Identification and detection of phosphorylated proteins by laser induced breakdown spectroscopy

Thesis (Master)--Izmir Institute of Technology, Chemistry, Izmir, 2011Includes bibliographical references (leaves: 53-56)Text in English; Abstract: Turkish and Englishx, 56 leavesLaser-Induced Breakdown Spectroscopy (LIBS) is an optical atomic emission spectroscopic technique that uses an energetic laser source to generate a luminous plasma. Spectrochemical analysis of the light emitted from the plasma reveals information about the elemental composition of the sample. Phosphorylation is an important regulatory mechanism that activates or deactivates many proteins and enzymes in a wide range of cellular process. Identification and detection of phosphoproteins have a crucial importance in phosphopeptide mapping. This study is based on the assessment of the capabilities and limitations of LIBS as a quick and simple method for in-gel identification and determination of phosphorylated proteins, specifically casein and ovalbumin before mass spectrometric analysis for the elucidation of phosporylation sites. For this purpose, an optical LIBS set-up was constructed from its commercially available parts and the system was optimized for LIBS analysis of polyacrylamide gels. Nd:YAG laser operating at 532 nm wavelength and at 10 Hz frequency was used to create plasma on dry gel surfaces. Emitted light from a luminous plasma was analyzed and detected by an Echelle type spectrograph containing Intensified CCD, detector. With this study, LIBS detection of phosphorous proteins after electrophoretic separation of phosphorylated proteins has been shown, for the first time. After SDS-PAGE gel separation process, phosphoproteins were recognized from prominent P(I) lines (at 253.5 nm and 255.3 nm) in a plasma formed by the focused laser pulses on the gel, just in the center or in the vicinity of the electrophoretic spot. Spectral emission intensity of P(I) lines from LIBS data has been optimized with respect to laser energy and detector timing parameters by using standard Na2HPO4. It has been shown that phosphorylated proteins (casein and ovalbumin in mixture) can be identified by LIBS after both coomassie brilliant blue and silver staining procedures. Technique shows a great promise in microlocal spotting of phosphorylated proteins in gel before MS analysis for the determination of the phosphorylation sites

Methodology and applications of elemental mapping by laser induced breakdown spectroscopy

In the last few years, LIBS has become an established technique for the assessment of elemental concentrations in various sample types. However, for many applications knowledge about the overall elemental composition is not sufficient. In addition, detailed information about the elemental distribution within a heterogeneous sample is needed. LIBS has become of great interest in elemental imaging studies, since this technique allows to associate the obtained elemental composition information with the spatial coordinates of the investigated sample. The possibility of simultaneous multi-elemental analysis of major, minor, and trace constituents in almost all types of solid materials with no or negligible sample preparation combined with a high speed of analysis are benefits which make LIBS especially attractive when compared to other elemental imaging techniques. The first part of this review is aimed at providing information about the instrumental requirements necessary for successful LIBS imaging measurements and points out and discusses state-of-the-art LIBS instrumentation and upcoming developments. The second part is dedicated to data processing and evaluation of LIBS imaging data. This chapter is focused on different approaches of multivariate data evaluation and chemometrics which can be used e.g. for classification but also for the quantification of obtained LIBS imaging data. In the final part, current literature of different LIBS imaging applications ranging from bioimaging, geoscientific and cultural heritage studies to the field of materials science is summarized and reviewed. 2020 The Authors. Published by Elsevier B.V

