Supplementary Table and Figure Legends

<p>Table S1. Summary of patient, tumour and explant characteristics. Figure S1. LA-ICP-MS analysis of FFPE sections to determine Pt ion distribution. Figure S2. Characteristics of viable and non�viable tumours. Figure S3. Individual explant responses to cisplatin. Figure S4. Identification of cisplatin sensitive/resistant samples. Figure S5. LA-ICP-MS analysis of cisplatin sensitive and resistant explants.</p>

Gaps in acquisition between integration times for MC-ICP-MS can introduce isotope ratio bias with a mixed detector array.

Oxidative stress is imbalance between oxidant and antioxidant levels in living systems. Human cells are protected from reactive oxygen species (ROS) by endogenous enzymatic antioxidants, such as superoxide dismutase (SOD) and catalase. Most of these compounds require particular redox metals in their structures as cofactors to allow them to scavenge the free radicals, in particular Cu, Zn or Mn-with SOD and Fe with catalase. The aim of this study was to quantify these metals in human cells to evaluate their effectiveness as novel biomarkers for measuring oxidative stress. The metals (Zn, Cu, Fe, Mn) were measured in vitro in skeletal muscle cells (C2C12) which were incubated under hypoxia or hyperoxia conditions generated by varying oxygen levels from 1% - 60% for 24 and 48 hours. Two methods were used to perform the analyses. Solution-based inductively coupled plasma mass spectrometry (ICP-MS) was applied to quantify Zn, Cu, Fe and Mn in cell populations, and laser ablation (LA)-ICP-MS was employed to compare their relative levels in individual cells. The data acquired from both techniques were positively correlated confirming the validity of the two approaches. The results showed that the concentration of the measured elements increased dramatically in cells grown at 25% - 60% O2, the most significant increase being in Cu at 60% O2. None showed any increase at 5% - 15% O2, indicating normoxia states. At 1% O2, all elements, except Fe, showed a significant increase and the most remarkable growth was in Mn. Increasing the incubation time to 48 hours had differing effects on the elements. Zn and Cu concentrations were unaffected by increasing incubation time except at 60% O2 where they showed further growth. In contrast, Mn concentration grew sharply for oxygen levels of 30% - 50% with no further effect at 1%, while Fe concentration decreased at 1% O2 and grew steadily for oxygen levels of 5% - 60%. It can be concluded that all four elements were significantly affected by stress conditions applied to cells, but at different rates. Importantly, this paper describes a novel method for estimating oxidative stress in cells based on the determination of redox elements in single cells and cell populations using ICP-MS.

A high-throughput, sensitive and rapid method was developed for the determination of Zn, Cu and Fe in small volumes (30 μL) of human serum using inductively coupled plasma mass spectrometry (ICP-MS). The sample preparation procedure employed simple 100-fold dilution of the serum samples with 1.0% butanol, 0.5% v/v ammonia, 0.02% v/v Triton X-100 and 0.01% v/v HNO3. The reliability of the method was evaluated using serum UTAK certified reference material, and the results matched well with the certified values. The method was applied to determine Zn, Cu and Fe in 81 human serum samples from participants in Alzheimer disease (AD) and age-related macular degeneration (AMD) studies. No significant differences were found in Zn and Cu levels between age matched controls, AD and AMD patients. Whilst iron levels appeared marginally higher in the AMD group, compared with the AD group, iron showed larger overall variability than the other two elements.

Measurement uncertainty of Pt–GG quantification in human lung cells by species-specific double IDMS.

Abstract To improve treatment outcomes in non–small cell lung cancer (NSCLC), preclinical models that can better predict individual patient response to novel therapies are urgently needed. Using freshly resected tumor tissue, we describe an optimized ex vivo explant culture model that enables concurrent evaluation of NSCLC response to therapy while maintaining the tumor microenvironment. We found that approximately 70% of primary NSCLC specimens were amenable to explant culture with tissue integrity intact for up to 72 hours. Variations in cisplatin sensitivity were noted with approximately 50% of cases responding ex vivo. Notably, explant responses to cisplatin correlated significantly with patient survival (P = 0.006) irrespective of tumor stage. In explant tissue, cisplatin-resistant tumors excluded platinum ions from tumor areas in contrast to cisplatin-sensitive tumors. Intact TP53 did not predict cisplatin sensitivity, but a positive correlation was observed between cisplatin sensitivity and TP53 mutation status (P = 0.003). Treatment of NSCLC explants with the targeted agent TRAIL revealed differential sensitivity with the majority of tumors resistant to single-agent or cisplatin combination therapy. Overall, our results validated a rapid, reproducible, and low-cost platform for assessing drug responses in patient tumors ex vivo, thereby enabling preclinical testing of novel drugs and helping stratify patients using biomarker evaluation. Cancer Res; 77(8); 2029–39. ©2017 AACR.

