Kevin J. Welham

The proteomic analysis of cisplatin resistance in breast cancer cells.

Resistance to cisplatin represents a major obstacle in the effective management of many cancers, including metastatic breast cancer. We aimed to gain further understanding of the mechanisms underlying development of cisplatin resistance using an in vitro cell line model. The MCF-7 breast cancer cell line and a novel derivative displaying significant resistance to cisplatin were analyzed using two-dimensional gel electrophoresis. The protein profiles were compared and 15 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The downregulation of beta-tubulin type 3, cytokeratin 17, tropomyosin 1-alpha, peroxiredoxin 4, heat shock 27-kDa protein 1, glutathione-S-transferase mu 3, ribosomal protein P0, isocitrate dehydrogenase 3, and peptidyl-prolyl isomerase A isoform 1 was associated with cisplatin-resistant cells. In contrast, the expression of hydroxyprostaglandin dehydrogenase 15-(NAD), matrix metalloproteinase 9, heterogeneous nuclear ribonucleoprotein A3, proteasome beta 1 subunit, electron transfer flavoprotein beta-polypeptide isoform 1, and peptidyl-propyl isomerase B precursor was upregulated in cisplatin-resistant cells. The downregulation (at least twofold) of glutathione-S-transferase mu 3, cytokeratin 17, and peroxiredoxin 4 was confirmed by Western blotting. We have identified alterations in the expression levels of several proteins that may be associated with cisplatin resistance and are candidates for further validation in clinical samples.

Cancer proteomics and its application to discovery of therapy response markers in human cancer

The administration of chemotherapy either alone or in combination with radiotherapy is an important factor in reducing the mortality and morbidity of cancer patients. Resistance to both chemotherapy and radiotherapy represents a major obstacle to a successful outcome. The identification of novel biomarkers that can be used to predict treatment response would allow therapy to be tailored on an individual patient basis. Although the mechanisms are unclear, it is accepted that development of therapy resistance is a multifactorial phenomenon involving alterations in several cellular pathways. Proteome analysis methods are powerful tools for identifying factors associated with resistance to anticancer therapy because they facilitate the simultaneous analysis of whole proteomes. The current review describes the plethora of existing proteomic approaches and details the studies that have identified biomarkers that may be useful in the prediction of clinical response to anticancer therapy. Cancer 2006.

The Sesame Seed Oil Constituent, Sesamol, Induces Growth Arrest and Apoptosis of Cancer and Cardiovascular Cells

Abstract: Sesamol is a potent inhibitor of fungal fatty acid biosynthesis. This effect is apparently due to inhibition of malic enzyme and the supply of NADPH that is required for this biosynthetic pathway. It is the ability of sesamol to reduce the synthesis of the coenzyme, NADPH, that makes it attractive for use in studying the effect of oxidants on tumor and vascular endothelial cells. By conducting preliminary studies on the effect of sesamol alone, it was clear that the compound demonstrated marked cytotoxicity. This paper describes the experiments performed.

Identification, release and olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (Solea senegalensis)

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in sufficient quantities to be detected is unclear. The current study identified bile salts in the intestinal and bile fluids of Solea senegalensis by mass spectrometry–liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identified in the bile were taurocholic acid (342xa0mM) and taurolithocholic acid (271xa0mM) plus a third, unidentified, bile salt of 532.3xa0Da. These three were also present in the intestinal fluid (taurocholic acid, 4.13xa0mM; taurolithocholic acid, 0.4xa0mM). In sole-conditioned water, only taurocholic acid (0.31xa0μM) was released in sufficient quantities to be measured (release rate: 24xa0nmolxa0kg−1xa0min−1). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal fluid and account for the difference in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.

Isothiocyanato Boron Dipyrromethenes—The First BODIPY Analogues of Fluorescein Isothiocyanate

Abstract Two boron complexes of 5-phenyldipyrromethenes bearing isothiocyanate groups on the phenyl ring have been synthesized for the first time. The utility of these new fluorescence probes for labeling biologically relevant proteins is demonstrated on two monoclonal antibodies that bind to antigens overexpressed on cancer cells. Spectral comparison of the two structures reveals significant photophysical differences, including bathochromically shifted excitation and emission bands, increased molar absorptivity and a large increase in fluorescence quantum yield of approximately 10 times. Differences in photophysical parameters are linked to hindered rotation of the phenyl ring in one of the probes.

Fabrication of an octadecylated silica monolith inside a glass microchip for protein enrichment.

Silica-based monolithic materials have shown great promise for use as sorbent materials due to their large surface area and bimodal pore size distribution. In this paper, a new process for the fabrication of a silica-based monolith inside a glass microchip and its modification with octadecylsilyl ligands was successfully developed for use in the microchip-based solid phase extraction of proteins. Monolithic porous silica without cracks was prepared by a sol-gel process, followed by placement of the monolithic silica disk inside the extraction chamber in the base plate of the microchip. The two plates of the glass microchip were then thermally bonded at 575 °C for 3 hours. The silica-based monolith was not affected by the thermal bonding of the two plates of the microchip. This process completely avoids the problem of shrinkage in the silica skeleton during preparation. The monolithic silica disk inside the glass microchip was subsequently modified with octadecylsilyl (C(18)) moieties for increased protein binding capacity. The performance of the microchip was evaluated using the extraction of six proteins varying in molecular weight and isoelectric point, namely insulin, cytochrome C, lysozyme, myoglobin, β-lactoglobulin, and hemoglobin at a concentration of 60 μM. The standard protein was mixed with a double concentration of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The results show that the octadecylated silica monolith was permeable, has the ability to remove impurities, and achieved a high extraction recovery of the proteins (94.8-99.7%) compared with conventional octadecylated silica particles (48.3-91.3%). The chip-to-chip reproducibility was assessed by calculating the relative standard deviations (RSDs) for the six proteins during extraction. The intra-batch and inter-batch RSDs were in the range of 2.0-4.5% and 2.9-6.4%, respectively. This new microfluidic device for protein extraction may find an application in the area of proteomic research.

Proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia reveals a possible role for kininogen

UNLABELLEDnCLL is an incurable disease with variable prognosis. The hyper reactivity of the B-cell receptor (BCR) to unknown antigen ligation plays a pivotal role in CLL-cell survival. We aimed to investigate the BCR signalling pathway using proteomics to identify novel proteins which may have clinical relevance in this disease. Three CLL samples were selected based upon BCR responsiveness, demonstrated by upregulation of phospho-ERK following in vitro stimulation. The differential expression of proteins, upon artificial stimulation of the BCR, was examined in these samples using two-dimensional gel electrophoresis in combination with mass spectrometry. Proteins of interest were subsequently examined using immunoblotting. Proteomic analysis revealed that kininogen, a critical protein of kinin-kallikrein system, was upregulated in all 3 clinical samples upon BCR stimulation. There are 2 forms of kininogen: HMWK and LMWK. The upregulation of LMWK upon BCR stimulation was confirmed by immunoblotting in all 3 of these samples. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases (147months versus 253months for LMWK negative cases; p=0.125). Kininogen may be a novel therapeutic target in CLL and the possible association with prognosis warrants further investigation.nnnBIOLOGICAL SIGNIFICANCEnWe have identified the upregulation of LMWK upon BCR stimulation of CLL samples. There is no previous published research to suggest a link between kininogen and normal B-cells or CLL cells. In 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases. The absence of LMWK protein expression on normal B-cells suggests that this could be a biomarker for CLL and further research should be undertaken.

Fabrication of a TCEP-immobilised monolithic silica microchip for reduction of disulphide bonds in proteins

Protein identification by mass spectrometry forms the cornerstone of proteomics. Commonly, the identification of proteins is based on digestion of proteins into peptides using proteolytic enzymes. Cleavage of the disulphide bonds in proteins before enzymatic digestion and mass spectral analysis is important in order to facilitate the accessibility of the enzyme to cleave the proteins into peptides. As a result, the protein sequence coverage will be increased. In this study, a novel approach for immobilisation of the reducing reagent on the surface of a silica-based monolith in order to use it for reduction of disulphide bonds in proteins was successfully developed. This was carried out by silanisation of the surface of the silica-based monolith with (3-aminopropyl)triethoxysilane (APTES), followed by immobilisation of the reducing reagent, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) on the surface of the amino-bonded silica monolith. The fabricated monolith was characterised using SEM analysis, EDX analysis, IR spectroscopy, and BET model. The performance of the fabricated glass microchip containing the TCEP-immobilised silica monolith to reduce the disulphide bonds in proteins was checked by injection of 100 μL of denatured insulin inside the microchip using a syringe pump at a flow rate of 10 μL min−1, followed by sealing both ends of the ETFE tubes with Blu-Tak. The microchip was kept in a humidified chamber for 30 min at 60 °C. After the reduction reaction, the reduced cysteine residues were alkylated with IAA at 60 °C for 30 min, followed by using a MALDI-TOF-MS instrument for qualitative confirmation. The results show that the fabricated microchip-based silica monolith has the ability to reduce disulphide bonds in insulin. In addition, the method is simple, reduces the risk of contamination, and results in lower amounts of the sample and reagents compared with the conventional techniques for proteomics sample preparation. A future study investigating reduction of the disulphide bonds in proteins from a real sample using this new microfluidic device would be very interesting.

Preconcentration of milk proteins using octadecylated monolithic silica microchip

Sample preparation is a bottleneck in systems for chemical analysis and it is a required step in order to remove interference and preconcentrate the target analytes. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transfer compared with common packed beds. This study has focused on the use of a glass microchip containing an inorganic silica-based monolithic material modified with octadecyl groups for preconcentration of milk proteins from skimmed cows milk that vary in molecular weight, hydrophobicity, and abundance. Comparison between the fabricated device and a commercial cartridge for the preconcentration of proteins in skimmed cows milk showed the ability of the device to successfully enrich protein mixtures from the sample. The three replicate experiments showed that the RSD of the mass to charge ratio of milk proteins ranged from 0.01 to 0.46%. In addition, it was found that there were no significant differences between the observed and reported masses of the milk proteins and the relative percentage error of the molecular masses ranged between 0.03 and 0.90%. The fact that the small amounts of sample required and short sample preparation time suggest that this new microfluidic device may be a viable alternative to existing procedures for protein extraction from real samples.

Reply to “Analysis of NO and its metabolites by mass spectrometry. Comment on ‘Detection of nitric oxide in tissue samples by ESI-MS’”

Comment by Tsikas et al. (Analyst, 2011, DOI: 10.1039/c0an00411a) on the preliminary work by Shen and colleagues (Analyst, 2010, 135, 302) describing the use of ESI-MS/MS for the detection of methylpiperazinobenzenediamine, as a probe for NO in tissue samples, have been addressed in this communication. The article concludes that the points raised by Tsikas represent a logical progression to the research reported and indicate the way forward in realising appropriate analytical methodology.