Laser Induced Breakdown Spectroscopy

This review describes the fundamentals, instrumentation, applications and future trends of an analytical technique that is in its early stages of consolidation and is establishing its definitive niches among modern spectrometric techniques. The technique has been named Laser Induced Breakdown Spectroscopy (LIBS) and its main characteristic stands in the use of short laser pulses as the energy source to vaporize samples and excite the emission of electromagnetic radiation from its elements and/or molecular fragments. The emitted radiation is analyzed by high resolution optics and the intensities are recorded, usually by fast triggered solid state detectors. Together, these devices allow producing and registering a wide ranging emission spectrum of the short-lived phenomenon induced by the laser pulse. The spectrum contains qualitative and quantitative information which can be correlated with sample identity or can be used to determine the amount of its constituents. This review is divided in four parts. First, the relevant historical and theoretical concepts associated with LIBS are presented; then the main practical aspects of the several experimental and instrumental approaches employed for implementation of the technique are critically described; the applications related in the literature, including those making use of chemometrics, are classified and exemplified with relevant and recently published work. Finally, an attempt to portray an overall evaluation and future perspectives of the technique are presented.Esta revisão descreve os aspectos fundamentais, a instrumentação, as aplicações e tendências futuras de uma técnica analítica que se encontra em seu estágio de consolidação e que está em vias de estabelecer o seu nicho entre as técnicas espectrofotométricas modernas. A técnica é denominada Espectroscopia de Emissão em Plasma Induzido por Laser (em inglês, Laser Induced Breakdown Spectroscopy, LIBS) e sua principal característica está no uso de pulsos de laser como fonte de energia para vaporizar a amostra e excitar a emissão de radiação eletromagnética, a partir de seus elementos e/ou fragmentos moleculares. A radiação emitida é analisada por meio de instrumentos ópticos de alta resolução e as suas intensidades são medidas, usualmente com detectores rápidos de estado sólido. Em conjunto, esses dispositivos permitem a geração e a medida de um espectro de emissão de faixa ampla do fenômeno induzido pelo pulso de laser. O espectro registrado contém informação qualitativa e quantitativa que pode ser correlacionada com a identidade da amostra ou empregada na determinação da quantidade de seus constituintes. Essa revisão é dividida em quatro partes. A primeira aborda aspectos históricos da técnica e os conceitos teóricos relevantes associados com LIBS; então, os aspectos práticos de diversas abordagens experimentais e instrumentais empregadas na implementação da técnica são revistos de forma crítica; as aplicações encontradas na literatura, incluindo aquelas que empregam quimiometria, são classificadas e exemplificadas por meio de trabalhos relevantes recentemente publicados. Finalmente, uma tentativa de estabelecer uma avaliação global e as perspectivas futuras para a técnica é apresentada.463512Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Rare Earth Elements analysis to identify anthropogenic signatures at Valle del Serpis (Spain) Neolithic settlements

Due to their particular geochemical properties and stability Rare Earth Elements (REE) can act as a ‘fingerprint’ for soils, and as a consequence have been employed in a variety of different archaeological scenarios in order to identify past human activities.In this study, for the first time, we apply REE signatures in different Spanish Neolithic settlements, all located in the Valle del Serpis region. More than 100 Neolithic settlements have been identified in this area, and most of these open sites are characterised by dark brown strata that are in contrast with the light brown soils of the valley. These dark brown deposits are usually covered by paleosols and have been interpreted as markers of anthropogenic activities. However, in order to demonstrate whether these strata are anthropogenic or natural features requires a better understand-ing of soil development processes. A total of fifty samples were taken across six different sites, and from each site the sam-pling was carried out at different depths through 3m deep sections. Four sites are clearly associated with archaeological findings (sites BF, LP, PB and AC); another one is from a natural section near the Neolithic site of Mas d’Is (MD) and has been radiocarbon dated to the beginning of the Holocene (7751-7611 cal BC); and the last corresponds to a place of uncertain attribution (BK). Major, minor and trace elements including REE were determined using XRF and ICP- MS, with Principal Components Analysis (PCA) used to statistically analyze these data. Results were then compared with the strata soil properties analysed by XRD and particle size analysis, and cross-referenced with archaeological data to aid interpretation. The results demonstrate that REE analyses provide significant details regarding anthropogenic activities and strata development history, and in this instance confirm and elaborate on the archaeological interpretation that these dark brown deposits are evidence of a region-wide agricultural system in the Neolithic Valle del Serpis

The source of the building stones from the Sagunto Castle archaeological area and its surroundings

A multidisciplinary study was carried out on the building stones of the masonries belonging to the Castle of Sagunto (Valencia, Spain), an important historical and archeological complex, characterized by several construction phases from the Roman Period to the Modern Ages. For the first time, the stones of the Sagunto Castle have been analysed to determine their chemical, mineralogical and petrographic features, the main physical and mechanical properties, and to understand their decay, use and recycling dynamics in the different building during the entire occupational period. Geochemical and mineralogical analyses employing X-ray fluorescence (XRF), inductively coupled plasma mass spectrometry (ICP-MS) and X-ray diffraction (XRD) were carried out together with optical and electronic microscope analysis to observe the stone macro- and micro-structures. The collected data were processed by Principal Component Analysis (PCA) to highlight differences among the studied structures. The results show that the stones employed to build Sagunto`s structures during the different historical periods are related to a specific quarried area located nearby Sagunto Castle hill and differences between the studied samples are mostly related to the conservation state of the buildings. Therefore, geochemical analyses confirm the origin of the raw materials, while petrographic and physical analyses have been useful to evaluate the conservation state of the studied Sagunto Castle structures

