Publications by authors named "Michael Cusack"

26 Publications

  • Page 1 of 1

Aeolian Prokaryotic Communities of the Global Dust Belt Over the Red Sea.

Front Microbiol 2020 12;11:538476. Epub 2020 Nov 12.

Red Sea Research Center and Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.

Aeolian prokaryotic communities (APC) are important components of bioaerosols that are transported freely or attached to dust particles suspended in the atmosphere. Terrestrial and marine ecosystems are known to release and receive significant prokaryote loads into and from the surrounded atmospheric air. However, compared to terrestrial systems, there is a lack of microbial characterization of atmospheric dust over marine systems, such as the Red Sea, which receives significant terrestrial dust loads and is centrally located within the Global Dust Belt. Prokaryotic communities are likely to be particularly important in the Global Dust Belt, the area between the west coast of North Africa and Central Asia that supports the highest dust fluxes on the planet. Here we characterize the diversity and richness of the APC over the Red Sea ecosystem, the only sea fully contained within the Global Dust Belt. MiSeq sequencing was used to target 16S ribosomal DNA of two hundred and forty aeolian dust samples. These samples were collected at ∼7.5 m high above the sea level at coastal and offshore sampling sites over a 2-year period (2015-2017). The sequencing outcomes revealed that the APC in the atmospheric dust is dominated by Proteobacteria (42.69%), Firmicutes (41.11%), Actinobacteria, (7.69%), and Bacteroidetes (3.49%). The dust-associated prokaryotes were transported from different geographical sources and found to be more diverse than prokaryotic communities of the Red Sea surface water. Marine and soil originated prokaryotes were detected in APC. Hence, depending on the season, these groups may have traveled from other distant sources during storm events in the Red Sea region, where the APC structure is influenced by the origin and the concentration of aeolian dust particles. Accordingly, further studies of the impact of atmospheric organic aerosols on the recipient environments are required.
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http://dx.doi.org/10.3389/fmicb.2020.538476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688470PMC
November 2020

Anthropogenic-induced acceleration of elemental burial rates in blue carbon repositories of the Arabian Gulf.

Sci Total Environ 2020 Jun 21;719:135177. Epub 2019 Nov 21.

Marine Studies Section, Center for Environment and Water, Research Institute, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia; Geosciences Department, College of Petroleum Engineering and Geosciences, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia; Deputy-Ministry for Environment, Ministry of Environment, Water and Agriculture, Riyadh, Saudi Arabia.

Along the past century, the Arabian Gulf has experienced a continuous and fast coastal development leading to increase the human pressures on the marine environment. The present study attempts to describe the historical changes of trace elements in the sediments of vegetated coastal habitats in the western Arabian Gulf. Pb-dated sediment cores collected from seagrass, mangrove and saltmarsh habitats were analyzed to evaluate historical variations in concentrations and burial rates of 20 trace elements (Al, As, Ba, Ca, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, P, Pb, S, Sr, V and Zn). The highest correlations (Spearman correlation coefficients ≥0.51) were found between crustal elements (Al, Fe, Co, Cr, K, Na, Mg, Mn, Ni, V, and P), suggesting a common crustal source in the Gulf. The increased concentrations of these crustal elements in modern marine sediments of the Arabian Gulf seem to be linked to increased mineral dust deposition in the area. Over the last century, both elemental concentrations and burial rates increased by factors of 1-9 and 1-15, respectively, with a remarkably fast increase occurring in the past six decades (~1960 - early 2000). This is most likely due to an increase in anthropogenic pressures along the Gulf coast. Our study demonstrates that sediments in vegetated coastal habitats provide long-term archives of trace elements concentrations and burial rates reflecting human activities in the Arabian Gulf.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135177DOI Listing
June 2020

Quantifying neurologic disease using biosensor measurements in-clinic and in free-living settings in multiple sclerosis.

NPJ Digit Med 2019 11;2:123. Epub 2019 Dec 11.

