We demonstrate how different normalization techniques in GC‐MS analysis impart unique properties to the data, influencing any biological inference. Using simulations, and empirical data, we compare the most commonly used techniques (Total Sum Normalization ‘TSN’; Median Normalization ‘MN’; Probabilistic Quotient Normalization ‘PQN’; Internal Standard Normalization ‘ISN’; External Standard Normalization ‘ESN’; and a compositional data approach ‘CODA’). When differences between biological classes are pronounced, ESN and ISN provides good results, but are less reliable for more subtly differentiated groups. MN, TSN, and (...) CODA approaches produced variable results dependent on the structure of the data, and are prone to false positive biomarker identification. In contrast, PQN exhibits the lowest false positive rate, though with occasionally poor model performance. Because ESN requires extensive pre‐planning, and offers only mixed reliability, and ISN, TSN, MN, and CODA approaches are prone to introducing artefactual differences, we recommend the use of PQN in GC–MS research. (shrink)

In this manuscript we describe Proteogenomics Viewer, a web‐based tool that collects MS peptide identification, indexes to genomic sequence and structure, assigns exon usage, reports the identified protein isoforms with genomic alignments and, most importantly, allows the inspection of MS2 information for proper peptide identification. It also provides all performed indexing to facilitate global analysis of the data. The relevance of such tool is that there has been an increase in the number of proteogenomic efforts to improve the annotation of (...) both genomics and proteomics data, culminating with the release of the two human proteome drafts. It is now clear that mass spectrometry‐based peptide identification of uncharacterized sequences, such as those resulting from unpredicted exon joints or non‐coding regions, is still prone to a higher than expected false discovery rate. Therefore, proper visualization of the raw data and the corresponding genome alignments are fundamental for further data validation and interpretation. (shrink)

Cells are the fundamental building blocks of organisms and their organization holds the key to our understanding of the processes that control Development and Physiology as well as the mechanisms that underlie disease. Traditional methods of analysis of subcellular structure have relied on the purification of organelles and the painstaking biochemical description of their components. The arrival of high‐throughput genomic and, more significantly, proteomic technologies has opened hereto unforeseen possibilities for this task. Recently two reports(1,2) show how much can be (...) gleaned from the combination of analytical centrifugation, mass spectrometry and advanced statistical techniques focused on a high‐throughput analysis of the content and organization of plant and animal cells. The results reveal intriguing possibilities for the future and the possibility of mapping much of the known proteome onto our current map of the cell. BioEssays 28: 780–784, 2006. © 2006 Wiley Periodicals, Inc. (shrink)

While mass spectrometry (MS)‐based quantification of small molecules has been successfully used for decades, targeted MS has only recently been used by the proteomics community to investigate clinical questions such as biomarker verification and validation. Targeted MS holds the promise of a paradigm shift in the quantitative determination of proteins. Nevertheless, targeted quantitative proteomics requires improvisation in making sample processing, instruments, and data analysis more accessible. In the backdrop of the genomic era reaching its zenith, certain questions arise: (...) is the proteomic era about to come? If we are at the beginning of a new future for protein quantification, are we prepared to incorporate targeted proteomics at the benchside for basic research and at the bedside for the good of patients? Here, an overview of the knowledge required to perform targeted proteomics as well as its applications is provided. A special emphasis is placed on upcoming areas such as peptidomics, proteoform research, and mass spectrometry imaging, where the utilization of targeted proteomics is expected to bring forth new avenues. The limitations associated with the acceptance of this technique for mainstream usage are also highlighted. (shrink)

Novel methods such as mass‐spectrometry enable a view of the proteomes of cells in unprecedented detail. Recently, these efforts have culminated in quantitative measurements of the number of copies per cell for most expressed proteins in organisms ranging from bacteria to mammalian cells. Here, we estimate the expected total number of proteins per unit of cell volume using known parameters related to the composition of cells such as the fraction of cell mass that is protein, and the (...) average protein length. Using simple arguments, we estimate a range of 2–4 million proteins per cubic micron (i.e. 1 fL) in bacteria, yeast, and mammalian cells. Interestingly, we find that measured values that are reported for fission yeast and mammalian cells are often about 3–10 times lower. We discuss this apparent discrepancy and how to use the estimate as benchmark to recalibrate proteome‐wide quantitative censuses or to revisit assumptions about cell composition. (shrink)

