Proteogenomic Analysis of Human Colon Cancer Reveals New Therapeutic Opportunities.

We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.

Reimagining infrastructure for a biodiverse future.

Civil infrastructure will be essential to face the interlinked existential threats of climate change and rising resource demands while ensuring a livable Anthropocene for all. However, conventional infrastructure planning largely neglects the contributions and maintenance of Earth's ecological life support systems, which provide irreplaceable services supporting human well-being. The stability and performance of these services depend on biodiversity, but conventional infrastructure practices, narrowly focused on controlling natural capital, have inadvertently degraded biodiversity while perpetuating social inequities. Here, we envision a new infrastructure paradigm wherein biodiversity and ecosystem services are a central objective of civil engineering. In particular, we reimagine infrastructure practice such that 1) ecosystem integrity and species conservation are explicit objectives from the outset of project planning; 2) infrastructure practices integrate biodiversity into diverse project portfolios along a spectrum from conventional to nature-based solutions and natural habitats; 3) ecosystem functions reinforce and enhance the performance and lifespan of infrastructure assets; and 4) civil engineering promotes environmental justice by counteracting legacies of social inequity in infrastructure development and nature conservation. This vision calls for a fundamental rethinking of the standards, practices, and mission of infrastructure development agencies and a broadening of scope for conservation science. We critically examine the legal and professional precedents for this paradigm shift, as well as the moral and economic imperatives for manifesting equitable infrastructure planning that mainstreams biodiversity and nature's benefits to people. Finally, we set an applied research agenda for supporting this vision and highlight financial, professional, and policy pathways for achieving it.

Revisiting the Preparation and Catalytic Performance of a Phosphine-Modified Co(II) Hydroformylation Precatalyst.

In light of recent conflicting reports regarding the hydroformylation catalytic activity derived from cationic Co(II) precatalysts of the form [Co(acac)(bis(phosphine))]BF4, the synthetic procedures and characterization of [Co(acac)(dppBz)]BF4, 1, are evaluated. Leveraging calibrated ESI-TOF MS methodologies, substantial quantities of Co(acac)2(dppBz), 2, were observed within samples of 1. The source of the impurity, 2, is determined to derive from incomplete protonolysis of the Co(acac)2 precursor and ligand scrambling occurring during the synthesis of 1. Revised synthetic procedures using lower temperature conditions and longer reaction times afford analytically pure samples of 1 based on ESI-TOF MS and NMR spectroscopic analysis. Complex 1 is demonstrated to act as a hydroformylation precatalyst for the conversion of 1-hexene to 1-heptanal under relatively mild conditions at 51.7 bar and 140 °C. The presence of impurity 2 is shown to dramatically decrease the catalytic performance derived from 1.

A Galaxy of informatics resources for MS-based proteomics.

INTRODUCTION: Continuous advances in mass spectrometry (MS) technologies have enabled deeper and more reproducible proteome characterization and a better understanding of biological systems when integrated with other 'omics data. Bioinformatic resources meeting the analysis requirements of increasingly complex MS-based proteomic data and associated multi-omic data are critically needed. These requirements included availability of software that would span diverse types of analyses, scalability for large-scale, compute-intensive applications, and mechanisms to ease adoption of the software. AREAS COVERED: The Galaxy ecosystem meets these requirements by offering a multitude of open-source tools for MS-based proteomics analyses and applications, all in an adaptable, scalable, and accessible computing environment. A thriving global community maintains these software and associated training resources to empower researcher-driven analyses. EXPERT OPINION: The community-supported Galaxy ecosystem remains a crucial contributor to basic biological and clinical studies using MS-based proteomics. In addition to the current status of Galaxy-based resources, we describe ongoing developments for meeting emerging challenges in MS-based proteomic informatics. We hope this review will catalyze increased use of Galaxy by researchers employing MS-based proteomics and inspire software developers to join the community and implement new tools, workflows, and associated training content that will add further value to this already rich ecosystem.

The Role of Anionic Ligands on Photoreactivity in Mo(VI) Dioxo Complexes of the Form MoO2 X2 (NN).

The photoreactivity of d0 metal dioxo complexes in activating C-H bonds has been recently studied.[1-3] We have previously reported that MoO2 Cl2 (bpy-t Bu) is an effective platform for light initiated C-H activation with unique product selectivity for the overall functionalization.[1] Herein we expand on these studies and report the synthesis and photoreactivity of several new Mo(VI) dioxo complexes with the general formula MoO2 (X)2 (NN); where X=F- , Cl- , Br- , CH3 - , PhO- , t BuO- and NN=2,2'-bipyridine (bpy) or 4,4'-tert-butyl-2,2'bipyridine (bpy-t Bu). Among these compounds, MoO2 Cl2 (bpy-t Bu) and MoO2 Br2 (bpy-t Bu) are able to participate in bimolecular photoreactivity with several substrates containing C-H bonds of various types such as allyls, benzyls, aldehydes (RCHO) and alkanes. MoO2 (CH3 )2 bpy and MoO2 (PhO)2 bpy do not participate in bimolecular photoreactions and instead they undergo photodecompositions. Computational studies indicate that the nature of the HOMO and LUMO is critical in supporting photoreactivity, with access to an LMCT (bpy→Mo) being necessary for tractable hydrocarbon functionalization.

Light-Initiated C-H Activation via Net Hydrogen Atom Transfer to a Molybdenum(VI) Dioxo.

MoO2Cl2(bpy-tBu) (1) is shown to be a potent one-electron oxidant upon irradiation with 365 nm light in various solvents, while being a weak two-electron oxidant in the dark. Complex 1 is characterized to activate various types of C-H bonds photochemically, including allylic and benzylic positions as well as alkanes and aldehydes. In all of these oxidations, 1 ultimately forms a bimetallic Mo(V)/Mo(V) species with a µ-oxo ligand (2). Depending on the substrate, the major organic product is identified as either an oxygenated or a C-C coupled (homodimerized) compound along with a minor chlorinated species. The product selectivity is proposed to be dependent upon the relative values between the bond dissociation enthalpy (BDE) of a potentially new C-OH bond within the product versus the BDE of a Mo-OH motif within a Mo(V)O(OH) intermediate. Based on this, we can estimate the BDE for Mo-OH to be 83-93 kcal/mol. Mechanistic studies suggest that the C-H activation occurs via a net hydrogen atom transfer (HAT) from 1* occurring either asynchronously or via a stepwise electron-proton transfer (ET-PT) process. Complex 2 is further demonstrated to reform dioxo 1 in the presence of chemical oxidants.

In Situ and Ex Situ X-ray Diffraction and Small-Angle X-ray Scattering Investigations of the Sol-Gel Synthesis of Fe3N and Fe3C.

Iron nitride (Fe3N) and iron carbide (Fe3C) nanoparticles can be prepared via sol-gel synthesis. While sol-gel methods are simple, it can be difficult to control the crystalline composition, i.e., to achieve a Rietveld-pure product. In a previous in situ synchrotron study of the sol-gel synthesis of Fe3N/Fe3C, we showed that the reaction proceeds as follows: Fe3O4 → FeOx → Fe3N → Fe3C. There was considerable overlap between the different phases, but we were unable to ascertain whether this was due to the experimental setup (side-on heating of a quartz capillary which could lead to thermal gradients) or whether individual particle reactions proceed at different rates. In this paper, we use in situ wide- and small-angle X-ray scattering (wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS)) to demonstrate that the overlapping phases are indeed due to variable reaction rates. While the initial oxide nanoparticles have a small range of diameters, the size range expands considerably and very rapidly during the oxide-nitride transition. This has implications for the isolation of Rietveld-pure Fe3N, and in an extensive laboratory study, we were indeed unable to isolate phase-pure Fe3N. However, we made the surprising discovery that Rietveld-pure Fe3C nanoparticles can be produced at 500 °C with a sufficient furnace dwell time. This is considerably lower than the previous reports of the sol-gel synthesis of Fe3C nanoparticles.

Evolution of the Local Structure in the Sol-Gel Synthesis of Fe3C Nanostructures.