Together with the former Chairs, I have the pleasure to introduce the first 2016 issue of JAAS to you. All of our past/present Chairs have contributed to the content of this themed issue to commemorate that in 2015, JAAS celebrated its 30th anniversary! To introduce the issue a number of our Chairs have written a few words. We hope that you enjoy their recollections and the papers that have been included in this themed issue.

Improving time resolution in sector-field ICP-MS through use of a plug-in data acquisition board.

A strategy, systematically developed, is reported for the quantitative analysis of the Fe spatial distribution in biological tissue using laser ablation with ICP-MS and on-line double isotope dilution analysis.

In recent years, laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) has gained increasing importance for biological analysis, where ultratrace imaging at micrometer resolution is required. However, while undoubtedly a valuable research tool, the washout times and sensitivity of current technology have restricted its routine and clinical application. Long periods between sampling points are required to maintain adequate spatial resolution. Additionally, temporal signal dispersion reduces the signal-to-noise ratio, which is a particular concern when analyzing discrete samples, such as individual particles or cells. This paper describes a novel, two-volume laser ablation cell and integrated ICP torch designed to minimize aerosol dispersion for fast, efficient sample transport. The holistic design utilizes a short, continuous diameter fused silica conduit, which extends from the point of ablation, through the ICP torch, and into the base of the plasma. This arrangement removes the requirement for a dispersive component for argon addition, and helps to keep the sample on axis with the ICP cone orifice. Hence, deposition of sample on the cones is theoretically reduced with a resulting improvement in the absolute sensitivity (counts per unit mole). The system described here achieved washouts of 1.5, 3.2, and 4.9 ms for NIST 612 glass, at full width half, 10%, and 1% maximum, respectively, with an 8-14-fold improvement in absolute sensitivity, compared to a single volume ablation cell. To illustrate the benefits of this performance, the system was applied to a contemporary bioanalytical challenge, specifically the analysis of individual biological cells, demonstrating similar improvements in performance.

This review covers developments in the design of Laser Ablation (LA) cells, the associated transport tubing assembly, and their coupling to Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) instrumentation. Recent ablation cell designs have reduced the pulse response duration for a single laser shot to <10 ms, using the criterion of the full peak width at 1% of the height of the maximum signal intensity. The evolution towards these low dispersion systems has been profoundly influenced by our understanding of processes driving the initial dispersion, of the design aspects of the cell and tubing that influence transport-induced dispersion and transport efficiency, and of limitations imposed by the temporal resolution of ICP-MS instruments, all of which are discussed. Rapid response LA-ICP-MS systems greatly benefit throughput and sensitivity, which are key parameters in 2D and 3D imaging at high lateral resolution. The analysis and imaging of biological material has come to the forefront as a key application of LA-ICP-MS. The impact of the technical developments in LA-ICP-MS systems on emerging applications, including multiplexed metal-tagged antibody detection (for immunohistochemistry), nanoparticle and compound hypo- and hyperaccumulation, and (intra-) cellular/histological studies, is also discussed.

This note presents a comparison of the use of saliva versus leukocytes for the determination of Pt-DNA adducts obtained from patients undergoing platinum-based chemotherapy. Samples of both blood and saliva were taken pre- and post-treatment and were analysed via sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) to determine the level of Pt-DNA adducts formed. As expected, significant inter-patient variability was seen; however, a lack of correlation between the levels of adducts observed in saliva and blood samples was also observed (Pearson correlation coefficient r = −0.2598). A high yield of DNA was obtained from saliva samples, but significant difficulties were experienced in obtaining patient adherence to the saliva sampling procedure. In both leukocyte and saliva samples, not only was Pt from previous chemotherapy cycles detected, but the rapid appearance of Pt in the DNA was noted in both sample types 1 h after treatment.

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 109 particles/ml for 1 h incorporated an average of 3.39 × 108 Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 105 counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific 154Sm-tagged anti–HLA-DR or 174Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of hematopoietic stem cell (HSC) transplantation, autoimmune disease, and solid organ transplantation. Determining the persistence of cell-based therapies in vivo is crucial to understanding their regulatory function and requires the combination of an extremely sensitive detection technique and a stable, long-lifetime cell labeling agent. This paper reports the first application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform single cell detection of T cell populations relevant to cellular immunotherapy. Purified human CD4(+) T cells were labeled with commercially available Gd-based magnetic resonance imaging (MRI) contrast agents, Omniscan and Dotarem, which enabled passive loading of up to 10(8) Gd atoms per cell. In mixed preparations of labeled and unlabeled cells, LA-ICP-MS was capable of enumerating labeled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained a sufficient label to remain detectable for up to 10 days post-labeling both in vitro and in vivo in an immunodeficient mouse model.

