Full-scan Mass Spectra Research Articles

High-Throughput and Comprehensive Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry.

New analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is presented in this work as an alternative approach to established shotgun MS or high-performance liquid chromatography-MS. The lipid class separation is performed by UHPSFC method based on 1.7 μm particle-bridged ethylene hybrid silica column with a gradient of methanol-water-ammonium acetate mixture as a modifier. All parameters of UHPSFC conditions are carefully optimized and their influence on the chromatographic behavior of lipids is discussed. The final UHPSFC/ESI-MS method enables a fast separation of 30 nonpolar and polar lipid classes within 6 min analysis covering 6 main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive and negative-ion full-scan and tandem mass spectra measured with high mass accuracy and high resolving power. Developed UHPSFC/ESI-MS method is applied for the analysis of porcine brain extract as a complex lipidomic sample, where 24 lipid classes containing 436 lipid species are identified. The method is validated for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in the clinical research.

Screening of tricyclic quinazoline alkaloids in the alkaloidal fraction of Adhatoda beddomei and Adhatoda vasica leaves by high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Adhatoda beddomei and Adhatoda vasica are popular Ayurvedic medicinal plants in India, belonging to the family Acanthaceae. Tricyclic quinazoline alkaloids are found to be the most abundant in these plants which are responsible for broad-spectrum medicinal properties. This study aims to seek identification and characterization of those alkaloids based on their fragmentation patterns. A method was developed to elucidate the main fragmentation pathways of tricyclic quinazoline alkaloids in positive ion mode using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). Chromatographic separation was carried on a Supelco Discovery HS C18 column (15 cm × 4.6 mm, 3 µm) with 0.1% formic acid aqueous solution and acetonitrile as a mobile phase. In full scan mass spectra, protonated molecules were observed for all the quinazoline alkaloids. Ring cleavages of the tricyclic quinazoline moiety were observed in MS(2) spectra and the characteristic ions provide valuable structural information of these alkaloids. Fragmentation pathways and fragment ion structures were proposed in two groups of quinazoline alkaloids. The established fragmentation patterns have been successfully used to identify 23 tricyclic quinazoline alkaloids in the alkaloidal fraction of A. beddomei and A. vasica.

UPLCMS Method Development and Validation of Amlodipine, Hydrochlorthiazide and Losartan in Combined Tablet Dosage Form

A simple, rapid, sensitive and specific UPLCMS method was developed and validated following ICH guidelines for simultaneous estimation of tablet dosage form containing amlodipine (AMLO) hydrochlorothiazide (HCT) and losartan (LOSAT) using telmisartan (TELMI) as an internal standard (IS). The separation was achieved using Waters ACQUITY BEH C18 (1.7 μm, 2.1 × 50 mm) column with gradient mode, mobile phase containing acetonitrile (A) & 1% ammonium acetate (B) pH adjusted to 2.8 with trifluoro acetic acid with gradient mode. The flow rate was 0.4 mL·mL﹣1 and the injection volume 2 μl. The retention time for amlodipine, hydrochlorothiazide and losartan was found to be 3.7, 2.5 and 3.9 min, respectively. The developed method was found to be linear over the concentration range of 50 - 300 ng·mL﹣1, 125 - 750 ng·mL﹣1 and 500 - 3000 ng·mL﹣1 for AMLO, HCT and LOSAT respectively. The signal intensities obtained in ion mode for amlodipine, hydrochlorothiazide, losartan and telmisartan (IS) they were found to be much higher positive ion mode (M+)﹣ parent ion at m/z, 409.02, 297.97, 422.91 and 515.03, respectively, in QUATTROZQ full scan mass spectra.

Cell cycle arrest and cell survival induce reverse trends of cardiolipin remodeling.

