Program

Session 2

2.1 Protein Quantification through Targeted Mass Spectrometry: The Way Out of Biomarker Purgatory? S. A. Carr Broad Institute of MIT and Harvard, Cambridge, MA The enormous potential of biomarkers to revolutionize clinical practice and improve patient care has been well documented. Given their high potential therapeutic and financial impact it is, on the surface, surprising that so few new protein biomarkers have been introduced into widespread clinical use recently. The reasons for the dearth of new protein biomarkers relate to the high false discovery rate of discovery “omics” methods (regardless of technology used), together with a lack of robust methods for biomarker verification in large clinical sample sets. It is now common for differential analysis of tissue or plasma by multidimensional LC-MS/MS (the workhorse tool for unbiased discovery) to provide confident identification of 1000's of proteins, 100's of which can vary 5-fold or more between case and control samples in discovery studies. Due to the extent of sample fractionation required to access proteins at lower abundance, it is not uncommon for the analysis of a single case/control sample pair to take up to two weeks of on-instrument time. This limits the numbers of samples that can be practically analyzed to typically 10 (or fewer) case vs control comparisons. These numbers are very small relative to the high dimensionality of the proteome (100,000's or more possible components when posttranslational modifications and other variants are taken into account), and the scale of normal variation in the human population. Thus a very large fraction, possibly exceeding 95% of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery “omics” experiments do not lead to biomarkers of immediate clinical utility, but rather produce “candidates” that must be “qualified” and “verified” (1,2). Lack of robust quantitative methods with sufficient sensitivity, reproducibility and throughput has significantly hampered our ability to credential candidates coming from unbiased proteomic discovery efforts since useful Ab reagents for the vast majority do not exist. Our laboratory has focused on addressing this serious barrier by developing robust targeted assay methods employing mass spectrometry to screen and quantify low abundance proteins in plasma. We have recently demonstrated that multiplexed assays for proteins at the low ng/mL level in plasma can be configured using Stable Isotope Dilution (SID) - Multiple Reaction Monitoring (MRM) Mass Spectrometry. Large-scale interlaboratory studies conducted under the auspices of the National Cancer Institute's Clinical Proteomics Assessment for Cancer (CPTAC) program have demonstrated that these assays can be reproducibly configured, deployed and run in multiple laboratories with assay CV's approaching clinical performance. Further improvements will come from the use of peptide immunoaffinity enrichment, referred to as SISCAPA, which holds particular promise for simplifying sample preparation and increasing both throughput and sensitivity of MRM-based assays. Using SISCAPA, assays can be readily configured that enable quantitation of proteins present at low ng/mL levels directly from plasma. This presentation will focus on the further development and application of MRM-MS and SISCAPA technologies in the context of cancer and cardiovascular disease. References 1. Rifai, N., Gillette, M. A., and Carr, S. A. (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat. Biotechnol. 24, 971–983. 2. Paulovich, A. G., Whiteaker, J. R., Hoofnagle, A. N., and Wang, P. (2008) The interface between biomarker discovery and clinical validation: The tar pit of the protein biomarker pipeline. Proteom. Clin. Appl. 2, 1386–1402. 2.2 Proteomics Targeted to Sub-Cellular Compartments and Integration with Genomics for Candidate Biomarker Discovery in Colorectal Cancer C. R. Jimenez OncoProteomics Laboratory, Department of Medical Oncology, VUmc-Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands Mass spectrometry-based proteomics applied to biomarker-rich sub-cellular compartments in (pre)-clinical samples is emerging as a powerful approach for discovery of tissue-derived biomarkers with close association to the disease. We have applied in-depth proteomics to sub-nuclear compartments, cell surface fractions and tumor secretomes for discovery of colorectal cancer-related proteins with potential use as imaging- and serum-based biomarkers. To this end, we analyzed preclinical (cell lines and genetic mouse model) as well as clinical samples. We have optimized the label-free GeLC-LTQ-FTMS/MS pipeline to allow for quantitation of individual proteins by spectral counting with good reproducibility. Integration of in-depth discovery proteomics with genomics data (microarray and arrayCHG) allows for a powerful approach to prioritize candidates for follow-up and validation by targeted strategies, including immunohistochemistry of tissue microarrays. Promising imageable biomarker candidates have been validated and validation of selected nuclear proteins and secretome differential proteins as serum and stool-based markers is underway. 2.3 Towards the Discovery of Biomarkers in Cerebrospinal Fluid by Combining Peptide Ligand Library Treatment and Label Free Protein Quantification on a LTQ-Orbitrap F. Roux-Dalvai1, E. Mouton-Barbosa1, A. Gonzalez de Peredo1, D. Bouyssié1, L. Guerrier2, E. Boschetti2, F. Berger3, O. Burlet-Schiltz1, and B. Monsarrat1 1CNRS IPBS, Toulouse University, France; 2BioRad, Gif-sur-Yvette, France; 3INSERM, Université Joseph Fourier, Grenoble, France Cerebrospinal Fluid (CSF) is the biological fluid in closest contact with the brain and thus contains some proteins and other products of neural cell origin, lending itself to proteomic analysis for potential biomarkers of neurological diseases. However, as in the case of other biological fluids, the main analytical challenge in proteomic characterization of the CSF is the very wide concentration range of proteins, largely exceeding the dynamic range of current analytical approaches. The most abundant protein, human serum albumin, constitutes alone around 45% of the total protein content in CSF, which renders extremely difficult the detection of low-abundant species. Here, we used the peptide ligand library technology Proteominer™ to reduce the dynamic range of protein concentration in CSF and unmask previously undetected proteins by nanoLC-MS/MS analysis on an LTQ-Orbitrap mass spectrometer. This method was first applied on a large pool of CSF from different sources, with the aim to better characterize the protein content of this fluid, especially for the low abundance components. We were able to identify 1189 proteins in CSF, and among these, 755 were only detected after Proteominer™ treatment. One drawback of this method is the large amount of biological fluid that has to be applied on conventional Proteominer™ columns, precluding its potential use for treatment of patient samples. The method has thus been optimized for clinical studies. First, the Proteominer™ treatment has been miniaturized to be compatible with the low CSF volume typically obtained after lumbar puncture. We could show that the treatment is still efficient with this miniaturized protocol, and that the dynamic range of protein concentration is actually reduced even with small amounts of beads, leading to an increase of more than 80% of the number of identified proteins in one LC-MS/MS run. Moreover, the reproducibility required for protein quantification has been checked for this new protocol in replicate experiments. Finally, a labelfree quantitative proteomic strategy dedicated to the analysis of large series of samples has been developed. For such studies, fractionation of the samples is generally not possible, but the analysis in one run of a complex sample, even after Proteominer™ treatment, limits the number of identified and quantified proteins. In order to increase this number, we implemented an approach based on the comparison of MS signals in individual runs with a previously generated MS/MS identification database containing m/z and retention time values associated with peptide sequences. The MFPaQ software was used to assign nonsequenced peptides MS signals to the identification database and to quantify them. The combination of ProteominerTM sample treatment with the bioinformatics workflow developed for data quantification allowed us to increase by a factor 3.4 the number of proteins identified and quantified in the same CSF sample. This work opens the way to future studies aiming at discovering biomarkers in CSF among the low abundance proteins.