Protein phosphorylation and de-phosphorylation, provide one of the most common signalling pathways within cells, being involved in regulating cellular processes, mediating enzyme inhibition, protein protein recognition and protein degradation. Compared with normal proteomics, phosphoproteomics poses some additional challenges requiring more initial separation and additional sensitivity to detect and quantify potentially ultra-low abundance species. In this work, the selective detection of phosphopeptides is described based on the incorporation of a metal tag, gallium N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). Experimental and theoretical characterisation of the resulting Ga-phosphopeptide complex is presented based on linear ion trap electrospray ionisation mass spectrometry (ESI-MS), Fourier transform mass spectrometry (FT-MS) and molecular modelling data. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (beta-PDGF), a small phosphopeptide. In addition high resolution Fourier transform mass spectrometry (FT-MS) was used for accurate mass determination and multistage tandem mass spectrometry of the gallium-beta-PDGF complex identified the fragmentation pathway. Finally, molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the beta-PDGF-Ga-LysNTA complex. (C) 2013 Elsevier B.V. All rights reserved.

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.

A detailed structural analysis of the complexes resulting from the association of the neutral, mono- and di-anionic forms cytoplasmic thiol containing tripeptide glutathione (GSH) to the post water substituted hydrolysed form of the anti-cancer drug cisplatin, [Pt(NH3)2]2+, in the gas phase and in solution is presented. These structures which are formed from a 1:1 molar ratio of the glutathione ligand and the Pt-drug, have been proposed as possible intermediates for several postulated disulfide bridged polymeric species. Ten gas phase structures on the [GSH−H+Pt(NH3)2]+ potential energy surface are presented and the coordination of the Pt metal is discussed. The [GSH+Pt(NH3)2]2+ potential energy surface was also explored and twelve gas phase structures on this surface are presented. In order to describe the complexes examined here in solution, water solvation effects were considered for eight and nine of the most energetically favored structures of the [GSH−H+Pt(NH3)2]+ and [GSH+Pt(NH3)2]2+ potential energy surfaces, respectively. The enthalpy and free energy, based on the most thermodynamically favored conformers of the reactants and products, for the addition reaction [Pt(NH3)2]2++[GSH−H]−→[GSH−H+Pt(NH3)2]+ in the gas phase at 298K were determined to be −331.8 and −314.8kcalmol−1, respectively. Similarly, the enthalpy and free energy of the gas phase reaction [Pt(NH3)2]2++GSH→[GSH+Pt(NH3)2]2+ at 298K were determined to be −185.0 and −167.4kcalmol−1.

The complexation of the Pt-based anti-cancer drug oxaliplatin (OxPt) with biological ligands other than DNA is believed to be a major cellular sink for the drug reducing its therapeutic potential and acting as a potential cause of toxicity. In this paper, the very first hypothesis driven investigation of the role of the naturally abundant cytoplasmic dipeptide ligand β-alanyl-l-histidine dipeptide (carnosine) in OxPt detoxification is presented. In vitro studies on hepatocellular carcinoma HepG2 cells suggest that carnosine may inhibit the cytotoxic action of OxPt most likely through the formation of complexes that are less cytotoxic than OxPt alone. Evidence is provided to suggest that pre-exposure of HepG2 cells to elevated levels of carnosine appears to have a lasting effect on reducing the cytotoxicity of OxPt even after the removal of the externally added carnosine. This effect, however, is likely under kinetic control as its magnitude was shown not to vary significantly with the level of carnosine exposure within the concentration range used in this study. Various mass spectrometry techniques employing electrospray ionization and chip nanospray were employed to study the interaction of oxaliplatin with carnosine as well as two of its derivatives β-alanyl-N-methylhistidine (anserine) and N-acetylcarnosine (NAC). Evidence of complexation between OxPt and each of the three ligands examined is presented. Most species observed were unambiguously assigned and compared to their theoretical isotopic patterns. Common fragmentation products due to the collisionally-activated protonated complexes of each of the ligands examined with OxPt, [M + OxPt + H](+), where M = carnosine, anserine or NAC, were reported. Density functional calculations at the B3LYP/LANL2DZ level were used to obtain structural information and relative free energies of different isomers of the observed precursor [Carnosine + OxPt + H](+) both in the gas phase and in solution as well as to probe its fragmentation, highlighting plausible fragmentation mechanisms that account for all the experimental results. Data are presented to show several binding modes between electron rich sites such as N and O centers of carnosine and the Pt metal of OxPt. Calculations were also employed to obtain proton affinities and free energies of key reactions. The proton affinities of carnosine, anserine and NAC at 298 K were calculated to be 254.4, 255.9 and 250.2 kcal mol(-1) respectively. To the best of our knowledge the proton affinities of anserine and N-acetyl-carnosine are the first reported values in the literature.