Optimising nutrient use efficiency in crop production

Food security for a growing population presents a significant challenge for crop production, with increasing pressures upon agriculutral productivity. There is a vast need to improve crop yield and quality using an efficient approach that does not present negative environmental impacts. A novel interrogation technique that is able to provide information of the overall health of a plant, would be extremely beneficial in an agriculutral, as well as research, setting. This information could be utilised to better understand the mechanisms of plant functions, including stress responses. Vibrational spectroscopy encompasses a range of techniques that are able to derive chemically specific information from a biological sample in a rapid, nondestructive and cost-effective manner. Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy are two such approaches and have been readily implemented across biological samples. However, their applications in the field of plant science have been relatively underexploited. This is largely associated with the presence of water and fluorescent metabolites found in plant tissues. The application of attenuated total reflectance (ATR)-FTIR and spontaneous Raman spectroscopy for in vivo plant monitoring to elucidate spectral alterations indicative of healthy plant growth in a non-destructive manner. These approaches are able to characterise the biochemical signature of leaves at distinct developmental stages, and correspond to known biological processes within the leaf such as cell wall expansion. This information is useful prior to monitoring studies as normal leaf growth could be considered background variance. No significant local or systemic effects manifest as a consequence of interrogation with these techniques, establishing this as a non-destructive approach for plant system investigations. Raman microspectroscopy as a tool for monitoring nutrient uptake at the leaf surface is also considered, alongside complementary ion probe and elemental analysis. Such a technique is useful in the agrochemical production of foliar fertilisers, where the efficiency of specific formulae can be rapidly compared. This can also further the current understanding of nutrient transport into plant tissues, as well as translocation. Agriculturally relevant levels of calcium were applied to the leaf surface and uptake was successfully illustrated at concentrations as low as 15 mM using Raman microspectroscopy. Ion probe analysis also complemented these findings, with elemental analysis unable to detect this subtle uptake of nutrients. This assay is now being implemented in agrochemical practise as a fertiliser screening method. Deficiencies in essential nutrients such as calcium are detrimental to crop yield and thus are a potential target for improving crop production. A range of spectroscopic methods, including the use of synchrotron radiation, were utilised to presymptomatically detect these deficiencies prior to their onset in live samples. Coupled with multivariate analysis, these techniques discriminate between deficient and control samples with high sensitivity and specificity, without extensive sample preparation that traditional analytical techniques require. These results suggest that Raman and ATRFTIR spectroscopic approaches could highly valuable in the field, where plant health and nutrient status could be assessed rapidly in situ. Here it is shown that these issues can be overcome and that qualitative spectral measurements can be obtained from plant samples. Due to the non-destructive nature of these approaches, they can be applied for a wider range of crop screening investigations, including the efficiency of nutrient uptake, as well as distinguishing nutrient deficiencies presymptomatically. As such, these spectroscopic methods may be implemented to unearth further details regarding nutrient use efficiency during crop production

Laser Induced Breakdown Spectroscopy

This review describes the fundamentals, instrumentation, applications and future trends of an analytical technique that is in its early stages of consolidation and is establishing its definitive niches among modern spectrometric techniques. The technique has been named Laser Induced Breakdown Spectroscopy (LIBS) and its main characteristic stands in the use of short laser pulses as the energy source to vaporize samples and excite the emission of electromagnetic radiation from its elements and/or molecular fragments. The emitted radiation is analyzed by high resolution optics and the intensities are recorded, usually by fast triggered solid state detectors. Together, these devices allow producing and registering a wide ranging emission spectrum of the short-lived phenomenon induced by the laser pulse. The spectrum contains qualitative and quantitative information which can be correlated with sample identity or can be used to determine the amount of its constituents. This review is divided in four parts. First, the relevant historical and theoretical concepts associated with LIBS are presented; then the main practical aspects of the several experimental and instrumental approaches employed for implementation of the technique are critically described; the applications related in the literature, including those making use of chemometrics, are classified and exemplified with relevant and recently published work. Finally, an attempt to portray an overall evaluation and future perspectives of the technique are presented