Verily Life Sciences, South San Francisco, CA USA.

Technological advances in passive digital phenotyping present the opportunity to quantify neurological diseases using new approaches that may complement clinical assessments. Here, we studied multiple sclerosis (MS) as a model neurological disease for investigating physiometric and environmental signals. The objective of this study was to assess the feasibility and correlation of wearable biosensors with traditional clinical measures of disability both in clinic and in free-living in MS patients. This is a single site observational cohort study conducted at an academic neurological center specializing in MS. A cohort of 25 MS patients with varying disability scores were recruited. Patients were monitored in clinic while wearing biosensors at nine body locations at three separate visits. Biosensor-derived features including aspects of gait (stance time, turn angle, mean turn velocity) and balance were collected, along with standardized disability scores assessed by a neurologist. Participants also wore up to three sensors on the wrist, ankle, and sternum for 8 weeks as they went about their daily lives. The primary outcomes were feasibility, adherence, as well as correlation of biosensor-derived metrics with traditional neurologist-assessed clinical measures of disability. We used machine-learning algorithms to extract multiple features of motion and dexterity and correlated these measures with more traditional measures of neurological disability, including the expanded disability status scale (EDSS) and the MS functional composite-4 (MSFC-4). In free-living, sleep measures were additionally collected. Twenty-three subjects completed the first two of three in-clinic study visits and the 8-week free-living biosensor period. Several biosensor-derived features significantly correlated with EDSS and MSFC-4 scores derived at visit two, including mobility stance time with MSFC-4 z-score (Spearman correlation -0.546;  = 0.0070), several aspects of turning including turn angle (0.437;  = 0.0372), and maximum angular velocity (0.653;  = 0.0007). Similar correlations were observed at subsequent clinic visits, and in the free-living setting. We also found other passively collected signals, including measures of sleep, that correlated with disease severity. These findings demonstrate the feasibility of applying passive biosensor measurement techniques to monitor disability in MS patients both in clinic and in the free-living setting.
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http://dx.doi.org/10.1038/s41746-019-0197-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906296PMC
December 2019

Functional metagenomic analysis of dust-associated microbiomes above the Red Sea.

Sci Rep 2019 09 24;9(1):13741. Epub 2019 Sep 24.

Red Sea Research Centre (RSRC) and Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology (KAUST), Thuwal, 23955, Saudi Arabia.

Atmospheric transport is a major vector for the long-range transport of microbial communities, maintaining connectivity among them and delivering functionally important microbes, such as pathogens. Though the taxonomic diversity of aeolian microorganisms is well characterized, the genomic functional traits underpinning their survival during atmospheric transport are poorly characterized. Here we use functional metagenomics of dust samples collected on the Global Dust Belt to initiate a Gene Catalogue of Aeolian Microbiome (GCAM) and explore microbial genetic traits enabling a successful aeolian lifestyle in Aeolian microbial communities. The GCAM reported here, derived from ten aeolian microbial metagenomes, includes a total of 2,370,956 non-redundant coding DNA sequences, corresponding to a yield of ~31 × 10 predicted genes per Tera base-pair of DNA sequenced for the aeolian samples sequenced. Two-thirds of the cataloged genes were assigned to bacteria, followed by eukaryotes (5.4%), archaea (1.1%), and viruses (0.69%). Genes encoding proteins involved in repairing UV-induced DNA damage and aerosolization of cells were ubiquitous across samples, and appear as fundamental requirements for the aeolian lifestyle, while genes coding for other important functions supporting the aeolian lifestyle (chemotaxis, aerotaxis, germination, thermal resistance, sporulation, and biofilm formation) varied among the communities sampled.
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http://dx.doi.org/10.1038/s41598-019-50194-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760216PMC
September 2019

Airborne Prokaryote and Virus Abundance Over the Red Sea.

Front Microbiol 2019 31;10:1112. Epub 2019 May 31.

Division of Biological and Environmental Science and Engineering, Red Sea Research Centre and Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.

