Laser Desorption Time-of-flight Research Articles

The Fruit Proteome Response to the Ripening Stages in Three Tomato Genotypes.

The tomato is a horticultural crop that appears in various colors as it ripens. Differences in the proteome expression abundance of a tomato depend on its genotype and ripening stage. Thus, this study aimed to confirm the differences in changes in the proteome according to four ripening stages (green, breaker, turning, and mature) of three tomato genotypes, i.e., yellow, black, and red tomatoes, using a gel-based proteomic technique. The number of protein spots shown as two-dimensional electrophoresis (2-DE) gels differed according to tomato genotype and ripening stage. A total of 286 variant proteins were determined using matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, confirming 233 identified protein functions. In three tomato genotypes in each ripening stage, grouping according to the Munich Information Center for Protein Sequences (MIPS) functional categories confirmed the variant proteins involved in the following: energy processes (21%); metabolism (20%); protein fate (15%); protein synthesis (10%); a protein with a binding function or cofactor requirement (8%); cell rescue, defense, and virulence (8%); cellular transport, transport facilitation, and transport routes (6%); the biogenesis of cellular components (5%); cell cycle and DNA processing (2%); others (5%). Among the identified protein spots in the function category, two proteins related to metabolism, four related to energy, four related to protein synthesis, and two related to interaction with the cellular environment showed significantly different changes according to the fruit color by the ripening stage. This study reveals the physiological changes in different types of tomatoes according to their ripening stage and provides information on the proteome for further improvement.

Insufficient sensitivity of laser desorption-time of flight mass spectrometry-based detection of hemozoin for malaria screening

Laser desorption-time of flight (LD-TOF) mass spectrometry-based detection of hemozoin was assessed for its performance characteristics as a rapid screening test for malaria. In spite of good specificity of >95%, poor sensitivity of 80.2% for microscopically positive samples makes the easy-to-apply and rapid approach unsuitable for the routine diagnostic setting.

Chemical detection of cysteine-rich circular petides in selected tropical Violaceae and Moringaceae families using modified G-250 and mass spectrometry

Cysteine-rich circular peptides (CRCs) comprise a large family of gene encoded and low molecular weight polypeptides that has recently engaged the attention of scientists. This class of peptides exhibit a continuous circular configuration and a cystine knot backbone, which defines their resilient nature-directed structural design. Many CRCs have been reported in medicinal plants the first of which is kalata B1 cyclotide from the traditional African plant Oldenlandia affinis. Their detection and isolation can be very challenging and evasive. Only about 1% of plant species have so far been reportedly screened. A modified preliminary chemo-microscopic/macroscopic method involving the use of G-250 stain was applied followed by thin layer chromatographic protosite reaction for plant selection. This was followed by the Matrix Assisted Laser Desorption Time of Flight (MALDI-TOF) Mass spectrometry guided experiment for cyclotide discovery. The blue colouration produced upon sample reaction with modified G-250 led to the selection of potential circular peptide containing plant samples. A further MALDI-TOF MS-guided screening resulted in the detection of circular peptides and cyclotides in Moringa oleifera, Rinorea dentata, R. oblongifolia and R. brachypatela. Viola odorata and Viola tricolor (positive controls) indicated the presence of cyclotides. Results from this study can serve as proof-of-concept for plant selection based on preliminary cysteine-rich circular peptide detection in plants especially with the use of G-250 stain.Keywords: Cysteine-rich Cyclotides, Violaceae, Moringaceae, TLC, Microscopy, MALDI-TOF MS

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings.Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC–MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.

