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The identification and quantification of functional cytochromes P450 (CYPs) in biological samples is proving important for robust analyses of drug efficacy and metabolic disposition. In this study, a novel CYP activity-based probe was rationally designed and synthesised, demonstrating selective binding of CYP isoforms. The dependence of probe binding upon the presence of NADPH permits the selective detection of functionally active CYP. This allows the detection and analysis of these enzymes using biochemical and proteomic methodologies and approaches.

Rationale 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a low molecular weight drug of the flavonoid group, which has an anti-vascular effect in tumours causing endothelial cell apoptosis and activation of cytokines. Flavonoid-based compounds have been reported to lead to an upregulation in the expression of lysophosphatidylcholines (LPC)-type lipids in solid tumours. A study employing TLC/MALDI-MS and MALDI-MS imaging to examine LS174T colorectal adenocarcinoma xenografts following administration of DMXAA has been conducted into this effect. Methods LS174T colorectal adenocarcinoma xenografts grown in male immune-deficient mice were treated with 27.5 mg/kg DMXAA. The control (before treatment) and 4 h and 24 h post-treatment tumours were excised and divided into two. MALDI-MS imaging experiments were carried out on 12 µm cryosections sections taken from one half of the tumours and from the other half the lipids were extracted and analysed by TLC/MALDI-MS. These experiments were carried out in triplicate. Results Statistical analysis of the MALDI-MS imaging data set indicated an increased amount of LPC in the 24 h post-treated sample and a decreased amount of PC in the 24 h post-treated sample, compared with the 4 h post-treated sample and the control. These effects were confirmed by the TLC/MALDI-MS data. The lipid extracts were separated into six spots on the TLC plate. These were identified as arising from different lipids classes, i.e. LPC, sphingomyelins (SM), phosphatidylcholines (PC) and phosphatidylethanolamines (PE). The TLC/MALDI-MS data indicated that LPC were highly expressed in the 4 h and 24 h post-treated tumour samples compared with the control. Examination of the mass spectrometric images confirms this increase and demonstrates additionally that the increase in the signals arising from LPC appears to be localised primarily within the central areas of the xenograft. Conclusions An increase in expression of LPC lipids in solid tumours treated with DMXAA has been demonstrated and shown to be localised in the central area of the tumour. Copyright © 2015 John Wiley & Sons, Ltd.

There has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancer-related deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The emergence of “omics” strategies over the last 20 years, enabling the characterisation and understanding of breast cancer at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies, such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for routine breast health monitoring. The current status of established and newly emerging imaging techniques for early detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those applied to high-risk, premenopausal women, will be reviewed.

Background: miRNAs (microRNAs) play a key role in triple-negative breast cancer (TNBC) progression, and its heterogeneity at the expression, pathological and clinical levels. Stratification of breast cancer subtypes on the basis of genomics and transcriptomics profiling, along with the known biomarkers&rsquo, receptor status, has revealed the existence of subgroups known to have diverse clinical outcomes. Recently, several studies have analysed expression profiles of matched mRNA and miRNA to investigate the underlying heterogeneity of TNBC and the potential role of miRNA as a biomarker within cancers. However, the miRNA-mRNA regulatory network within TNBC has yet to be understood. Results and Findings: We performed model-based integrated analysis of miRNA and mRNA expression profiles on breast cancer, primarily focusing on triple-negative, to identify subtype-specific signatures involved in oncogenic pathways and their potential role in patient survival outcome. Using univariate and multivariate Cox analysis, we identified 25 unique miRNAs associated with the prognosis of overall survival (OS) and distant metastases-free survival (DMFS) with &ldquo, risky&rdquo, and &ldquo, protective&rdquo, outcomes. The association of these prognostic miRNAs with subtype-specific mRNA genes was established to investigate their potential regulatory role in the canonical pathways using anti-correlation analysis. The analysis showed that miRNAs contribute to the positive regulation of known breast cancer driver genes as well as the activation of respective oncogenic pathway during disease formation. Further analysis on the &ldquo, risk associated&rdquo, miRNAs group revealed significant regulation of critical pathways such as cell growth, voltage-gated ion channel function, ion transport and cell-to-cell signalling. Conclusion: The study findings provide new insights into the potential role of miRNAs in TNBC disease progression through the activation of key oncogenic pathways. The results showed previously unreported subtype-specific prognostic miRNAs associated with clinical outcome that may be used for further clinical evaluation.

Matched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.