The sol-gel synthesis of iron carbide (Fe3C) nanoparticles proceeds through multiple intermediate crystalline phases, including iron oxide (FeOx) and iron nitride (Fe3N). The control of particle size is challenging, and most methods produce polydisperse Fe3C nanoparticles of 20-100 nm in diameter. Given the wide range of applications of Fe3C nanoparticles, it is essential that we understand the evolution of the system during the synthesis. Here, we report an in situ synchrotron total scattering study of the formation of Fe3C from gelatin and iron nitrate sol-gel precursors. A pair distribution function analysis reveals a dramatic increase in local ordering between 300 and 350 °C, indicating rapid nucleation and growth of iron oxide nanoparticles. The oxide intermediate remains stable until the emergence of Fe3N at 600 °C. Structural refinement of the high-temperature data revealed local distortion of the NFe6 octahedra, resulting in a change in the twist angle suggestive of a carbonitride intermediate. This work demonstrates the importance of intermediate phases in controlling the particle size of a sol-gel product. It is also, to the best of our knowledge, the first example of in situ total scattering analysis of a sol-gel system.

Proteogenomic characterization of human colon and rectal cancer.

Extensive genomic characterization of human cancers presents the problem of inference from genomic abnormalities to cancer phenotypes. To address this problem, we analysed proteomes of colon and rectal tumours characterized previously by The Cancer Genome Atlas (TCGA) and perform integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. Messenger RNA transcript abundance did not reliably predict protein abundance differences between tumours. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA 'microsatellite instability/CpG island methylation phenotype' transcriptomic subtype, but had distinct mutation, methylation and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates, including HNF4A (hepatocyte nuclear factor 4, alpha), TOMM34 (translocase of outer mitochondrial membrane 34) and SRC (SRC proto-oncogene, non-receptor tyrosine kinase). Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology.

Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity.

Alternative splicing dramatically increases transcriptome complexity but its contribution to proteome diversity remains controversial. Exon-exon junction spanning peptides provide direct evidence for the translation of specific splice isoforms and are critical for delineating protein isoform complexity. Here we found that junction-spanning peptides are underrepresented in publicly available mass spectrometry-based shotgun proteomics data sets. Further analysis showed that evolutionarily conserved preferential nucleotide usage at exon boundaries increases the occurrence of lysine- and arginine-coding triplets at the end of exons. Because both lysine and arginine residues are cleavage sites of trypsin, the nearly exclusive use of trypsin as the protein digestion enzyme in shotgun proteomic analyses hinders the detection of junction-spanning peptides. To study the impact of enzyme selection on splice junction detectability, we performed in-silico digestion of the human proteome using six proteases. The six enzymes created a total of 161,125 detectable junctions, and only 1,029 were common across all enzyme digestions. Chymotrypsin digestion provided the largest number of detectable junctions. Our experimental results further showed that combination of a chymotrypsin-based human proteome analysis with a trypsin-based analysis increased detection of junction-spanning peptides by 37% over the trypsin-only analysis and identified over a thousand junctions that were undetectable in fully tryptic digests. Our study demonstrates that detection of proteome diversity resulted from alternative splicing is limited by trypsin cleavage specificity, and that complementary digestion schemes will be essential to comprehensively analyze the translation of alternative splicing isoforms.

Probing Zika Virus Neutralization Determinants with Glycoprotein Mutants Bearing Linear Epitope Insertions.