Good and bad opposites

The goal of this paper is to combine corpus methodology with experimentalmethods to gain insights into the nature of antonymy as a lexico-semantic relationand the degree of antonymic canonicity of word pairs in language and inmemory. Two approaches to antonymy in language are contrasted, the lexicalcategorical model and the cognitive prototype model. The results of the investigationsupport the latter model and show that different pairings have differentlevels of lexico-semantic affinity. At this general level of categorization, empiricalmethods converge; however, since they measure slightly different aspect of lexico-semantic opposability and affinity, and since the techniques of investigationare different in nature, we obtain slightly conflicting results at the more specificlevels. We conclude that some antonym pairs can be diagnosed as “canonical”on the strength of three indicators: textual co-occurrence, individual judgementabout “goodness” of opposition, and elicitation evidence

Sustainability of food, energy and environment with biofuels.

Growing reliance on food-based biofuels has created considerable controversy about its impact on food prices and the environment and led to scepticism about its sustainability. This review describes the concept of sustainability in the context of biofuels and then discusses the factors affecting the economic viability of current and next-generation biofuels and their environmental and social sustainability. Cellulosic biofuels from dedicated energy crops offer considerable promise for reducing the competition for land and avoiding many of the negative environmental impacts associated with corn-ethanol. But the production of any type of biofuel is likely to involve trade-offs among the multi-dimensional aspects of sustainability. Technological innovation and policy incentives are needed to develop more sustainable biofuels and to guide the mix of feedstocks, their methods and locations of production.

Cutting a Pie Is Not a Piece of Cake

Is there a division among n players of a cake using n-1 parallel vertical cuts, or of a pie using n radial cuts, that is envy-free (each player thinks he or she receives a largest piece and so does not envy another player) and undominated (there is no other allocation as good for all players and better for at least one)? David Gale first asked this question for pies. We provide complete answers for both cakes and pies. The answers depend on the number of players (two versus three or more players) and whether the players' preferences satisfy certain continuity assumptions. We also give some simple algorithms for cutting a pie when there are two or more players, but these algorithms do not guarantee all the properties one might desire in a division, which makes pie-cutting harder than cake-cutting. We suggest possible applications and conclude with two open questions.