Topologies of amyloidogenic proteins in Congo red-positive sliced sections of formalin-fixed paraffin embedded tissues by MALDI-MS imaging coupled with on-tissue tryptic digestion

ObjectivesMatrix-assisted laser desorption time-of flight ionization (MALDI)-imaging MS (IMS) with MSMS analysis using on-tissue tryptic digests is a powerful tool for identification of disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections. We applied this novel IMS technique, not only to identify tryptic peptides of deposited amyloidogenic proteins but also to clarify topologies of these proteins in amyloidosis tissue sections. MethodsSequence determinations of tryptic peptides derived from amyloidogenic proteins were performed using MALDI-MSMS analysis directly from Congo red positive regions in tissue sections with/without procedure for retrieval of epitopes before on-tissue digestion. ResultsTryptic peptides, m/z=1073.5 and 1924.3 were identified with the sequences, from 48th to 56th and 1st to 19th positions of Ig lambda V-III region, respectively. Other peptides, m/z=1365.5 and 1523.5 were with the sequences, from 22nd to 34th and 36th to 48th positions of TTR, respectively. Heat-map images of all four tryptic peptides were overlapped with Congo red positive regions. Immunohistochemistry of FFPE tissue sections was confirmed to only react with anti-λ chain antibody in a case of AL-type amyloidosis or anti-TTR antibody in two cases of TTR-type amyloidosis. ConclusionIMS with MSMS analysis using on-tissue tryptic digestion enables us not only to identify amyloidogenic molecule in a sliced tissue section but also to play a complementary role with the conventional pathological examination.

Synthesis, Isolation, and Spectroscopic Characterization of Holmium-Based Mixed-Metal Nitride Clusterfullerenes: HoxSc3−xN@C80 (x=1, 2)

The synthesis, isolation and spectroscopic characterization of holmium-based mixed metal nitride clusterfullerenes Ho(x) Sc(3-x) N@C(80) (x=1, 2) are reported. Two isomers of Ho(x) Sc(3-x) N@C(80) (x=1, 2) were synthesized by the reactive gas atmosphere method and isolated by multistep recycling HPLC. The isomeric structures of Ho(x) Sc(3-x) N@C(80) (x=1, 2) were characterized by laser-desorption time-of-flight (LD-TOF) mass spectrometry and UV/Vis/NIR, FTIR and Raman spectroscopy. A comparative study of M(x) Sc(3-x) N@C(80) (M=Gd, Dy, Lu, Ho) demonstrates the dependence of their electronic and vibrational properties on the encaged metal. Despite the distinct perturbation induced by 4f(10) electrons, we report the first paramagnetic (13) C NMR study on Ho(x) Sc(3-x) N@C(80) (I; x=1, 2) and confirm I(h) -symmetric cage structure. A (45) Sc NMR study on HoSc(2) N@C(80) (I, II) revealed a temperature-dependent chemical shift in the temperature range of 268-308 K.

Proteomic biomarkers predicting lymph node involvement in serum of cervical cancer patients. Limitations of SELDI-TOF MS

BackgroundLymph node status is not part of the staging system for cervical cancer, but provides important information for prognosis and treatment. We investigated whether lymph node status can be predicted with proteomic profiling.Material & methodsSerum samples of 60 cervical cancer patients (FIGO I/II) were obtained before primary treatment. Samples were run through a HPLC depletion column, eliminating the 14 most abundant proteins ubiquitously present in serum. Unbound fractions were concentrated with spin filters. Fractions were spotted onto CM10 and IMAC30 surfaces and analyzed with surface-enhanced laser desorption time of flight (SELDI-TOF) mass spectrometry (MS). Unsupervised peak detection and peak clustering was performed using MASDA software. Leave-one-out (LOO) validation for weighted Least Squares Support Vector Machines (LSSVM) was used for prediction of lymph node involvement. Other outcomes were histological type, lymphvascular space involvement (LVSI) and recurrent disease.ResultsLSSVM models were able to determine LN status with a LOO area under the receiver operating characteristics curve (AUC) of 0.95, based on peaks with m/z values 2,698.9, 3,953.2, and 15,254.8. Furthermore, we were able to predict LVSI (AUC 0.81), to predict recurrence (AUC 0.92), and to differentiate between squamous carcinomas and adenocarcinomas (AUC 0.88), between squamous and adenosquamous carcinomas (AUC 0.85), and between adenocarcinomas and adenosquamous carcinomas (AUC 0.94).ConclusionsPotential markers related with lymph node involvement were detected, and protein/peptide profiling support differentiation between various subtypes of cervical cancer. However, identification of the potential biomarkers was hampered by the technical limitations of SELDI-TOF MS.

