Mass Spectrometry Imaging Suggests That Cisplatin Affects Exocytotic Release by Alteration of Cell Membrane Lipids

We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effect of cisplatin, the first member of the platinum-based anticancer drugs, on the membrane lipid composition of model cells to see if lipid changes might be involved in the changes in exocytosis observed. Platinum-based anticancer drugs have been reported to affect neurotransmitter release resulting in what is called the “chemobrain”; however, the mechanism for the influence is not yet understood. TOF-SIMS imaging was carried out using a high energy 40 keV (CO₂)₆₀₀₀⁺ gas cluster ion beam with improved sensitivity for intact lipids in biological samples. Principal components analysis showed that cisplatin treatment of PC12 cells significantly affects the abundance of different lipids and their derivatives, particularly phosphatidylcholine and cholesterol, which are diminished. Treatment of cells with 2 μM and 100 μM cisplatin showed similar effects on induced lipid changes. Lipid content alterations caused by cisplatin treatment at the cell surface are associated with the molecular and bimolecular signaling pathways of cisplatin-induced apoptosis of cells. We suggest that lipid alterations measured by TOF-SIMS are involved, at least in part, in the regulation of exocytosis by cisplatin

Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 °C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation

An Electrochemical Method for Investigation of Conformational Flexibility of Active Sites of <i>Trametes versicolor</i> Laccase Based on Sensitive Determination of Copper Ion with Cysteine-Modified Electrodes

This study demonstrates a facile yet effective electrochemical method to investigate the conformational flexibility of the active sites of <i>Trametes versicolor</i> (<i>Tv</i>) laccase based on sensitive determination of copper ions (Cu²⁺) dissociated from the enzyme with the cysteine-modified Au electrodes. In the native state, the multicopper active sites are deeply buried in the polypeptide of <i>Tv</i> laccase and are thus not electrochemically detectable even at the cysteine-modified Au electrodes. Upon the unfolding of <i>Tv</i> laccase induced by guanidine hydrochloride (GdnHCl), copper ions dissociate from the peptide chain and, as a consequence, are electrochemically reduced and thus detected at the cysteine-modified Au electrodes. Such a property could be used to investigate the conformational flexibility of multicopper active sites of <i>Tv</i> laccase in a simple way. We find that both the conformation of the multicopper active sites in <i>Tv</i> laccase and the enzyme activity change with the presence of a low concentration of GdnHCl denaturant (midpoint, where 50% of the enzyme is unfolded, at 0.7 M). This concentration is lower than that required to induce the conformational changes of <i>Tv</i> laccase molecule as a whole (midpoint at 3.4 M), as investigated by the intrinsic fluorescence of <i>Tv</i> laccase. This observation suggests that the multicopper active sites are formed by relatively weak interactions and hence may be conformationally more flexible than the intact enzyme. The electrochemical method demonstrated in this study is technically simple yet effective and could be potentially useful for investigation on the thermodynamics and kinetics of the conformational changes of multicopper oxidases induced by different denaturants

Transferrin Serves As a Mediator to Deliver Organometallic Ruthenium(II) Anticancer Complexes into Cells

We report herein a systematic study on interactions of organometallic ruthenium­(II) anticancer complex [(η⁶-arene)­Ru­(en)­Cl]⁺ (arene = <i>p</i>-cymene (<b>1</b>) or biphenyl (<b>2</b>), en = ethylenediamine) with human transferrin (hTf) and the effects of the hTf-ligation on the bioavailability of these complexes with cisplatin as a reference. Incubated with a 5-fold excess of complex <b>1</b>, <b>2</b>, or cisplatin, 1 mol of diferric hTf (holo-hTf) attached 0.62 mol of <b>1</b>, 1.01 mol of <b>2</b>, or 2.14 mol of cisplatin. Mass spectrometry revealed that both ruthenium complexes coordinated to N-donors His242, His273, His578, and His606, whereas cisplatin bound to O donors Tyr136 and Tyr317 and S-donor Met256 in addition to His273 and His578 on the surface of both apo- and holo-hTf. Moreover, cisplatin could bind to Thr457 within the C-lobe iron binding cleft of apo-hTf. Neither ruthenium nor platinum binding interfered with the recognition of holo-hTf by the transferrin receptor (TfR). The ruthenated/platinated holo-hTf complexes could be internalized via TfR-mediated endocytosis at a similar rate to that of holo-hTf itself. Moreover, the binding to holo-hTf well preserved the bioavailability of the ruthenium complexes, and the hTf-bound <b>1</b> and <b>2</b> showed a similar cytotoxicity toward the human breast cancer cell line MCF-7 to those of the complexes themselves. However, the conjugation with holo-hTf significantly reduced the cellular uptake of cisplatin and the amount of platinated DNA adducts formed intracellularly, leading to dramatic reduction of cisplatin cytotoxicity toward MCF-7. These findings suggest that hTf can serve as a mediator for the targeting delivery of Ru­(arene) anticancer complexes while deactivating cisplatin

Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current–time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction

Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current–time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction

Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current–time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction

Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current–time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction

Mass Spectrometric Proteomics Reveals that Nuclear Protein Positive Cofactor PC4 Selectively Binds to Cross-Linked DNA by a <i>trans</i>-Platinum Anticancer Complex

An MS-based proteomic strategy combined with chemically functionalized gold nanoparticles as affinity probes was developed and validated by successful identification and quantification of HMGB1, which is well characterized to interact selectively with 1,2-cross-linked DNA by cisplatin, from whole cell lysates. The subsequent application of this method to identify proteins responding to 1,3-cross-linked DNA by a <i>trans</i>-platinum anticancer complex, <i>trans</i>-PtTz (Tz = thiazole), revealed that the human nuclear protein positive cofactor PC4 selectively binds to the damaged DNA, implying that PC4 may play a role in cellular response to DNA damage by <i>trans</i>-PtTz

Quantitative Mass Spectrometry Combined with Separation and Enrichment of Phosphopeptides by Titania Coated Magnetic Mesoporous Silica Microspheres for Screening of Protein Kinase Inhibitors

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO₂/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with <i>r</i>² being 0.99. The IC₅₀ values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents