Lipid imaging for visualizing cilastatin amelioration of cisplatin-induced nephrotoxicity.

Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species.

Dual Internal Standards with Metals and Molecules for MALDI Imaging of Kidney Lipids.

The quest for internal standards useful in MALDI imaging studies goes on to get not only lateral distribution but also reliable relative quantitative information. We developed a method based on application of matrix and dual internal standards to allow intra- and intersample normalization of lipids intensities in kidney sections of control and cisplatin-treated Wistar rats. An inkjet printer was used to deposit a custom-prepared ink with DHB as MALDI matrix, a primary lipids-based internal standard, and a spiked lanthanide as a secondary internal standard.

MALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy.

Imaging techniques for mapping molecular distributions in tissue sections can reveal valuable information on biomolecules involved in relevant biochemical processes. A method has been developed for comprehensive, reproducible and sensitive lipid imaging by matrix-assisted laser/desorption ionization-LTQ-Orbitrap mass spectrometry in kidney sections, showing the benefits of exact mass determination. Matrix deposition parameters for positive and negative lipid ion imaging using different matrices such as 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA) or α-cyano-4-hydroxycinnamic acid (CHCA) have been optimized for the broadest detection and identification of renal lipids.

Printing metal-spiked inks for LA-ICP-MS bioimaging internal standardization: comparison of the different nephrotoxic behavior of cisplatin, carboplatin, and oxaliplatin.

The study of the distribution of the cytostatic drugs cisplatin, carboplatin, and oxaliplatin along the kidney may help to understand their different nephrotoxic behavior. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the acquisition of trace element images in biological tissues. However, results obtained are affected by several variations concerning the sample matrix and instrumental drifts.

Application of MeCAT-Click labeling for protein abundance characterization of E. coli after heat shock experiments.

Unlabelled: In a proof of concept study, metal-coded affinity tags based on click chemistry (MeCAT-Click) were used to analyze the proteome of Escherichia coli (E. coli) in response to heat stress. This allows high labeling efficiency, high detection sensitivity, and multiplex capabilities, which are pivotal for its application to protein quantification.

Gadolinium-uptake by aquatic and terrestrial organisms-distribution determined by laser ablation inductively coupled plasma mass spectrometry.

Gadolinium (Gd) based contrast agents (CA) are used to enhance magnetic resonance imaging. As a consequence of excretion by patients and insufficient elimination in wastewater treatment plants they are detected in high concentrations in surface water. At present, little is known about the uptake of these species by living organisms in aquatic systems.

Investigation of a combined microdroplet generator and pneumatic nebulization system for quantitative determination of metal-containing nanoparticles using ICPMS.

In this work, a routinely applicable approach is presented to characterize metal NPs. Individual droplets generated from a microdroplet generator (MDG) were merged into an aerosol generated by a pneumatic nebulizer (PN) and introduced into an ICPMS. The MDG offers high transport efficiency of individual and discrete droplets and was therefore used to establish a calibration function for mass quantification of NPs which were introduced through the PN following the single particle procedure as described elsewhere.

A shotgun approach for the identification of platinum-protein complexes.

A shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin-, oxaliplatin-, and carboplatin-protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation.

Novel approach for labeling of biopolymers with DOTA complexes using in situ click chemistry for quantification.

In this work, we present a two-step labeling approach for the efficient tagging with lanthanide-containing complexes. For this purpose, derivatization of the cysteine residues with an alkyne group acting as linker was done before the DOTA complex was introduced using in situ click chemistry. The characterization of this new methodology is presented including the optimization of the labeling process, demonstration of the quantitative capabilities using both electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS) detection, and study of the fragmentation behavior of the labeled peptides by collision-induced dissociation (CID) for identification purposes.

Bridging the gap between molecular and elemental mass spectrometry: higher energy collisional dissociation (HCD) revealing elemental information.

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared.

Identification of the major ACE-inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate from cuttlefish wastewater.

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC₅₀ = 76.

Inductively coupled plasma mass spectrometry-based method for the specific quantification of sulfenic acid in peptides and proteins.

A robust ICPMS-based method is introduced to obtain relative and absolute quantification of sulfenic acid (SA) in peptides and proteins. A new metal-containing reagent (Ln-DOTA-Dimedone) devised to react specifically with SA has been developed. The lanthanide-containing metal-coded affinity tag (Ln-MeCAT) was used to quantify thiol residues.

On the complexity and dynamics of in vivo Cisplatin-DNA adduct formation using HPLC/ICP-MS.

In this work we present a methodology to measure the complex adduct spectrum caused by the interaction of Cisplatin with DNA. By using an optimized DNA digestion procedure we were able to show that the adduct spectrum in in vivo duplex DNA is much more complex than described so far. For the first time a high abundance of interstrand adducts has been detected by using HPLC/ESI-MS.

Double spike isotope dilution GC-ICP-MS for evaluation of mercury species transformation in real fish samples using ultrasound-assisted extraction.

Sample preparation continues being a key factor to obtain fast and reliable quantification of Hg species. Assisted procedures enhance the efficiency and reduce the extraction time; however, collateral species transformations have been observed. Moreover, differential interconversions have been observed even between similar matrixes, which introduce an important uncertainty for real sample analysis.

MeCAT peptide labeling for the absolute quantification of proteins by 2D-LC-ICP-MS.

Metal-Coded Affinity Tags (MeCAT) reagents were devised for the absolute quantification of labeled proteins and peptides using inductively coupled plasma mass spectrometry (ICP-MS). After the recent publication of quantification approaches for digested proteins, this work presents a multidimensional strategy for the application of MeCAT to samples which require higher chromatographic resolution. Two-dimensional separations based on strong cation exchange (SCX) and reversed-phase (RP) chromatography, were used for the quantification of lysozyme, bovine serum albumin and transferrin after tryptic digestion.

Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics.

In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy.

Elemental bioimaging in kidney by LA-ICP-MS as a tool to study nephrotoxicity and renal protective strategies in cisplatin therapies.

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters.

Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling.

Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification. Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest and scope of elemental mass spectrometry techniques for quantitative proteomics.

Analytical methodologies for metallomics studies of antitumor Pt-containing drugs.

Pt-containing drugs are nowadays essential components in cancer chemotherapy. However, drug resistance and side effects limit the efficiency of the treatments. In order to improve the response to Pt-based drugs, different administration strategies or new Pt-compounds have been developed with little success.