Aeolian dust exerts a considerable influence on atmospheric and oceanic conditions negatively impacting human health, particularly in arid and semi-arid regions like Saudi Arabia. Aeolian dust is often characterized by its mineral and chemical composition; however, there is a microbiological component of natural aerosols that has received comparatively little attention. Moreover, the amount of materials suspended in the atmosphere is highly variable from day to day. Thus, understanding the variability of atmospheric dust loads and suspended microbes throughout the year is essential to clarify the possible effects of dust on the Red Sea ecosystem. Here, we present the first estimates of dust and microbial loads at a coastal site on the Red Sea over a 2-year period, supplemented with measurements from dust samples collected along the Red Sea basin in offshore waters. Weekly average dust loads from a coastal site on the Red Sea ranged from 4.6 to 646.11 μg m, while the abundance of airborne prokaryotic cells and viral-like particles (VLPs) ranged from 77,967 to 1,203,792 cells m and from 69,615 to 3,104,758 particles m, respectively. To the best of our knowledge, these are the first estimates of airborne microbial abundance in this region. The elevated concentrations of resuspended dust particles and suspended microbes found in the air indicate that airborne microbes may potentially have a large impact on human health and on the Red Sea ecosystem.
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http://dx.doi.org/10.3389/fmicb.2019.01112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554326PMC
May 2019

Accelerated burial of petroleum hydrocarbons in Arabian Gulf blue carbon repositories.

Sci Total Environ 2019 Jun 6;669:205-212. Epub 2019 Mar 6.

King Abdullah University of Science and Technology, Red Sea Research Centre, Thuwal 23955-6900, Saudi Arabia.

Massive consumption of petroleum since the past century has led to considerable emissions into marine ecosystems. Marine sediments may accumulate substantial quantities of petroleum and associated contaminants in oil-producing areas. Here, we report accelerated accumulation of total petroleum hydrocarbons (TPH) in 'blue carbon' vegetated ecosystems of the Arabian Gulf - the world's most important region for oil production. In addition to increased accumulation with the onset of oil exploitation, sediment records reflect a large depositional event associated with the 1991 Gulf War, with the magnitude of these maxima varying across habitats, depending on their elevation along the shoreline. Blue carbon ecosystems of the Arabian Gulf currently bury about 2300 megagrams (Mg) of TPHs annually and have accumulated TPH stocks of 59,799 Mg over the past 25 years alone. Massive burial and sequestration of TPH by blue carbon ecosystems is an important, but thus far unrecognized, removal mechanism in the Arabian Gulf. Conserving these ecosystems is important to avoid possible remobilization of sequestered TPH into the surrounding environment.
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http://dx.doi.org/10.1016/j.scitotenv.2019.01.437DOI Listing
June 2019

Single Usage of a Kitchen Degreaser Can Alter Indoor Aerosol Composition for Days.

Environ Sci Technol 2017 Jun 10;51(11):5907-5912. Epub 2017 May 10.

Institute of Chemical Process Fundamentals of the CAS , Prague CZ-165 02, Czech Republic.

To the best of our knowledge, this study represents the first observation of multiday persistence of an indoor aerosol transformation linked to a kitchen degreaser containing monoethanol amine (MEA). MEA remaining on the cleaned surfaces and on a wiping paper towel in a trash can was able to transform ammonium sulfate and ammonium nitrate into (MEA)SO and (MEA)NO. This influence persisted for at least 60 h despite a high average ventilation rate. The influence was observed using both offline (filters, impactors, and ion chromatography analysis) and online (compact time-of-flight aerosol mass spectrometer) techniques. Substitution of ammonia in ammonium salts was observed not only in aerosol but also in particles deposited on a filter before the release of MEA. The similar influence of other amines is expected based on literature data. This influence represents a new pathway for MEA exposure of people in an indoor environment. The stabilizing effect on indoor nitrate also causes higher indoor exposure to fine nitrates.
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http://dx.doi.org/10.1021/acs.est.6b06050DOI Listing
June 2017

Detailed comparison of OC/EC aerosol at an urban and a rural Czech background site during summer and winter.

Sci Total Environ 2015 Jun 13;518-519:424-33. Epub 2015 Mar 13.

Institute of Chemical Process Fundamentals of the CAS, v.v.i., Rozvojová 2/135, 165 02, Prague 6 - Suchdol, Czech Republic.

Winter and summer measurements of organic carbon and elemental carbon (OC and EC) in PM2.5 were performed in parallel at two sites, the rural background station Košetice and the Prague-Suchdol urban background site, with a 2-h time resolution using semi-online field OC/EC analysers. Seasonal and site differences were found in the OC and EC contents of PM2.5. Overall, the highest concentrations of both OC and EC were during winter at the urban site. The average urban impact was 50% for OC and 70% for EC. The summer season gives similar concentrations of OC at both sites. However, higher concentrations of EC, caused by higher traffic, were found at the urban site with an average urban increase of 50%. Moreover, an analysis of four OC fractions depending on the volatility (OC1 - most volatile, OC4 - least volatile) and pyrolytic carbon (PC) is provided. A similar level of each OC fraction at both sites was found in summer, except for higher OC1 at urban and higher PC at the rural site. In winter, the differences between the urban and rural sites were dominated by a large increase of the OC1 fraction in comparison with the rural site. A diurnal pattern of concentration and share of OC1 and PC suggests a prevailing influence of local sources on their concentrations at the urban site in winter. The OC3 and OC4 diurnal cycles suggest their more regional or long range transport origin in both seasons. The prevalent influence of OC1 at any urban site has not been previously reported. The minimisation of semi-volatile carbon losses during semi-continuous sampling and analysis, in comparison with off-line sampling methods, is a probable reason for the observed differences.
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http://dx.doi.org/10.1016/j.scitotenv.2015.03.029DOI Listing
June 2015

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.

Mol Cell Proteomics 2015 Sep 18;14(9):2357-74. Epub 2015 Feb 18.

Department of Biochemistry, Vanderbilt University School of Medicine, and the Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232;

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.
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http://dx.doi.org/10.1074/mcp.M114.047050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563721PMC
September 2015

Urban NH3 levels and sources in six major Spanish cities.

Chemosphere 2015 Jan 6;119:769-777. Epub 2014 Sep 6.

Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Barcelona, Spain.

A detailed spatial and temporal assessment of urban NH3 levels and potential emission sources was made with passive samplers in six major Spanish cities (Barcelona, Madrid, A Coruña, Huelva, Santa Cruz de Tenerife and Valencia). Measurements were conducted during two different periods (winter-autumn and spring-summer) in each city. Barcelona showed the clearest spatial pattern, with the highest concentrations in the old city centre, an area characterised by a high population density and a dense urban architecture. The variability in NH3 concentrations did not follow a common seasonal pattern across the different cities. The relationship of urban NH3 with SO2 and NOX allowed concluding on the causes responsible for the variations in NH3 levels between measurement periods observed in Barcelona, Huelva and Madrid. However, the factors governing the variations in A Coruña, Valencia and Santa Cruz de Tenerife are still not fully understood. This study identified a broad variability in NH3 concentrations at the city-scale, and it confirms that NH3 sources in Spanish urban environments are vehicular traffic, biological sources (e.g. garbage containers), wastewater treatment plants, solid waste treatment plants and industry. The importance of NH3 monitoring in urban environments relies on its role as a precursor of secondary inorganic species and therefore PMX. Further research should be addressed in order to establish criteria to develop and implement mitigation strategies for cities, and to include urban NH3 sources in the emission inventories.
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http://dx.doi.org/10.1016/j.chemosphere.2014.07.097DOI Listing
January 2015

Variation and quantification among a target set of phosphopeptides in human plasma by multiple reaction monitoring and SWATH-MS2 data-independent acquisition.

Electrophoresis 2014 Dec 10;35(24):3487-97. Epub 2014 Jul 10.