Zika virus (ZIKV) glycoproteins are the primary target of the humoral immune response. In this study, we explored the capacity of these glycoproteins to tolerate insertion of linear epitope sequences and the potential of antibodies that bind these epitopes to inhibit infection. We first created a panel of ZIKV mutants with the FLAG epitope inserted in the premembrane (prM) and envelope (E) glycoprotein regions. The insertion locations were based on the results of our recent transposon insertional mutagenesis screen. Although FLAG insertions in prM greatly impaired viral fitness, this sequence was tolerated in numerous surface-exposed E protein sites. We observed that mutants bearing FLAG epitopes in E domains I and II and the E domain I-II hinge region were all neutralized by FLAG antibody; however, the neutralization sensitivity varied highly. We measured the antibody binding efficiency and found that this closely matched the pattern of neutralization sensitivity. We determined that E glycosylation did not affect antibody binding to a nearby epitope or its capacity to serve as a neutralization target. Although we could not generate infectious viruses with FLAG epitope insertions in a buried region of E protein domain III, we found that the V5 epitope could be inserted at this site without greatly impacting fitness. Furthermore, this virus was efficiently neutralized by V5 antibodies, highlighting that even buried epitopes can function as neutralization targets. Finally, we analyzed the timing of antibody neutralization activity during cell entry and found that all antibodies blocked a step after cell attachment.IMPORTANCE Zika virus (ZIKV) infections are associated with severe birth defects and neurological disease. The structure of the mature ZIKV particle reveals a virion surface covered by the envelope glycoprotein, which is the dominant target of the humoral immune response. It is unclear if all regions of the envelope protein surface or even buried epitopes can function as neutralization targets. To test this, we created a panel of ZIKV mutants with epitope insertions in different regions of the envelope protein. In characterizing these viruses, we found that the strength of antibody binding to an epitope is the major determinant of the neutralization potential of an antibody, that even a buried region of the envelope protein can be efficiently targeted, and that the sole potential envelope glycan does not impact nearby epitope antibody binding and neutralization. Furthermore, this work provides important insights into our understanding of how antibodies neutralize ZIKV.

Average and Local Structure of Apatite-Type Germanates and Implications for Oxide Ion Conductivity.

Materials with the apatite structure have a range of important applications in which their function is influenced by details of their local structure. Here, we describe an average and local structural study to probe the origins of high-temperature oxide ion mobility in La10(GeO4)6O3 and La8Bi2(GeO4)6O3 oxygen-excess materials, using the low-conductivity interstitial oxide-free La8Sr2(GeO4)6O2 as a benchmark. For La10 and La8Bi2, we locate the interstitial oxygen, Oint, responsible for conductivity by Rietveld refinement and relate the P63/m to P1̅ phase transitions on cooling to oxygen ordering. Local structural studies using neutron total scattering reveal that well-ordered GeO5 square pyramidal groups form in the structure at low temperature, but that Oint becomes significantly more disordered in the high-conductivity, high-temperature structures, with a transition to more trigonal-bipyramid-like average geometry. We relate the higher conductivity of Bi materials to the presence of several Oint sites of similar energy in the structure, which correlates with its less-distorted low-temperature average structure.

Colorectal Cancer Cell Line Proteomes Are Representative of Primary Tumors and Predict Drug Sensitivity.

BACKGROUND AND AIMS: Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. METHODS: Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. RESULTS: Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. CONCLUSIONS: Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.

Thermodynamic Hydricity of Transition Metal Hydrides.

Transition metal hydrides play a critical role in stoichiometric and catalytic transformations. Knowledge of free energies for cleaving metal hydride bonds enables the prediction of chemical reactivity, such as for the bond-forming and bond-breaking events that occur in a catalytic reaction. Thermodynamic hydricity is the free energy required to cleave an M-H bond to generate a hydride ion (H(-)). Three primary methods have been developed for hydricity determination: the hydride transfer method establishes hydride transfer equilibrium with a hydride donor/acceptor pair of known hydricity, the H2 heterolysis method involves measuring the equilibrium of heterolytic cleavage of H2 in the presence of a base, and the potential-pKa method considers stepwise transfer of a proton and two electrons to give a net hydride transfer. Using these methods, over 100 thermodynamic hydricity values for transition metal hydrides have been determined in acetonitrile or water. In acetonitrile, the hydricity of metal hydrides spans a range of more than 50 kcal/mol. Methods for using hydricity values to predict chemical reactivity are also discussed, including organic transformations, the reduction of CO2, and the production and oxidation of hydrogen.

proBAMsuite, a Bioinformatics Framework for Genome-Based Representation and Analysis of Proteomics Data.

To facilitate genome-based representation and analysis of proteomics data, we developed a new bioinformatics framework, proBAMsuite, in which a central component is the protein BAM (proBAM) file format for organizing peptide spectrum matches (PSMs)(1) within the context of the genome. proBAMsuite also includes two R packages, proBAMr and proBAMtools, for generating and analyzing proBAM files, respectively. Applying proBAMsuite to three recently published proteomics datasets, we demonstrated its utility in facilitating efficient genome-based sharing, interpretation, and integration of proteomics data. First, the interpretation of proteomics data is significantly enhanced with the rich genomic annotation information. Second, PSMs can be easily reannotated using user-specified gene annotation schemes and assembled into both protein and gene identifications. Third, using the genome as a common reference, proBAMsuite facilitates seamless proteomics and proteogenomics data integration. Finally, proBAM files can be readily visualized in genome browsers and thus bring proteomics data analysis to a general audience beyond the proteomics community. Results from this study establish proBAMsuite as a useful bioinformatics framework for proteomics and proteogenomics research.

