expected, DOX–TRF treated cells have a higher drug accumulation compared to DOX treated cells. DOX–TRF was predominantly cytoplasmic. In addition, TfR-mediated transportation had a significant impact on the transformation of DOX to DOXol in the cells. This study provided the evidence that immunoisolation together with LC/MS/MS is an effective technique in subcellular investigations.

Several signal transduction pathways have been reported being involved in the acquisition of P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) upon exposure to anti-cancer drugs, whereas there is evidence indicating that the expression and activity of P-gp were not equally or even reversely modulated by different drugs.To further illustrate this drug-specific effect, possible mechanisms that enable breast cancer cells MCF-7 to acquire MDR to either paclitaxel (PTX) or doxorubicin (DOX) were investigated in a time-dependent manner.The results suggested that at least two pathways participated in this process. One was the short and transient activation of NF-κB, the second one was the relatively prolonged induction of PXR. Both PXR and NF-κB pathways took part in the PTX drug resistance acquisition, whereas DOX did not exert a significant effect on the PXR-mediated induction of P-gp. Furthermore, the property of NF-κB activation shared by DOX and PTX was not identical. An attempt made in the present study demonstrated that the acquired resistance to DOX was via or partially via NF-κB activation but not its upstream receptor TLR4, while PTX can induce the drug resistance via TLR4-NF-κB pathway.To our knowledge, this report is among the first to directly compare the time dependence of NF-κB and PXR pathways. The current study provides useful insight into the distinct ability of DOX and PTX to induce P-gp mediated MDR in breast cancer. Different strategies may be required to circumvent MDR in the presence of different anti-cancer drugs.

•A novel and advanced LC/MS/MS-based targeted proteomics assay was developed and validated.•Protein digestion efficiency and membrane extraction recovery were discussed.•The LC/MS/MS assay was compared with the conventional analytical methods.•The LC/MS/MS assay can allow the measurement of the P-gp expression level in a more accurate manner.P-glycoprotein (P-gp) is the most frequently proposed factor for multi-drug resistance. It is traditionally measured using antibody-based methods. While these techniques can provide relative quantification values for P-gp levels, the important information that is usually missing is its amount in the biological system. In this study, a novel and advanced liquid chromatography–tandem mass spectrometry (LC/MS/MS)-based targeted proteomics assay was developed and validated for the determination of P-gp in the breast cancer drug sensitive cell line MCF-7/WT and the drug resistant cell line MCF-7/ADR. Three tryptic peptides (434STTVQLMQR442, 674GSQAQDR680 and 368IIDNKPSIDSYSK380) can specifically represent P-gp. Among these peptides, 434STTVQLMQR442 was selected as the surrogate analyte for quantification, and a stable isotope-labeled synthetic peptide with the same sequence was used as an internal standard. The calibration range was validated from 10 to 1000 ng/mL. The intra- and inter-day precisions were within 5.9% and 3.7%, respectively. The accuracy for the quality control (QC) samples was within 8.0%. Using this assay, the amounts of P-gp were accurately quantified as 3.53 fg/cell (∼2.08 × 10−2 amol/cell) in the MCF-7/WT cells and 34.5 fg/cell (∼2.02 × 10−1 amol/cell) in the MCF-7/ADR cells. This outcome was then compared with those obtained by conventional analytical methods including confocal microscopy, western blotting and flow cytometry. The comparative results show that not only is the LC/MS/MS-based targeted proteomics assay able to monitor the protein levels in a more accurate manner, but the large discrepancy observed between the other methods was most likely due to the lack of specificity and the semi-quantitative nature of the conventional assays

Currently, the absolute quantification of human transferrin (hTRF) is based on several techniques other than mass spectrometry. Although these techniques provide valuable information on protein levels and can be extremely sensitive, they often lack the specificity and reproducibility that can be provided by mass spectrometry. In this study, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) based targeted proteomics assay was developed and validated for the determination of transferrin in human serum. We selected the tryptic peptide 108EDPQTFYYAVAVVK121 as the surrogate analyte for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Sample cleanup and enrichment were achieved using solid phase extraction. The validated calibration range was from 500 to 5000 ng/mL. The intra- and inter-day precisions were less than 4.9% and 9.0%, respectively. The bias for the quality control (QC) samples was less than 5.4%. Finally, this assay was successfully applied to the quantitative analysis of transferrin in clinical samples. The obtained values were assessed by independently measuring transferrin in the same samples using a commercially available immunoturbidimetric assay. As a result, the absolute concentrations determined by the LC/MS/MS assay compared well with those obtained with the immunoturbidimetric method; however, the LC/MS/MS assay afforded more reliable transferrin values at low concentrations.Highlights► A novel LC/MS/MS-based targeted proteomics assay was developed and validated. ► Protein digestion efficiency and surrogate peptide selection were discussed. ► The LC/MS/MS assay was compared with a commercial immunoturbidimetric method. ► Clinical monitoring of prospective protein biomarkers could be achieved by LC/MS/MS.

Manganese superoxide dismutase (MnSOD) has been identified as an important scavenger of reactive oxygen species (ROS), which can cause oxidative stress followed by breast cancer. A number of subsequent population-based studies have investigated the association between MnSOD Ala-9Val polymorphism and the risk of breast cancer. However, these studies have yielded conflicting results. This fact implies that the effect of MnSOD Ala-9Val polymorphism on the susceptibility to breast cancer may be modified by other risk factors. To provide a more definitive conclusion, a full meta-analysis combining and summarizing 16 studies was first performed using both traditional and Bayesian approaches. During this step, a recessive inheritance mode was determined after a biological justification. The capability of the Bayesian method was highlighted in the estimation of a pooled odds ratio and 95% confidence interval. As a result, no significant association was observed (OR = 0.978, CI = 0.914–1.046). Bayesian meta-regression and subgroup analysis were then conducted to find possible risk modifications by other factors, including menopausal status, ethnicity effect, use of oral contraceptives, use of hormone replacement therapy, fruits and vegetables intake, vitamin supplement, and body mass index. While the power of most subgroups may be insufficient to make a statistical statement, an evidence-based sample size calculation based upon updated meta-analysis was performed to power a future trial. For example, approximately 5,000 subjects are required for a new Asian study (2,500 cases and 2,500 controls) to achieve 80% power.