Ligand-Coated Vanadium Oxide Clusters: Capturing Gas-Phase Magic Numbers in Solution

Subnanometer vanadium oxide clusters in the 10−30 atom size range are produced in the gas phase with laser vaporization, coated with gas-phase ligands, and then captured in solution using a laser vaporization flow reactor. Acetonitrile (MeCN) and tetrahydrofuran (THF) ligands form complexes efficiently with the oxide clusters, rendering them soluble in solutions of these same ligands and other solvents. The structures and compositions of clusters captured in solution are investigated with laser desorption time-of-flight (LD-TOF) mass spectrometry, UV−visible and IR spectroscopy, and density functional theory computations. MeCN forms oxide clusters having the exact stoichiometries found previously to be stable in the gas phase (V3O6, V4O9, and V5O12), in a simple ligand-addition process. THF produces similar oxide cores, but with terminal oxygens displaced. Infrared spectra are consistent with the presence of terminal oxygens for the MeCN complexes and their absence for the THF species. In either case, DFT computations show that the vanadium oxide cores are minimally perturbed by ligand addition. Solutions of both samples exhibit visible photoluminescence with only minor dependence on the ligand, indicating that the core oxide is the source of the emission.

Laser-Induced Oxidation of Cholesterol Observed During MALDI-TOF Mass Spectrometry

Conditions for the detection of three odd-electron cholesterol oxidation peaks were determined and these peaks were shown to be artifacts of the matrix-assisted laser desorption time of flight (MALDI-TOF) process. Matrix choice, solvent, laser intensity and cholesterol concentration were systematically varied to characterize the conditions leading to the highest signals of the radical cation peaks, and it was found that initial cholesterol solution concentration and resultant density of solid cholesterol on the MALDI target were important parameters in determining signal intensities. It is proposed that hydroxyl radicals, generated as a result of laser irradiation of the employed 2,5-dihydroxybenzoic acid (DHB) matrix, initiate cholesterol oxidation on the MALDI target. An attempt to induce the odd-electron oxidation peaks by means of adding an oxidizing agent succeeded using an acetonitrile solution of DHB, cholesterol, and cumene hydroperoxide. Moreover, addition of free radical scavengers reduced the abundances of some oxidation products under certain conditions. These results are consistent with the mechanism of oxidation proposed herein involving laser-induced hydroxyl radical production followed by attack on neutral cholesterol. Hydroxyl radical production upon irradiation of dithranol matrix may also be responsible for generation of the same radical peaks observed from cholesterol in dithranol by an analogous mechanism.

ChemInform Abstract: Mass Spectroscopic and ESR Characterization of Soluble Yttrium- Containing Metallofullerenes YC82 and Y2C82.

Solvent soluble, yttrium-containing fullerenes are extracted from yttrium/carbon soot produced by the carbon-arc fullerene generation method. An yttrium-graphite composite yields solvent extractable molecules such as YC{sub 82} and Y{sub 2}C{sub 82} as evidenced by a laser desorption time-of-flight (LD-TOF) mass spectroscopic analysis of the extracts. This is consistent with the very recent results by Smalley et al. where YC{sub 82} and Y{sub 2}C{sub 82} are prepared by laser vaporization of yttrium-graphite rods. The present extracts are souble by both toluene and carbon disulfide, but the solubility is much higher is CS{sub 2} than toluene. LD-TOF mass spectra also show the presence of a series of peaks due to the dimetallofullerens Y{sub 2}C{sub 2n}(2n = 84; 90 {le} 2n {le}140). The metallofullerenes YC{sub 60} and YC{sub 70} are not observed in the mildest possible LD conditions under which YC{sub 82} and the dimetallofullerenes Y{sub 2}C{sub 82} and Y{sub 2}C{sub 2n} are readily observed. ESR spectral results are also reported for the present yttrium-fullerene complexes and indicate the charge separation state of Y{sup 3+}C{sub 82}{sup 3-} as has been found for La{sup 3+}C{sub 82}{sup 3-} by Johnson et. al. 13 refs., 4 figs.