Buck Institute for Research on Aging, Novato, CA, USA.

Human plasma contains proteins that reflect overall health and represents a rich source of proteins for identifying and understanding disease pathophysiology. However, few studies have investigated changes in plasma phosphoproteins. In addition, little is known about the normal variations in these phosphoproteins, especially with respect to specific sites of modification. To address these questions, we evaluated variability in plasma protein phosphorylation in healthy individuals using multiple reaction monitoring (MRM) and SWATH-MS2 data-independent acquisition. First, we developed a discovery workflow for phosphopeptide enrichment from plasma and identified targets for MRM assays. Next, we analyzed plasma from healthy donors using an analytical workflow consisting of MRM and SWATH-MS2 that targeted phosphopeptides from 58 and 68 phosphoproteins, respectively. These two methods produced similar results showing low variability in 13 phosphosites from 10 phosphoproteins (CVinter < 30%) and high interpersonal variation of 16 phosphosites from 14 phosphoproteins (CVinter > 30%). Moreover, these phosphopeptides originate from phosphoproteins involved in cellular processes governing homeostasis, immune response, cell-extracellular matrix interactions, lipid and sugar metabolism, and cell signaling. This limited assessment of technical and biological variability in phosphopeptides generated from plasma phosphoproteins among healthy volunteers constitutes a reference for future studies that target protein phosphorylation as biomarkers.
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http://dx.doi.org/10.1002/elps.201400167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565165PMC
December 2014

Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma.

Mol Cell Proteomics 2014 Apr 6;13(4):1034-49. Epub 2014 Feb 6.

Buck Institute for Research on Aging, 8001 Redwood Blvd., Novato, California 94945;

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.
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http://dx.doi.org/10.1074/mcp.M113.035485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977182PMC
April 2014

Neural network model for the prediction of PM10 daily concentrations in two sites in the Western Mediterranean.

Sci Total Environ 2013 Oct 17;463-464:875-83. Epub 2013 Jul 17.

Department of Chemistry University of Bari, via Orabona 4, 70126 Bari, Italy. Electronic address:

An artificial neural network (ANN) was developed and tested to forecast PM10 daily concentration in two contrasted environments in NE Spain, a regional background site (Montseny), and an urban background site (Barcelona-CSIC), which was highly influenced by vehicular emissions. In order to predict 24-h average PM10 concentrations, the artificial neural network previously developed by Caselli et al. (2009) was improved by using hourly PM concentrations and deterministic factors such as a Saharan dust alert. In particular, the model input data for prediction were the hourly PM10 concentrations 1-day in advance, local meteorological data and information about air masses origin. The forecasted performance indexes for both sites were calculated and they showed better results for the regional background site in Montseny (R(2)=0.86, SI=0.75) than for urban site in Barcelona (R(2)=0.73, SI=0.58), influenced by local and sometimes unexpected sources. Moreover, a sensitivity analysis conducted to understand the importance of the different variables included among the input data, showed that local meteorology and air masses origin are key factors in the model forecasts. This result explains the reason for the improvement of ANN's forecasting performance at the Montseny site with respect to the Barcelona site. Moreover, the artificial neural network developed in this work could prove useful to predict PM10 concentrations, especially, at regional background sites such as those on the Mediterranean Basin which are primarily affected by long-range transports. Hence, the artificial neural network presented here could be a powerful tool for obtaining real time information on air quality status and could aid stakeholders in their development of cost-effective control strategies.
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http://dx.doi.org/10.1016/j.scitotenv.2013.06.093DOI Listing
October 2013

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

Mol Cell Proteomics 2013 Sep 20;12(9):2623-39. Epub 2013 May 20.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.
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http://dx.doi.org/10.1074/mcp.M112.027078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769335PMC
September 2013

Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways.

Proc Natl Acad Sci U S A 2013 Apr 1;110(16):6601-6. Epub 2013 Apr 1.