Purpose Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopatholigical features. Our aim, in this pilot study, was to investigate the intra- and inter-patient protein composition of nipple secretions and the implications for their use as liquid biopsies. Experimental design Matched pairs of NAF (n = 15) were characterised for physicochemical properties and SDS PAGE. Four pairs were selected for semi-quantitative proteomic profiling and trypsin-digested peptides analysed using 2D LC Orbitrap Fusion mass spectrometry. The resulting data was subject to bioinformatics analysis and statistical evaluation for functional significance. Results A total of 1990 unique proteins were identified many of which are established cancer associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals was observed. Conclusions This was the most complete proteomic study of NAF to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort. This article is protected by copyright. All rights reserved

Traditionally, proteomics is the high-throughput characterization of the global complement of proteins in a biological system using cutting-edge technologies (robotics and mass spectrometry) and bioinformatics tools (Internet-based search engines and databases). As the field of proteomics has matured, a diverse range of strategies have evolved to answer specific problems. Chemical proteomics is one such direction that provides the means to enrich and detect less abundant proteins (the ‘hidden’ proteome) from complex mixtures of wide dynamic range (the ‘deep’ proteome). In pharmacology, chemical proteomics has been utilized to determine the specificity of drugs and their analogues, for anticipated known targets, only to discover other proteins that bind and could account for side effects observed in preclinical and clinical trials. As a consequence, chemical proteomics provides a valuable accessory in refinement of second- and third-generation drug design for treatment of many diseases. However, determining definitive affinity capture of proteins by a drug immobilized on soft gel chromatography matrices has highlighted some of the challenges that remain to be addressed. Examples of the different strategies that have emerged using well-established drugs against pharmaceutically important enzymes, such as protein kinases, metalloproteases, PDEs, cytochrome P450s, etc., indicate the potential opportunity to employ chemical proteomics as an early-stage screening approach in the identification of new targets.

Cancer significantly affects millions of people worldwide. It is possible to use proteomic techniques to aid in detection, monitoring of treatment and progression, as well as gaining an increased understanding of cancer. Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry can be utilised to detect the presence of proteins and peptides within various samples from the body, including blood, biological fluids and tumour tissue. This review aims to introduce MALDI mass spectrometry and discuss a range of applications in the field of cancer research, from quantitative to qualitative methods. Also described is MALDI imaging mass spectrometry which differs from typical sample preparation methods, as analytes are ionised directly from the tissue. Finally, presented is a brief summary of the status of biomarker discovery using blood/serum and biological fluids samples, and the implications in the clinic.

AQ4N (banoxatrone) (1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione) is an example of a bioreductive prodrug in clinical development. In hypoxic cells AQ4N is reduced to the topoisomerase II inhibitor AQ4 (1,4-bis- {[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione). By inhibition of topoisomerase II within these hypoxic areas, AQ4N has been shown to sensitise tumours to existing chemo- and radiotherapy treatments. In this study the distribution of AQ4N and AQ4 in treated H460 human tumour xenografts has been examined by imaging matrix-assisted laser desorption/ionisation mass spectrometry. Images of the distribution of AQ4N and AQ4 have been produced that show little overlap. The distribution of ATP in the tumour xenografts was also studied as an endogenous marker of regions of hypoxia since concentrations of ATP are known to be decreased in these regions. The distribution of ATP was similar to that of AQ4N, i.e. in regions of abundant ATP there was no evidence of conversion of AQ4N into AQ4. This indicates that the cytotoxic metabolite AQ4 is confined to hypoxic regions of the tumour as intended.

The use of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry on intact cell microorganisms, Intact Cell MALDI (ICM), has been shown by numerous workers to yield effective species level identification. Early work highlighted the significant effect that variation in culture media, incubation conditions and length of incubation had on the spectra produced. Therefore, in order to achieve reliable and reproducible species level identification and sub-typing of microorganisms from ICM fingerprints, it has been essential to develop standardised methods. For methicillin-resistant Staphylococcus aureus (MRSA), a major nosocomial pathogen, we have developed such a standardised method. In this paper we present the experimental parameters, namely, the incubation period, the number of passages required from lyophilised or stored isolates, the method of deposition of the bacterial cells, the concentration of matrix solution, the drying time of bacterial cells prior to the addition of the matrix solution, the time between preparation of the bacterial/matrix sample and analysis and the MALDI pulsed extraction setting, which were considered during the development of defined methods.

Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.