Strategy for Identification by Mass Spectrometry of a New Human Hemoglobin Variant with Two Mutations in Cis in the β-Globin Chain: Hb S-Clichy [β6(A3)Glu→Val; β8(A5)Lys→Thr

Hemoglobinopathies are the most frequent genetic diseases in the world. Among them, the Hb S variant [β6(A3)Glu→Val], which, in the homozygous state, produces a severe disease known as sickle cell anemia with polymerization of Hb S inside red blood cells under hypoxic conditions. Additional mutations, in cis or in trans of the βS-globin chain, may inhibit or enhance the polymerization process. We describe here a new hemoglobin (Hb) variant (Hb S-Clichy) which carries the βS‐globin chain and an additional mutation β8(A5)Lys→Thr. The variant was detected by routine electrophoretic techniques and cation exchange liquid chromatography (CE-LC). Globin chain separation by reversed phase LC (RP-LC) showed normal and abnormal β chains, confirming that the additional abnormality was located in cis to Hb S. Electrospray ionization mass spectrometry (ESI-MS) gave a 57 Da mass decrease for the abnormal globin chain. The abnormal chain was isolated and submitted to trypsin digestion. Normal peptides βT-1 and βT-2 were not observed on the matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrum but a new peptide βT-1,2 was detected. Nano LC-ESI-MS/MS of the new peptide showed that the glutamic acid at codon 6 was replaced by a valine residue, and the lysine at codon 8 was replaced by a threonine residue, as confirmed by DNA sequencing. This example demonstrates that in a population where Hb S is present, every unidentified Hb needs to be clearly characterized to prevent major sickle cell syndromes. In addition, the identification of these variants must be considered in newborn screening for sickle cell disease, using either classical biochemical methods or MS techniques.

Synthesis, Characterization, and Gene Delivery of Poly-l-lysine Octa(3-aminopropyl)silsesquioxane Dendrimers: Nanoglobular Drug Carriers with Precisely Defined Molecular Architectures

Macromolecules with defined nanosizes--nanoglobules--were synthesized and characterized as novel drug carriers with precise molecular architectures. Poly-L-lysine dendrimers with a cubic octa(3-aminopropyl)silsesquioxane (OAS) core, (L-lysine 8-OAS, (L-lysine) 16-(L-lysine) 8-OAS, (L-lysine) 32-(L-lysine) 16-(L-lysine) 8-OAS, and (L-lysine) 64-(L-lysine) 32-(L-lysine) 16-(L-lysine) 8-OAS, were divergently synthesized by solution phase peptide chemistry in good yield and purity. Matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry showed complete substitution of the surface amino groups of lower generation dendrimers during synthesis, as well as precisely defined molecular architectures of the nanoglobules. The structures of the nanoglobules were further characterized by (1)H- and (13)C-NMR and 2D-NMR (correlation spectroscopy (COSY) and pulsed-field-gradient heteronuclear multiple quantum correlation (gHMQC)) spectroscopy. The (1)H-NMR spectroscopy revealed that the nanoglobules had a relatively rigid molecular architecture. Cytotoxicity studies showed that these nanoglobules exhibited a size-dependent toxicity, but it was much lower than that of linear poly-L-lysine. Preliminary in vitro nucleic acid delivery studies have shown that these globular dendrimers can efficiently deliver plasmid DNA to MDA-MB-231 cells. These nanoglobules hold much promise as safe drug carriers with precisely defined molecular architecture.