Cell survival from the arrested state can be a cause of the cancer recurrence. Transition from the arrest state to the growth state is highly regulated by mitochondrial activity, which is related to the lipid compositions of the mitochondrial membrane. Cardiolipin is a critical phospholipid for the mitochondrial integrity and functions. We examined the changes of cardiolipin species by LC-MS in the transition between cell cycle arrest and cell reviving in HT1080 fibrosarcoma cells. We have identified 41 cardiolipin species by MS/MS and semi-quantitated them to analyze the detailed changes of cardiolipin species. The mass spectra of cardiolipin with the same carbon number form an envelope, and the C64, C66, C68, C70 C72 and C74 envelopes in HT1080 cells show a normal distribution in the full scan mass spectrum. The cardiolipin quantity in a cell decreases while entering the cell cycle arrest, but maintains at a similar level through cell survival. While cells awakening from the arrested state and preparing itself for replication, the groups with short acyl chains, such as C64, C66 and C68 show a decrease of cardiolipin percentage, but the groups with long acyl chains, such as C70 and C72 display an increase of cardiolipin percentage. Interestingly, the trends of the cardiolipin species changes during the arresting state are completely opposite to cell growing state. Our results indicate that the cardiolipin species shift from the short chain to long chain cardiolipin during the transition from cell cycle arrest to cell progression.

Unravelling associations between unassigned mass spectrometry peaks with frequent itemset mining techniques.

BackgroundMass spectrometry-based proteomics experiments generate spectra that are rich in information. Often only a fraction of this information is used for peptide/protein identification, whereas a significant proportion of the peaks in a spectrum remain unexplained. In this paper we explore how a specific class of data mining techniques termed “frequent itemset mining” can be employed to discover patterns in the unassigned data, and how such patterns can help us interpret the origin of the unexpected/unexplained peaks.ResultsFirst a model is proposed that describes the origin of the observed peaks in a mass spectrum. For this purpose we use the classical correlative database search algorithm. Peaks that support a positive identification of the spectrum are termed explained peaks. Next, frequent itemset mining techniques are introduced to infer which unexplained peaks are associated in a spectrum. The method is validated on two types of experimental proteomic data. First, peptide mass fingerprint data is analyzed to explain the unassigned peaks in a full scan mass spectrum. Interestingly, a large numbers of experimental spectra reveals several highly frequent unexplained masses, and pattern mining on these frequent masses demonstrates that subsets of these peaks frequently co-occur. Further evaluation shows that several of these co-occurring peaks indeed have a known common origin, and other patterns are promising hypothesis generators for further analysis. Second, the proposed methodology is validated on tandem mass spectrometral data using a public spectral library, where associations within the mass differences of unassigned peaks and peptide modifications are explored. The investigation of the found patterns illustrates that meaningful patterns can be discovered that can be explained by features of the employed technology and found modifications.ConclusionsThis simple approach offers opportunities to monitor accumulating unexplained mass spectrometry data for emerging new patterns, with possible applications for the development of mass exclusion lists, for the refinement of quality control strategies and for a further interpretation of unexplained spectral peaks in mass spectrometry and tandem mass spectrometry.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-014-0054-1) contains supplementary material, which is available to authorized users.

Isolation, identification, cytotoxic activity, and metabolic profiles of fungi isolated from San Martín volcano soil

As part of our continuing search for novel cytotoxic fungal compounds from unexplored habitats of Mexico, a series of 72 saprotrophic microscopic fungi were isolated from a soil sample collected at the San Martin Volcano, Veracruz, Mexico. The organic (CHCl3-MeOH) extracts from the axenic solid (moisture rice) cultures were tested against HCT-15 (colorectal), MCF-7 (breast), and HeLa (cervix) cell lines and dereplicated against a database containing more than 180 fungal secondary metabolites via recording UPLC retention times, UV data, and full-scan (high-resolution) mass spectra and MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. Fungal active extracts (growth inhibition > 70% at 20 µg/mL) with negative dereplication were scaled-up (10 ×) and submitted for chemical studies for bioactive compounds isolation. Taxonomic diversity of fungal isolates was assessed by nuclear ribosomal internal transcribed spacer (ITS) barcoding. The active strains belongs to the Phyla of Ascomycota and Basidiomycota, and some of the metabolites identified are aurofusarin, pycnidione, 2-hydroxyacoronene, pestalotin, roridins A, D, E, and H, muconomycin B, ternatin, and trichodermol.

Structural characterization of secoiridoid glycosides by high-performance liquid chromatography/electrospray ionization mass spectrometry.

The fragmentation behavior of seven secoiridoid glycosides (SIGs) isolated from Gentiana triflora Pall., a traditional Chinese medicine, was investigated by electrospray ionization ion trap mass spectrometry (ESI-IT-MS). Multistage electrospray ionization mass spectrometry (ESI-MS(n)) experiments both in positive- and negative-ion mode were used to elucidate the main fragmentation pathways of these compounds. In full scan mass spectra, different deprotonated or protonated molecules were observed for compounds with different basic structure. Ring cleavages of the aglycon moiety were observed in MS(2) spectra and the characteristic ions provided valuable information on the basic structural skeletons. Their fragmentation pathways and fragment ion structures were proposed. The established fragmentation patterns have been successfully used to identify eleven SIGs in the extract of Gentiana triflora Pall.

Towards automated discrimination of lipids versus peptides from full scan mass spectra

Although physicochemical fractionation techniques play a crucial role in the analysis of complex mixtures, they are not necessarily the best solution to separate specific molecular classes, such as lipids and peptides. Any physical fractionation step such as, for example, those based on liquid chromatography, will introduce its own variation and noise. In this paper we investigate to what extent the high sensitivity and resolution of contemporary mass spectrometers offers viable opportunities for computational separation of signals in full scan spectra. We introduce an automatic method that can discriminate peptide from lipid peaks in full scan mass spectra, based on their isotopic properties. We systematically evaluate which features maximally contribute to a peptide versus lipid classification. The selected features are subsequently used to build a random forest classifier that enables almost perfect separation between lipid and peptide signals without requiring ion fragmentation and classical tandem MS-based identification approaches. The classifier is trained on in silico data, but is also capable of discriminating signals in real world experiments. We evaluate the influence of typical data inaccuracies of common classes of mass spectrometry instruments on the optimal set of discriminant features. Finally, the method is successfully extended towards the classification of individual lipid classes from full scan mass spectral features, based on input data defined by the Lipid Maps Consortium.

Determination of Polyphenols in Commercial Extra Virgin Olive Oils from Different Origins (Mediterranean and South American Countries) by Liquid Chromatography–Electrospray Time-of-Flight Mass Spectrometry

The phenolic profiles of extra virgin olive oil samples from different origins and varieties have been studied and compared. The determination of 13 target polyphenols (gallic acid, hydroxytyrosol, tyrosol, vanillic acid, caffeic acid, syringic acid, gibberellic acid, p-coumaric acid, ferulic acid, oleuropein, resveratrol, luteolin, and apigenin) was carried out by liquid chromatography using a column (C18, 4.6 × 100 mm) with small particle size (1.8 μm) and mass spectrometric detection using electrospray time-of-flight mass spectrometry (LC–TOFMS). Prior to LC–MS analysis, a solid-phase extraction procedure using SPE-Diol cartridges was used to isolate the polyphenol fraction. The full-scan mass spectra acquisition by TOF analyzers offered the possibility of searching for a large number of compounds by means of accurate mass information of the molecules and allowed the detection of other phenolic compounds not included in the initial target list, namely ligstroside aglycon, oleuropein aglycon, and elenolic acid. The proposed method was successfully applied to the analysis of 78 samples of extra virgin olive oil collected from ten different countries: Argentina (5), Chile (6), France (1), Greece (1), Italy (1), Morocco (4), Peru (32), Portugal (1), Spain (26), and Syria (1). No qualitative differences in the composition of the phenolic fraction were found between samples of different origins. Only quantitative differences were observed for some phenolic compounds. Therefore, further studies are still needed to exploit the possibility of using phenolic profile as a marker for olive oil geographical origin.

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

Multiple reaction monitoring for the detection of disease-related synaptic proteins.

Multiple reaction monitoring for the detection of disease-related synaptic proteins.

UPLCMS Method Development and Validation of Tablet Dosage form Containing Glimepiride, Metformin and Pioglitazone Using Internal Standard

A simple, rapid, sensitive and specific Ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous estimation and validation of tablet dosage form containing glimepiride (GLIM), metformin (MET) and pioglitazone (PIO) using Tolzamide (TOLAZ) as an internal standard (IS). The chromatography separation was achieved with Waters ACQUITY HSS C18, 1.8 μm, 2.1X50 mm column with mobile phase containing acetonitrile (A) & 1% ammonium acetate buffer (B) (pH 2.5 adjusted with trifluoro acetic acid) with gradient mode Gradient mode [2 min: 20 A : 80% B, 2-4 min: 70% A : 30% B, 4-5 min,80% A : 20% B, 8-10 min 90% A: 10% B]. The flow rate was 0.4 mL min -1 column maintained at 25 0 C and the injection volume was 2 μl. The selected chromatographic condition were found to effectively separate glimepiride, metformin and pioglitazone with retention time of 3.17, 0.425, 2.3 min, respectively. The proposed method was found to be rectilinear over the range of 2-12 ng mL -1 , 500-3000 ng mL -1 and 15-90 ng mL -1 for glimepiride, metformin, and pioglitazone respectively. The signal intensities obtained in both positive and negative ion mode all drugs including internal standard found to be much higher in positive ion mode (M +H)+ parent ions at m/z, 491.11, m/z 129.87, m/z 357.04 and m/z 312.04 respectively in QUATTROZQ full scan mass spectra. The present method was validated as per ICH guidelines with respect to precision, specificity, linearity, limit of detection, limit of quantification, accuracy, and robustness and it can also be used for routine quality control analysis of these drugs in biological samples either alone or in combined pharmaceutical dosage forms.

Identification of combined conjugation of nabumetone phase I metabolites with glucuronic acid and glycine in minipig biotransformation using coupling high-performance liquid chromatography with electrospray ionization mass spectrometry

High-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) was applied for the analysis of nabumetone metabolites during the biotransformation in minipigs. In addition to known phase I metabolites, the identification of phase II metabolites was achieved on the basis of their full-scan mass spectra and subsequent MSn analysis using both positive-ion and negative-ion ESI mode. Some phase I metabolites are conjugated with both glucuronide acid and glycine, which is quite unusual type of phase II metabolite not presented so far for nabumetone. These metabolites were found in small intestine content, but they were absent in minipigs urine.

Differentiation of glucose-containing disaccharides isomers by fragmentation of the deprotonated non-covalent dimers using negative electrospray ionization tandem mass spectrometry

In this work, the glucose-containing disaccharide isomers were studied using negative electrospray ionization tandem mass spectrometry (ESI-MS/MS). Interestingly, the full-scan mass spectra of the disaccharides revealed that the deprotonated dimers were the predominant gas phase ions during ionization process. Importantly, several diagnostic fragment ions relative to linkage positions and anomeric configurations, arising from the covalent bond dissociation of dimers without breakdown of the non-covalent complexes, can be detected in the tandem mass spectra. Based on the scarce fragmentation characteristic, an original and simple approach for structural discrimination of disaccharide isomers was put forward. In addition, density functional theory (DFT) was employed to find out the reason why several fragmentations of intramolecular sugar bonds had preceded breakdown of the non-covalent complexes.

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Gaining understanding of common complex diseases and their treatments are the main drivers for life sciences. As we show here, comprehensive protein set analyses offer new opportunities to decipher functional molecular networks of diseases and assess the efficacy and side-effects of treatments in vivo. Using mass spectrometry, we quantitatively detected several thousands of proteins and observed significant changes in protein pathways that were (dys-) regulated in diet-induced obesity mice. Analysis of the expression and post-translational modifications of proteins in various peripheral metabolic target tissues including adipose, heart, and liver tissue generated functional insights in the regulation of cell and tissue homeostasis during high-fat diet feeding and medication with two antidiabetic compounds. Protein set analyses singled out pathways for functional characterization, and indicated, for example, early-on potential cardiovascular complication of the diabetes drug rosiglitazone. In vivo protein set detection can provide new avenues for monitoring complex disease processes, and for evaluating preclinical drug candidates.

Direct desorption/ionization of analytes by microwave plasma torch for ambient mass spectrometric analysis

Ambient ionization is the new revolution in mass spectrometry (MS). A microwave plasma produced by a microwave plasma torch (MPT) at atmospheric pressure was directly used for ambient mass spectrometric analysis. H3O(+) and NH4(+) and their water clusters from the background are formed and create protonated molecules and ammoniated molecules of the analytes. In the full-scan mass spectra, both the quasi-molecular ions of the analytes and their characteristic ionic fragments are obtained and provide evidence of the analyte. The successful detection of active compounds in both medicine and garlic proves that MPT has the efficient desorption/ionization capability to analyze solid samples. The obtained decay curve of nicotine in exhaled breath indicates that MPT-MS is a useful tool for monitoring gas samples in real time. These results showed that the MPT, with the advantages of stable plasma, minimal optimization, easy, solvent-free operation, and no pretreatment, is another potential technique for ambient MS.