The emerging field of lipidomics has identified lipids as key players in disease physiology. Their physicochemical diversity allows precise control of cell structure and signaling events through modulation of membrane properties and trafficking of proteins. As such, lipids are important regulators of brain function and have been implicated in neurodegenerative and mood disorders. Importantly, environmental chronic stress has been associated with anxiety and depression and its exposure in rodents has been extensively used as a model to study these diseases. With (...) the accessibility to modern mass‐spectrometry lipidomic platforms, it is now possible to snapshot the extensively interconnected lipid network. Here, we review the fundamentals of lipid biology and outline a framework for the interpretation of lipidomic studies as a new approach to study brain pathophysiology. Thus, lipid profiling provides an exciting avenue for the identification of disease signatures with important implications for diagnosis and treatment of mood disorders. (shrink)

Mitochondria have been fundamental to the eco‐physiological success of eukaryotes since the last eukaryotic common ancestor (LECA). They contribute essential functions to eukaryotic cells, above and beyond classical respiration. Mitochondria interact with, and complement, metabolic pathways occurring in other organelles, notably diversifying the chloroplast metabolism of photosynthetic organisms. Here, we integrate existing literature to investigate how mitochondrial metabolism varies across the landscape of eukaryotic evolution. We illustrate the mitochondrial remodelling and proteomic changes undergone in conjunction with major evolutionary transitions. We (...) explore how the mitochondrial complexity of the LECA has been remodelled in specific groups to support subsequent evolutionary transitions, such as the acquisition of chloroplasts in photosynthetic species and the emergence of multicellularity. We highlight the versatile and crucial roles played by mitochondria during eukaryotic evolution, extending from its huge contribution to the development of the LECA itself to the dynamic evolution of individual eukaryote groups, reflecting both their current ecologies and evolutionary histories. (shrink)

Earlier investigations of the hormonal control of vitellogenin (egg‐yolk protein) synthesis in mosquitoes relied on microsurgical manipulations to identify organs that secrete the various hormones that stimulate or inhibit vitellogenesis. Advances in the last 10 years, using radioimmunoassays, HPLC, mass spectrometry and tissue‐culture incubations, have shown that the control mechanism involves a complex interaction between ecdysteroids, juvenile hormones and peptide hormones.

Integrated developmental and elemental information in teeth provide a unique framework for documenting breastfeeding histories, physiological disruptions, and neurotoxicant exposure in humans and our primate relatives, including ancient hominins. Here we detail our method for detecting the consumption of mothers’ milk and exploring health history through the use of laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS) mapping of sectioned nonhuman primate teeth. Calcium‐normalized barium and lead concentrations in tooth enamel and dentine may reflect milk and formula consumption with minimal (...) modification during subsequent tooth mineralization, particularly in dentine. However, skeletal resorption during severe illness, and bioavailable metals in nonmilk foods, can complicate interpretations of nursing behavior. We show that explorations of the patterning of multiple elements may aid in the distinction of these important etiologies. Targeted studies of skeletal chemistry, gastrointestinal maturation, and the dietary bioavailability of metals are needed to optimize these unique records of human health and behavior. (shrink)

The human genome contains about 30,000 genes, each creating several transcripts per gene. Transcript structures and expression are studied by high‐throughput transcriptomic techniques using microarrays. Generally, transcripts are not directly operating molecules, but are translated into functional proteins, post‐translationally modified by proteolysis, glycosylation, phosphorylation, etc., sometimes with great functional impact. Proteins need to be analyzed by proteomic techniques, less suited for high‐throughput. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), separating thousands of proteins has developed slowly over the past quarter of a century. (...) This technique is now quite reproducible and suitable for differential proteomics, comparing normal and diseased cells/tissues revealing differentially regulated proteins. 2D‐PAGE is combined with protein‐identification methods, currently mass spectrometry (MS), which has been significantly improved over the last decade. Other proteomic techniques studying protein–protein interactions are now either established or still being developed, such as peptide or protein arrays, phage display, and the yeast two‐hybrid system. The strengths and weaknesses of these techniques are discussed. BioEssays 26:901–915, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

For over four decades, knowledge that symptoms of some inherited diseases can be prevented or reduced via early detection and treatment in newborns has underpinned state-funded screening programs in most developed countries. Conditions for which newborn screening is now a recognized preventative public health initiative include phenylketonuria, congenital hypothyroidism, and, more recently, cystic fibrosis and sickle cell disorder. The use of tandem mass spectrometry to detect conditions such as amino-acidopathies and fatty-acid oxidation defects is also becoming increasingly prevalent. (...) a. (shrink)