High Resolution Footprinting of the Hepatitis C Virus Polymerase NS5B in Complex with RNA

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

Carcinogen-induced histone alteration in normal human mammary epithelial cells

Little is known about early carcinogen-induced protein alterations in mammary epithelium. Detection of early alterations would enhance our understanding of early-stage carcinogenesis. Here, normal human mammary epithelial cells (HMECs) were exposed to dietary and environmental carcinogens [2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), 4-aminobiphenyl (ABP), benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin] individually or in combination. A phage display library of single-chain variable fragment antibodies was used to screen protein targets altered by the treatment. In combination with matrix-assisted laser desorption time of flight, we identified histone H3 as a target antigen. Although histone H3 total protein remained unchanged in control and treated HMEC, the methylation of lysine 4 was altered. A reduction in mono-methyl histone H3 (Lys 4) was observed in treated HMEC compared with control HMEC. This alteration was shown to be dependent on carcinogen concentration and specific for PhIP and ABP. To characterize potential histone demethylation mechanisms, localization and protein expression patterns of lysine-specific demethylase 1 (LSD1) were analyzed. In control HMEC, LSD1 was present at the nuclear periphery. However, following 72 h carcinogen treatment, LSD1 localized within the nucleus. Within 48 h after treatment, mono-methyl histone H3 (Lys 4) was restored and LSD1 localization was reversed. Protein expression levels of LSD1 were also increased in treated HMEC compared with control HMEC. Our data suggest that the induction of a single enzyme, LSD1, represents an early response to carcinogen exposure, which leads to the demethylation of histone H3 (Lys 4), which, in turn, may influence the expression of multiple genes critical in early-stage mammary carcinogenesis.

Characterization and Comparison of Major Urinary Proteins from the House Mouse, Mus musculus domesticus, and the Aboriginal Mouse, Mus macedonicus

Urine from the house mouse, Mus musculus domesticus, contains a high concentration of major urinary proteins (MUPs), which convey olfactory information between conspecifics. In wild populations, each individual expresses a different pattern of around 8 to 14 electrophoretically separable MUP isoforms. To examine whether other Mus species express MUPs and exhibit a similar level of individual heterogeneity, we characterized urinary proteins in urine samples from an aboriginal species, Mus macedonicus, captured from different sites in Turkey. Anion exchange chromatography and electrospray ionization mass spectrometry demonstrated that M. macedonicus urine contained a single major peak of mass 18,742 Da, and in contrast to M. m. domesticus, all individuals were the same. The M. macedonicus masses were not predicted from any known MUP gene sequence. Endoproteinase Lys-C (Lys-C) digestion of the purified M. macedonicus urinary protein followed by matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry demonstrated that it shared considerable, but not complete, sequence homogeneity with M. m. domesticus MUPs. Three M. macedonicus Lys-C peptides differed in mass from their M. m. domesticus counterparts. These three peptides were further characterized by tandem mass spectrometry. The complete sequences of two were determined, and in conjunction with methyl esterification, the amino acid composition of the third was inferred, and the sequence narrowed down to three permutations. The complete M. macedonicus sequence contained a maximum of seven amino acid substitutions, discernible by tandem mass spectrometry, relative to a reference M. m. domesticus sequence. Six of these were on the surface of the molecule. Molecular modeling of the M. macedonicus sequence demonstrated that the amino acid substitutions had little effect on the tertiary structure. The differences in the level of heterogeneity between the two species are discussed in relation to their environment and behavior. In addition, the differences in protein structure allow speculation into molecular mechanisms of MUP function.

C80 Encaging Four Different Atoms: The Synthesis, Isolation, and Characterizations of ScYErN@C80

The synthesis, isolation, and spectroscopic characterizations of an endohedral fullerene with four heteroatoms encapsulated (ScYErN@C80) are reported for the first time. The isomeric structure and electronic properties of this molecule are studied by various spectrometry methods such as high-performance liquid chromatography (HPLC), laser desorption time-of-flight (LD-TOF) mass spectroscopy, cyclic voltammetry, Fourier transform infrared (FTIR) spectroscopy, and visible-near infrared (vis-NIR) absorption spectroscopy. The carbon cage of ScYErN@C80 is assigned as Ih-C80, and the four-membered ScYErN cluster is suggested to rotate rapidly inside the fullerene cage. Six electrons are transferred from the nuclear cluster ScYErN to the fullerene cage, which leads to a closed-shell electronic structure of the Ih-C80 and results in excellent stability of this molecule.