Growth, HR-XRD, optical, thermal, luminescence and nonlinear optical studies of novel organic nonlinear optical crystal: l-Threonine formate

The NLO single crystals of l-threonine formate (LTF) were grown by slow evaporation method in the molar ratio 1:1. The grown crystal was characterized by single crystal XRD, Powder XRD, HR-XRD, mass, thermal and UV⿿vis⿿NIR spectral analysis. Single crystal XRD analysis revealed that the crystal system belongs to tetragonal with cell parameters a=7.86⿫, b=7.86⿫ and c=11.07⿫. The sharp and well defined Bragg peaks observed in the powder XRD pattern confirm the crystalline nature of LTF compound. The structural perfection of the grown crystals has been analyzed by high-resolution X-ray diffraction (HR-XRD) analysis. Mass spectroscopic analysis determined the compound molecular weight from the full scan mass spectra. The optical properties of the grown crystal were calculated from UV transmission spectral analysis. Thermo gravimetric and differential thermal analysis reveals the thermal stability of the grown crystal. Second harmonic generation (SHG) of powdered LTF sample was tested using Nd: YAG laser and is found to be 1.21 times that of potassium dihydrogen orthophosphate. The fluorescence spectral study was carried out in the range of 280⿿500nm and the optical band gap is estimated to be 3.042eV.

Transmission mode direct analysis in real time mass spectrometry for fast untargeted metabolic fingerprinting

Untargeted metabolic fingerprinting is a discovery tool to better understand biochemical processes involved in detecting and characterizing disease states and responses to environmental stressors. Although current mass spectrometric (MS) methods are very powerful, there is a clear need for more rapid, high-throughput MS approaches for metabolomics studies. A rapid metabolic fingerprinting method for human blood sera that utilizes a new transmission mode direct analysis in real time (TM-DART) sampling technique coupled with quadrupole time of flight mass spectrometry (QTOFMS) is presented. In this approach, the sample is deposited directly on a stainless steel mesh that is held in the ionization region by a custom-built module. As a result, the DART plasma gas stream interacts with the sample in a flow-through fashion, which maximizes the interaction between the sample and ionizing species and minimizes variance in sample positioning. The optimization of TM-DART parameters directly affecting metabolite desorption and ionization, such as sample position and ionizing gas desorption temperature, was critical in achieving high sensitivity and detecting a broad mass range of metabolites. Ramping the ionizing gas desorption temperature further enhanced analysis by adding a simple separation dimension to this ambient approach. In terms of reproducibility, TM-DART compared favorably with traditional probe mode (PM-) DART analysis, with coefficients of variation as low as 16%. The longer-lasting TM-DART signals enabled the acquisition of metabolite full scan and product ion accurate mass spectra in a single experiment, resulting in greater confidence in metabolite identification. TM-DART MS proved to be a powerful analytical technique for rapid metabolome analysis of human blood sera.

Abstract 618: Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker.

Abstract Accumulated evidence points to DNA repair capacity as an important therapeutic factor in cancer in predicting patient response to DNA-damaging agents such as chemotherapeutic drugs and ionizing radiation. Recent findings suggest that some types of malignant tumors possess increased DNA repair capacity that may affect the therapy and outcome of cancer. Thus, the knowledge of the over-expression or under-expression levels of DNA repair proteins in tumors and disease-free tissues will help develop and guide treatment strategies that will likely lead to the best treatment results for patients. In this context, DNA repair proteins are becoming predictive, prognostic and therapeutic factors in cancer, and also promising drug targets for cancer treatment as DNA repair inhibitors are being developed to increase the efficacy of cancer therapy. We developed assays for the mass spectrometric measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. There is strong evidence for a critical role of NEIL1 in maintaining the genetic stability and in prevention of diseases such as cancer and metabolic syndrome. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), using the fully 15N-labeled analogue of hNEIL1 (15N-hNEIL1) as an internal standard, which we produced, purified and characterized. Both hNEIL1 and 15N-hNEIL1 were hydrolyzed with trypsin. Eighteen tryptic peptides of each protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1, and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides of both proteins were recorded and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and 15N-hNEIL1 on the basis of product ions. We also showed the detection of these proteins following their separation by gel electrophoresis and in-gel trypsin digestion. Our results suggest that the assays developed would be highly suitable for the positive identification and accurate quantification of hNEIL1 in tissues in vivo as a potential cancer biomarker. . Citation Format: Miral Dizdaroglu. Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2013-618