Effects of Fuel Composition on High-Pressure Non-Premixed Natural Gas Combustion

Tectonic underplating of trench sediments beneath magmatic arcs: the central California example

We summarize the post Late Cretaceous regional tectonic evolution of the central California Coast Ranges, west of the San Andreas fault system. The Monterey terrane of North American origin was laterally transferred to the Pacific plate via the San Andreas fault. The Monterey terrane is an assembly of three tectonic units, Salinia, Nacimiento and Sierra de Salinas blocks, two of which have been previously identified as separate terranes. These blocks are separated by two regionally important thrust faults: the Sur fault as well as the Salinas shear zone. Based on thermobarometric and thermochronologic constraints and the existence of a common younger cover sequence, these blocks were juxtaposed together after the latest Cretaceous. The Salinian assemblage represents a crustal section through the continental interior side of the Mesozoic California arc and formed during the Late Cretaceous, primarily during a regionally significant magmatic flare‐up between 95 and 80 Ma. In the Santa Lucia Range, parts of the arc are exposed to palaeo‐depths in excess of 30 km. The Nacimiento and Sierra de Salinas assemblages comprise basement rocks representing Late Cretaceous variants of the Franciscan Complex and are interpreted to be correlative. They represent the lower plate of a regionally important thrust system; the upper plate is the Salinian assemblage, whereas the Sur and Salinas faults are local exposures of the structure. We concur with previous estimates of 150 to 180 km of shortening during a brief time span (3 cm/yr. This fault system corresponds to the megathrust of the Farallon subduction beneath North America during the early stages of the regionally extensive episode of shallow subduction (Laramide orogeny). As a result, trench sediment was thrust under North America and tectonically underplated to the lower crust of North America. The Salinas shear zone, in particular, is a ductile expression of shallow subduction; thermobarometry in the upper plate, lower plate and the shear zone itself indicate that this is the fossil subduction megathrust originating at depths of ∼35 km. The entire system collapsed extensionally soon after the trench sediment was underthrust, possibly because of the lack of strength of the lower plate. Arc magmatism in the upper plate ceased at the onset of underplating. This regional example illustrates the significance of tectonic underplating in shallow subduction systems. Accretion‐related trench sediment was shuffled from the trench to the sub‐arc region of the upper plate, but not recycled into the mantle. This process requires that the subduction megathrust be located solely within the North American crust. This geometry requires a sudden migration of the subduction interface toward the arc and may apply to other regional examples, including the modern shallow subduction of the Cocos plate beneath southern Mexico. The tectonically underplated trench sediment undergoes regional, Barrovian metamorphism, after initially following a high‐pressure/low‐temperature path. Moreover, the shear zone marking the fossil intracrustal megathrust was subject to granulite‐facies metamorphism and limited partial melting.

Conditions for triangular decoupling control

The main purpose of this article is to explore the relationship of two existing conditions for the triangular decoupling problem. The first one is the triangular-diagonal-dominance condition proposed by Hung and Anderson. The second one is the stable coprime factorisation-described condition proposed by Gomez and Goodwin, which has been proven as a necessary and sufficient condition for the triangular decoupling problem. This article proves that the two conditions are actually equivalent. It also provides easy-to-use criteria for assessment of the solvability of the triangular decoupling problem.

Origin of substituent effects in edge-to-face aryl–aryl interactions

Substituent effects in the edge-to-face configuration of the benzene dimer have been studied using modern density functional theory. An accurate interaction potential energy curve has been computed for the unsubstituted dimer using ab initio methods with large basis sets. The recommended binding energy for the edge-to-face benzene dimer is 2.31 kcal mol(-1), estimated at the counterpoise-corrected CCSD(T)/aug-cc-pVTZ level of theory. For both edge-ring and face-ring-substituted dimers, interaction energies correlate with sigma(m) for the substituents, indicating that substituent effects can be understood qualitatively in terms of simple electrostatic effects, although in the latter case dispersion results in some scatter in the data. In contrast to prevailing models of substituent effects in benzene dimers, polarization of the pi-system of the substituted ring does not induce substituent effects. For edge-ring-substituted dimers, substituent effects arise from differential electrostatic interactions between the hydrogens on the substituted ring and the pi-cloud of the face ring and direct interactions of the substituents with the unsubstituted ring. For face-ring-substituted dimers, substituent effects arise from direct electrostatic and dispersion interactions of the substituents with the edge ring. Substituents with sigma(m) > 0.12 favor edge ring substitution while for sigma(m) < 0.12 substitution on the face ring is preferred.

In Memoriam: Steven Alan Pullman (1947–2009)