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two‐dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High‐throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser‐induced fluorescence detection of labeled DNA strands have been of key importance for (...) the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH‐driven mobilization, provides efficient separations and on‐line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single‐cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single‐molecule detection. (shrink)

RésuméLe dosage de traces de molécules organiques dans les matrices biologiques fait appel à des techniques analytiques physico‐chimiques dont la chromatographie en phase gazeuse couplée à la spectrumétrie de masse est une des plus puissantes. Des possibilityés nouvelles pour augmenter la sensibilityé de la détection de ces substances sont presentees et mises en relation avec la signification physiologique des résultats obtenus.SummaryGas chromatography‐mass spectrometry is the method of choice for the quantitative determination of organic traces in biological systems. New (...) technical developments allow higher sensitivity and greater specificity which are discussed together with the physiological meaning of the data thus obtained.ZusammenfassungDie Dosierung der Spuren von organischen Molekülen in den biologischen Matrizen erfordert physico‐chemische analytische Techniken, deren Chromatographie in gasförmiger Phase ver‐bunden mit der Massenspektrometrie eine der mächtigsten ist. Es werden neue Möglichkeiten zur Steigerung der Empfindlichkeit der Aufweisung dieser Substanzen vorgelegt, und mit der physio‐logischen Bedeutung der erhaltenen Ergebnisse in Beziehung gesetzt. (shrink)

The primary transcripts, pre‐mRNAs, of almost all protein‐coding genes in higher eukaryotes contain multiple non‐coding intervening sequences, introns, which must be precisely removed to yield translatable mRNAs. The process of intron excision, splicing, takes place in a massive ribonucleoprotein complex known as the spliceosome. Extensive studies, both genetic and biochemical, in a variety of systems have revealed that essential components of the spliceosome include five small RNAs–U1, U2, U4, U5 and U6, each of which functions as a RNA, protein complex (...) called an snRNP (small nuclear ribonucleoprotein). In addition to snRNPs, splicing requires many non‐snRNP protein factors, the exact nature and number of which has been unclear. Technical advances, including new affinity purification methods and improved mass spectrometry techniques, coupled with the completion of many genome sequences, have now permitted a number of proteomic analyses of purified spliceosomes. These studies, recently reviewed by Jurica and Moore,1 reveal that the spliceosome is composed of as many as 300 distinct proteins and five RNAs, making it among the most complex macromolecular machines known. BioEssays 25:1147–1149, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

Lower plasma levels of high-density lipoproteins in adolescents with type 2 diabetes have been associated with a higher pulse wave velocity, a marker of arterial stiffness. Evidence suggests that HDL proteins or particle subspecies are altered in T2D and these may drive these relationships. In this work, we set out to reveal any specific proteins and subspecies that are related to arterial stiffness in youth with T2D from proteomics data. Plasma and PWV measurements were previously acquired from lean and T2D (...) adolescents. Each plasma sample was separated into 18 fractions and evaluated by mass spectrometry. Then, we applied a validated network-based computational approach to reveal HDL subspecies associated with PWV. Among 68 detected phospholipid-associated proteins, we found that seven were negatively correlated with PWV, indicating that they may be atheroprotective. Conversely, nine proteins show positive correlation with PWV, suggesting that they may be related to arterial stiffness. Intriguingly, our results demonstrate that apoA-I and histidine-rich glycoprotein may reverse their protective roles and become antagonistic in the setting of T2D. Furthermore, we revealed two arterial stiffness-associated HDL subspecies, each of which contains multiple PWV-related proteins. Correlation and disease association analyses suggest that these HDL subspecies might link T2D to its cardiovascular-related complications. (shrink)

The study of intrinsic phosphorylation dynamics and kinetics in the context of complex protein architecture in vivo has been challenging: Method limitations have prevented significant advances in the understanding of the highly variable turnover of phosphate groups, synergy, and cooperativity between P‐sites. However, over the last decade, powerful analytical technologies have been developed to determine the full catalog of the phosphoproteome for many species. The curated databases of phospho sites found by mass spectrometry analysis and the computationally predicted (...) sites based on the linear sequence of kinase motifs are valuable tools. They allow investigation of the complexity of phosphorylation in vivo, albeit with strong discrepancies between different methods. A series of hypothetical scenarios on combinatorial processive phosphorylation is proposed that are likely unverifiable with current methodologies. These proposed a priori postulates could be considered as possible extensions of the known schemes of the activation/inhibition signaling process in vivo. (shrink)