Molecular polypyridine-based metal complexes as catalysts for the reduction of CO2.

Polypyridyl transition metal complexes represent one of the more thoroughly studied classes of molecular catalysts towards CO2 reduction to date. Initial reports in the 1980s began with an emphasis on 2nd and 3rd row late transition metals, but more recently the focus has shifted towards earlier metals and base metals. Polypyridyl platforms have proven quite versatile and amenable to studying various parameters that govern product distribution for CO2 reduction. However, open questions remain regarding the key mechanistic steps that govern product selectivity and efficiency. Polypyridyl complexes have also been immobilized through a variety of methods to afford active catalytic materials for CO2 reductions. While still an emerging field, materials incorporating molecular catalysts represent a promising strategy for electrochemical and photoelectrochemical devices capable of CO2 reduction. In general, this class of compounds remains the most promising for the continued development of molecular systems for CO2 reduction and an inspiration for the design of related non-polypyridyl catalysts.

Maximizing the Photocatalytic Activity of Metal-Organic Frameworks with Aminated-Functionalized Linkers: Substoichiometric Effects in MIL-125-NH2.

Despite the promise of utilizing metal-organic frameworks (MOFs) as highly tunable photocatalytic materials, systematic studies that interrogate the relationship between their catalytic performances and the amount of functionalized linkers are lacking. Aminated linkers are known to enhance the absorption of light and afford photocatalysis with MOFs under visible-light irradiation. However, the manner in which the photocatalytic performances are impacted by the amount of such linkers is poorly understood. Here, we assess the photocatalytic activity of MIL-125, a TiO2/1,4-benzenedicarboxylate (bdc) MOF for the oxidation of benzyl alcohol to benzaldehyde when increasing amounts of bdc-NH2 linkers (0%, 20%, 46%, 70%, and 100%) are incorporated in the framework. Analytical TEM allowed assessing the homogeneous localization of bdc-NH2 in these mixed-linker MOFs. Steady state reaction rates reveal two regimes of catalytic performances: a first linear regime up to ∼50% bdc-NH2 into the hybrid framework whereby increased amounts of bdc-NH2 yielded increased photocatalytic rates, followed by a plateau up to 100% bdc-NH2. This unexpected "saturation" of the catalytic activity above ∼50% bdc-NH2 content in the framework whatever the wavelength filters used demonstrates that amination of all linkers of the MOF is not required to obtain the maximum photocatalytic activity. This is rationalized on the basis of mixed-valence Ti3+/Ti4+ intermediate catalytic centers revealed by electron spin resonance (ESR) measurements and recent knowledge of lifetime excited states in MIL-125-type of solids.

The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses.

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.

Role of pendant proton relays and proton-coupled electron transfer on the hydrogen evolution reaction by nickel hangman porphyrins.

The hangman motif provides mechanistic insights into the role of pendant proton relays in governing proton-coupled electron transfer (PCET) involved in the hydrogen evolution reaction (HER). We now show improved HER activity of Ni compared with Co hangman porphyrins. Cyclic voltammogram data and simulations, together with computational studies using density functional theory, implicate a shift in electrokinetic zone between Co and Ni hangman porphyrins due to a change in the PCET mechanism. Unlike the Co hangman porphyrin, the Ni hangman porphyrin does not require reduction to the formally metal(0) species before protonation by weak acids in acetonitrile. We conclude that protonation likely occurs at the Ni(I) state followed by reduction, in a stepwise proton transfer-electron transfer pathway. Spectroelectrochemical and computational studies reveal that upon reduction of the Ni(II) compound, the first electron is transferred to a metal-based orbital, whereas the second electron is transferred to a molecular orbital on the porphyrin ring.

Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts.

The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm ). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.