Balwir Matharoo-Ball

Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis.

A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.

Potassium channels in the human myometrium.

The contractility of the human uterus is under the fine control of a variety of interacting bioactive agents. During labour, the excitability of the uterus is drastically transformed in comparison with the non‐labour state and is manifest at the membrane level via the acivity of uterine ion channels. This article reviews the contribution of potassium (K+) channels to human uterine excitability.

Down-Regulation of the α- and β-Subunits of the Calcium-Activated Potassium Channel in Human Myometrium with Parturition

Abstract Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the α- and/or β-subunit or arises from a physical dissociation of the α-subunit from the β-subunit. The β-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using α- and β-subunit-specific antibodies, detected bands of 110–125 and 36 kDa, respectively. Protein expression levels of the α-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, α-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased β-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the α- and β-subunits in samples taken before or after the onset of labor. We conclude that during labor, the α-subunit is not physically uncoupled from the β-subunit, but a decline occurs in the level of β-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced β-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.

Diagnostic biomarkers differentiating metastatic melanoma patients from healthy controls identified by an integrated MALDI-TOF mass spectrometry/bioinformatic approach

The prognosis of advanced metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) remains dismal with a 5‐year survival rate of 6–18%. In the present study, an integrated MALDI mass spectrometric approach combined with artificial neural networks (ANNs) analysis and modeling has been used for the identification of biomarker ions in serum from stage IV melanoma patients allowing the discrimination of metastatic disease from healthy status with high specificities of 92% for protein ions and 100% for peptide biomarkers. Our ANNs model also correctly classified 98% of a blind validation set of AJCC stage I melanoma samples as nonstage IV samples, emphasizing the power of the newly defined biomarkers to identify patients with late‐stage metastatic melanoma. Sequence analysis identified peptides derived from metastasis‐associated proteins; alpha 1‐acid glycoprotein precursor‐1/2 (AAG‐1/2) and complement C3 component precursor‐1 (CCCP‐1). Furthermore, quantitation of serum AAG by an immunoassay showed a significant (p<0.001) increase in AAG serum concentration in stage IV patients in comparison with healthy volunteers; moreover; the quantity of AAG plotted against MALDI‐MS peak intensity classified the groups into two distinct clusters. Ongoing studies of other disease stages will provide evidence whether our strategy is sufficiently robust to give rise to stage‐specific protein/peptide signatures in melanoma.

Identification of SPARC-like 1 protein as part of a biomarker panel for Alzheimer's disease in cerebrospinal fluid.

We have used proteomic fingerprinting to investigate diagnosis of Alzheimers disease (AD). Samples of lumbar cerebrospinal fluid (CSF) from clinically-diagnosed AD cases (n = 33), age-matched controls (n = 20), and mild cognitive impairment (MCI) patients (n = 10) were used to obtain proteomic profiles, followed by bioinformatic analysis that generated a set of potential biomarkers in CSF samples that could discriminate AD cases from controls. The identity of the biomarker ions was determined using mass spectroscopy. The panel of seven peptide biomarker ions was able to discriminate AD patients from controls with a median accuracy of 95% (sensitivity 85%, specificity 97%). When this model was applied to an independent blind dataset from MCI patients, the intensity of signals was intermediate between the control and AD patients implying that these markers could potentially predict patients with early neurodegenerative disease. The panel were identified, in order of predictive ability, as SPARC-like 1 protein, fibrinogen alpha chain precursor, amyloid-β, apolipoprotein E precursor, serum albumin precursor, keratin type I cytoskeletal 9, and tetranectin. The 7 ion ANN model was further validated using an independent cohort of samples, where the model was able to classify AD cases from controls with median accuracy of 84.5% (sensitivity 93.3%, specificity 75.7%). Validation by immunoassay was performed on the top three identified markers using the discovery samples and an independent sample cohort which was from postmortem confirmed AD patients (n = 17).

Biobanking from the patient perspective

Plain English summaryBiobanks are collections of donations of biological material (DNA, cells, tissue etc.) and related data which are very valuable for research into human diseases. A variety of biobanks exist for example within hospitals, research institutes, pharmaceutical companies and patient organisations. The role of patients in biobanking is changing from being seen simply as donors, to actual collaborators in the design, development and the running of biobanks. In this article, we provide a number of examples of patients acting as partners at the heart of biobanking, where their voice and perspective is being seen and used as a valuable resource for the biobank. Our aim is that these examples can be used by those who work with patients in biobank-based research, to design future strategies for patient and public involvement in all biobanks.AbstractBiobanks and biobanking research plays an increasingly important role in healthcare research and delivery as health systems become more patient-centred and medicine becomes more personalised. There is also growing acceptance and appreciation of the value that patients, patient advocacy organisations and the public can bring as stakeholders in biobanking and more generally in research. Therefore, the importance of active, early and sustained engagement and involvement of patient and public representatives in biobanks will become increasingly relevant.Organising and facilitating patient and public involvement in biobanking takes considerable time and effort for all stakeholders involved. Therefore, for any biobank operator considering involving patients and the public in their biobanking activities, consideration of best practices, current guidance, ethical issues and evaluation of involvement will be important.In this article, we demonstrate that patients are much more than donors to biobanks—they are collaborators at the heart of biobanking with an important voice to identify perspective, which can be an extremely valuable resource for all biobanks to utilise. The case studies herein provide examples of good practice of patient involvement in biobanking as well as outcomes from these practices, and lessons learned. Our aim is to provide useful insights from these efforts and potential future strategies for the multiple stakeholders that work with patients and the public involved in biobank-based research.