The vertebrate immune system uses an impressive arsenal of mechanisms to combat harmful cellular states such as infection. One way is via cells delivering real‐time snapshots of their protein content to the cell surface in the form of short peptides. Specialized immune cells (T cells) sample these peptides and assess whether they are foreign, warranting an action such as destruction of the infected cell. The delivery of peptides to the cell surface is termed antigen processing and presentation, and decades of (...) research have provided unprecedented understanding of this process. However, predicting the capacity for a given peptide to be immunogenic—to elicit a T cell response—has remained both enigmatic and a long sought‐after goal. In the era of big data, a point is being approached where the steps of antigen processing and presentation can be quantified and assessed against peptide immunogenicity in order to build predictive models. This review presents new findings in this area and contemplates challenges ahead. (shrink)

Cet article présente les résultats des analyses radiochronologiques pratiquées sur sept échantillons d'ossements humains issus des fouilles de l'habitat mésohelladique de l'Aspis. Les analyses ont été réalisées au Centre de recherche sur les isotopes de l'université de Gröningen, par la méthode SMA (spectrométrie de masse par accélérateur). L'objectif principal des analyses est de dater plus précisément les phases d'occupation de l'habitat en comparant les dates absolues avec la chronologie relative fondée sur la stratigraphie complexe et la séquence céramique du site. (...) Les résultats suggèrent que le passage de l'HM III à l'HR I se situe aux alentours de 1700 av. J.-C. La méthode ne permet pas de dater avec précision le passage de l'HM II à l'HM III parce que ces deux phases sont de courte durée alors que la marge d'incertitude des datations par le radiocarbone est assez grande, étant donné la forme de la courbe de calibration pour cette période. (shrink)

This article deals with a set of images which were probably the first in history to be reproduced on four continents: the Jesuit Jerome Nadal's Evangelicae Historiae Imagines, first published in Antwerp in 1593. It begins with a brief discussion of what we know about Nadal's life and the production history of his book, as well as its relationship to his Adnotationes et Meditationes in Evangelia quae in sacrosanto missae sacrificio toto anno leguntur, which acts as a commentary to the (...) Imagines. Then follows a description of the structure of the two books, and how they narrate and expound the significance of the Gospels. The remaining sections discuss the adaptations of Nadal's imagery. While his work has received much attention from scholars around the world in recent decades, the aim of the present article is to bring together research on the use and re-use of these images in Europe, the Americas, India, China, Japan, Iran and Ethiopia, and to make clear their global impact in a way that has not been attempted before. (shrink)

Qu'entend-on par enchantement et désenchantement du monde? Quelles seraient les voies à suivre pour susciter un réenchantement du monde qui sera le fait de l’humain lui-même? La raison et la rationalité sont-elles les causes des maux de nos errements ou des outils pour les contrer? Suffira-t-il d’avoir « foi en la raison » pour surmonter les défis que l’humanité s’est elle-même imposés par négligence et irresponsabilité.

A conception of parental experience is proposed to enhance the move of the study of parenting into the interpersonal realm by describing parental subjectivity from the parent's point of view. Explanations are based on that which the parent can be accountable for, on parental dialogues with observers/clinicians about their dialogues with their infants. This conception of parental subjectivity is compared with other conceptions which define parental subjectivity as the mental apparatus of the parent and not as representing the evolving relation (...) of the parent with the infant, and with explanations which consider parental reports in terms of the parent's psychodynamics and cognitive abilities rather than in terms of the on-going dialogues between themselves and their infants. The paper introduces a typology of parental dialogues with observers/clinicians about their dialogues with their infants, within the context of the non-verbal nature of the infant's communication. The findings from empirical examinations of this typology are presented, and their implications for the proposal that the study of parental relations with their infants should consider the parent's accountability are discussed. (shrink)

The text confronts the elements of which Luhmann theorizes the educative system, from a critical form to center the suitable approach to know the multiplicity of elements from the educative system. Luhmann’s foundations are criticized and other levels are proposed, from a critical vision of social..