A growing number of peptides are being used today as biomarkers and therapeutic drugs. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), in providing a highly specific and reliable technique, is likely to have a substantial impact on peptide quantification. However, most of the applications developed in research laboratories have relied on capillary liquid chromatography, nanospray interfaces, and ion trap or other types of mass spectrometers, which are not commonly found in preclinical and clinical laboratories. Triple quadrupole mass spectrometers are not often considered for quantitative analysis of peptides, despite the fact that they are currently an increasingly common tool in preclinical and clinical laboratories. However, sensitive peptide analyses have been reported, as in the demonstration of structurally distinctive immonium ions that were formed in abundance. Thus, we have recently proposed that use of immonium ions might provide a potential pathway for the quantitative analysis of oligo-peptides with intra-chain disulfide bonds. In the present report, the capability of these ions in LC/MS/MS toward peptide determination was further explored using a polypeptide, human epidermal growth factor (hEGF). hEGF is much larger and more complex than the oligo-peptides previously investigated in our lab. Both the experiments and theoretical calculations were carried out. The results indicate that polypeptides with intra-chain disulfide bonds can increase fragmentation efficiency and raise the number of immonium product ions produced, on the condition that at least one of their intra-loops is sensitive to abrupt bond cleavage. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of hEGF in human plasma. Good linearity and accuracy were achieved.Fragmentation efficiency as a function of collision energy for the transition of m/z 1037 → 86.

A number of studies have been investigated the association between CYP17 T34C polymorphism and the risk of breast cancer; the results of these studies are inconsistent, however. This fact implies that the effect of CYP17 T34C polymorphism on susceptibility to breast cancer may be modified by other risk factors. In order to provide a more definitive conclusion, a full meta-analysis combining and summarizing 24 studies was first performed. Both traditional method and Bayesian approach were applied. Odds ratio was estimated using a dominant mode of inheritance after a biological justification for the choice of genetic model. The results of homogeneity analysis (H = 1.16, I2 = 25.4%, and P = 0.127) suggested the presence of heterogeneity across the studies. Thus, random effects models simulated by the DerSimonian–Laird method were employed. The capability of a Bayesian approach was highlighted in the estimation of a pooled odds ratio and 95% confidence interval. The results of meta-analysis (OR = 1.001, CI = 0.832–1.208) suggest no significant association in the combined populations. Furthermore, Bayesian meta-regression and subgroup analysis were conducted to investigate the sources of heterogeneity. The risk factors evaluated in the study were menopausal status, ethnicity, age at menarche, age at first birth, parity, use of oral contraceptives, body mass index (BMI), and use of hormone repair therapy (HRT). After these population stratifications, there was evidence indicating that a possible impact of menopausal status, age at menarche, and BMI on the association between CYP17 T34C polymorphism and the risk of breast cancer.

A growing number of peptides are being used today in bioanalytical laboratories. Because of this, there is an increasing interest in the development of highly sensitive, specific and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays for the quantitative analysis of peptides in biological samples. Among the mass spectrometers previously used for peptide quantification, triple quadrupole mass spectrometers are generally not considered the instrument of choice. With this instrumentation, collision cascades or multiple fragmentations tend to generate multiple peaks that have weak intensities. This leads to a loss in detection sensitivity. However, in cases where immonium product ions were formed in abundance, it was found that peptide quantification succeeded. A common feature of these peptides is their intra-loop structure. To elucidate the usefulness of this feature in fragmentation, several peptide analytes with intra-chain disulfide bonds were investigated in this study, including a newly synthesized analog having a single amino acid substitution. The results presented here indicate that abrupt bond cleavage from the intra-loop structure of peptides could be one of the premises for intense immonium ion generation. In contrast, any preferential cleavage of peptide bonds (e.g., proline effect) that gives rise to a linearized sequence would break the intactness of the loop and prevent it from completely dissociating. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of peptides with intra-chain disulfide bonds in biological fluids.Generation of abundant immonium ions in product ion spectrum of peptides with intra-chain disulfide bonds.

A number of studies have investigated the association between TNF-α −308G/A polymorphism and the risk of HCV infection; the results of these studies are conflict, however. To provide a more definitive conclusion, a meta-analysis combining and summarizing 12 studies was performed. The Mantel–Haenszel and DerSimonian–Laird methods were employed in traditional fixed effects and random effects meta-analysis, respectively. The capability of a Bayesian approach was highlighted in the estimation of a pooled odds ratio and 95% confidence interval, as well as in the calculation of a sample size for the new study design. Heterogeneity and publication bias across the studies were also explored. The results of the meta-analysis (OR = 1.179, CI = 0.833–1.649) suggest no significant association between TNF-α −308G/A polymorphism and susceptibility to HCV infection in the combined populations. However, there was evidence indicating a possible impact of ethnicity (Asian vs. non-Asian populations) on the association evaluated here (βethnicity = 0.293 ± 0.271). While the power of existing Asian studies was insufficient to make a statistical statement, the sample size of a new clinical study was estimated (500 subjects with 80% statistical power) for further assessment of an association between TNF-α −308G/A polymorphism and risk of HCV infection in Asians.