Expanding the Number of Stable Isomeric Structures of the C80 Cage: A New Fullerene Dy3N@C80

The production, isolation, and spectroscopic characterization of a new Dy3N@C80 cluster fullerene that exhibits three isomers (1-3) is reported for the first time. In addition, the third isomer (3) forms a completely new C80 cage structure that has not been reported in any endohedral fullerenes so far. The isomeric structures of the Dy3N@C80 cluster fullerene were analyzed by studying HPLC retention behavior, laser desorption time-of-flight (LD-TOF) mass spectrometry, and UV-Vis-NIR and FTIR spectroscopy. The three isomers of Dy3N@C80 were all large band-gap (1.51, 1.33, and 1.31 eV for 1-3, respectively) materials, and could be classified as very stable fullerenes. According to results of FTIR spectroscopy, the Dy3N@C80 (I) (1) was assigned to the fullerene cage C80:7 (I(h)), whereas Dy3N@C80 (II) (2) had the cage structure of C80:6 (D(5h)). The most probable cage structure of Dy3N@C80 (III) (3) was proposed to be C80:1 (D(5d)). The significant differences between Dy3N@C80 and other reported M3N@C80 (M = Sc, Y, Gd, Tb, Ho, Er, Tm) cluster fullerenes are discussed in detail, and the strong influence of the metal on the nitride cluster fullerene formation is concluded.

Synthesis and characterization of a new water-soluble endohedral metallofullerene for MRI contrast agents

Water-soluble amino acid derivatives of gadolinium (Gd) endohedral metallofullerenes (AAD-EMFs), Gd@C 82O m (OH) n (NHCH 2CH 2COOH) l ( m ≈ 6, n ≈ 16 and l ≈ 8) are synthesized by a direct reaction of the pure endohedral metallofullerene Gd@C 82 with an excess of alkaline solution of β-alanine. The structure of the AAD-EMFs is characterized by FTIR, XPS and laser-desorption time-of-flight (LD-TOF) mass spectrometries. Water proton relaxivity analysis indicates that the longitudinal relaxivity R 1 (the effect on 1/ T 1, 9.1 mM −1 s −1) of AAD-EMFs is higher than that of the commercial MRI contrast agent, Magnevist (gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA, 5.6 mM −1 s −1). The MRI phantom studies are performed to confirm the high efficiency of this sample as MRI contrast agents.

Epidermal Growth Factor Gene (EGF) Polymorphism and Risk of Melanocytic Neoplasia

A common single nucleotide polymorphism (SNP) in the 5' untranslated region (5'UTR) of the epidermal growth factor (EGF) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case-control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.

Pulsed laser deposition of c * axis oriented pentacene films

Pulsed laser desorption and film deposition behaviors have been investigated on pentacene as an organic molecule primarily due to its applications in electronics. The laser desorption time-of-flight (LDTOF) mass spectrum exhibited a single parent peak when a pressed pentacene pellet was ablated by an N2 laser beam of its fluence lower than 100 mJ/cm2, indicating that pentacene could be evaporated without an appreciable photodecomposition by the pulsed laser beam. Nd : YAG pulsed laser deposition of pentacene films was performed using such optimization parameters as laser fluences and wavelength (second, third and forth harmonic generations (SHG, THG, FHG)). The analyses with AFM, XRD and UV-Vis spectroscopy revealed the fabrication of c* axis oriented pentacene films on quartz, silicon, and CaF2 substrates by the SHG. The SHG films have a surface morphology superior to those of films deposited by THG and FHG.