The identification of human tumour antigens: current status and future developments

The biggest challenge facing us today in cancer control and prevention is the identification of novel biomarkers for detection and improved therapeutic interventions to reduce mortality and morbidity rates. Biomarkers are important indicators to inform us of the physiological state of the cell at a specific time. It is now clear that malignant transformation occurs by changes in cellular DNA and protein expression with subsequent clonal proliferation of the altered cells. The affected genes and their expressed protein products or biomarkers are those involved in the normal growth and maintenance of the cancerous cells. These biomarkers could prove pivotal for the identification of early cancer and people at risk of developing cancer. Altered proteins or changes in gene expression in malignant cells may lead to the expression of tumour antigens recognised by host immune system. In this review we discuss current research into the molecular technologies making possible the global genomic-wide analysis of changes in DNA (genotyping), RNA expression (transcriptomics) and protein expression (proteomics) that have accelerated the rate of new biomarker/tumour antigen discovery. To gain a comprehensive understanding of the physiology and pathophysiology of cancer an approach that harmoniously integrates the various ‘omic’ platforms are key to unraveling the complexity ‘needle-in-a-haystack’ quality of biomarker/tumour antigen discovery.

Antioxidant Enzyme Expression, Lipid Peroxidation, and Protein Oxidation in Human Myometrium With Parturition

Oxygen levels fluctuate considerably during human labor leading to hypoxia and reoxygenation of the uteroplacental unit and in some cases may compromise the progression of labor. Our aim was to assess the possible contribution of oxidative stress to the onset of labor. Thiobarbituric acid was used as a marker of lipid peroxidation along with Western blotting using anti-dinitrophenylhydrazine (DNPH) to assess protein carbonylation in myometrial samples obtained before and after the onset of term and preterm labor. Levels of key antioxidative enzymes were also compared. Higher levels of lipid peroxidation were observed in myometrial samples obtained during term or preterm labor. Reduced levels of glutathione peroxidase (GSHPx) were also encountered in these 2 groups. Conversely, protein carbonyl content was higher in laboring term and preterm myometrial samples. Levels of catalase (CAT) and superoxide dismutase (SOD) were unaltered across all 4 groups. Lipids in the laboring myometrium are susceptible to oxidative injury possibly due to diminished protection as a result of lower GSHPx activity. The reason for enhanced protein carbonylation suggests differential mechanisms governing protein turnover in the pregnant compared with the parturient uterus. Localized, oxidant damage of human myometrium may be a causal factor in difficult deliveries.

Serum biomarkers which correlate with failure to respond to immunotherapy and tumor progression in a murine colorectal cancer model

Purpose: To advance our understanding of mechanisms involved in tumor progression/regression, a CT26 colorectal mouse model treated intra‐tumorally with DISC‐herpes simplex virus as immunotherapy was used in the discovery and validation phases to investigate and ultimately identify biomarkers correlating with the failure to respond to immunotherapy.

Diagnostic Distinction of Malignant Melanoma and Benign Nevi by a Gene Expression Signature and Correlation to Clinical Outcomes

Background: Histopathologic examination alone can be inadequate for diagnosis of certain melanocytic neoplasms. Recently, a 23-gene expression signature was clinically validated as an ancillary diagnostic test to differentiate benign nevi from melanoma. The current study assessed the performance of this test in an independent cohort of melanocytic lesions against clinically proven outcomes. Methods: Archival tissue from primary cutaneous melanomas and melanocytic nevi was obtained from four independent institutions and tested with the gene signature. Cases were selected according to pre-defined clinical outcome measures. Malignant lesions were defined as stage I–III primary cutaneous melanomas that produced distant metastases (metastatic to sites other than proximal sentinel lymph node(s)) following diagnosis of the primary lesion. Melanomas that were metastatic at the time of diagnosis, all re-excisions, and lesions with <10% tumor volume were excluded. Benign lesions were defined as cutaneous melanocytic lesions with no adverse long-term events reported. Results: Of 239 submitted samples, 182 met inclusion criteria and produced a valid gene expression result. This included 99 primary cutaneous melanomas with proven distant metastases and 83 melanocytic nevi. Median time to melanoma metastasis was 18 months. Median follow-up time for nevi was 74.9 months. The gene expression score differentiated melanoma from nevi with a sensitivity of 93.8% and a specificity of 96.2%. Conclusions: The results of gene expression testing closely correlate with long-term clinical outcomes of patients with melanocytic neoplasms. Impact: Collectively, this provides strong evidence that the gene signature adds valuable adjunctive information to aid in the accurate diagnosis of melanoma. Cancer Epidemiol Biomarkers Prev; 26(7); 1107–13. ©2017 AACR.