Buck Institute for Research on Aging, Novato, CA 94945, USA.

Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD(+)-dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis.
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http://dx.doi.org/10.1073/pnas.1302961110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631688PMC
April 2013

Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.

Mol Cell Proteomics 2012 May 26;11(5):202-14. Epub 2012 Mar 26.

Buck Institute for Research on Aging, Novato, California 94945, USA.

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
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http://dx.doi.org/10.1074/mcp.M112.017707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418851PMC
May 2012

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers.

J Proteome Res 2012 Apr 13;11(4):2508-20. Epub 2012 Mar 13.

Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, 513 Parnassus Avenue, Box 0665, San Francisco, California 94143, United States.

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.
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http://dx.doi.org/10.1021/pr201206wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383053PMC
April 2012

Intense winter atmospheric pollution episodes affecting the Western Mediterranean.

Sci Total Environ 2010 Mar 18;408(8):1951-9. Epub 2010 Feb 18.

Institute of Environmental Assessment and Water Research (IDAEA), Department of Geosciences CSIC, LLuis Solé i Sabarís S/N, Barcelona, Spain.

The geographic location of the Western Mediterranean Basin and its peculiar topography, the climatic conditions and the intense anthropogenic and natural emissions of atmospheric pollutants are key factors necessary to interpret the atmospheric aerosol phenomenology over this area. During the cold season it is common to have severe atmospheric particulate matter (PM) pollution episodes (of an anthropogenic origin) affecting this region, not only in the urban and industrial areas but also in the regional and rural sites. During these episodes, the midday hourly PM(1) levels at regional background sites are in many cases higher than those at urban areas. Around 10% of the days under winter anticyclonic conditions registered similar PM(1) levels at the regional background than at the urban area and, sporadically the daily PM(1) levels at the regional background sites may exceed those at urban sites. Furthermore, the very high hourly PM(1) levels measured at regional background sites during these episodes are not regularly attained in the closest urban areas, which leads to the hypothesis that an important formation of secondary aerosols occurs during the transport of the polluted air masses towards the elevated rural sites. The interpretation of the variability of PM levels and composition (2002-2008) at one urban site (Barcelona) and at one regional background site (Montseny) allows us to illustrate the phenomenology of these scenarios, to quantify the mean annual contributions to the PM levels and to identify their main tracers. Ammonium nitrate appears to be the most abundant compound during these scenarios, although organic species and trace metals also increase markedly. Owing to the intensity, composition and recurrence of these atmospheric pollution episodes, important health, climatic and ecological implications may be derived.
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http://dx.doi.org/10.1016/j.scitotenv.2010.01.052DOI Listing
March 2010

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.

Nat Biotechnol 2009 Jul 28;27(7):633-41. Epub 2009 Jun 28.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.
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http://dx.doi.org/10.1038/nbt.1546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855883PMC
July 2009

Efficient identification of critical residues based only on protein structure by network analysis.

PLoS One 2007 May 9;2(5):e421. Epub 2007 May 9.

Buck Institute for Age Research, Novato, California, United States of America.

Despite the increasing number of published protein structures, and the fact that each protein's function relies on its three-dimensional structure, there is limited access to automatic programs used for the identification of critical residues from the protein structure, compared with those based on protein sequence. Here we present a new algorithm based on network analysis applied exclusively on protein structures to identify critical residues. Our results show that this method identifies critical residues for protein function with high reliability and improves automatic sequence-based approaches and previous network-based approaches. The reliability of the method depends on the conformational diversity screened for the protein of interest. We have designed a web site to give access to this software at http://bis.ifc.unam.mx/jamming/. In summary, a new method is presented that relates critical residues for protein function with the most traversed residues in networks derived from protein structures. A unique feature of the method is the inclusion of the conformational diversity of proteins in the prediction, thus reproducing a basic feature of the structure/function relationship of proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000421PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855080PMC
May 2007

Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity.

J Biol Chem 2006 Aug 16;281(33):23686-97. Epub 2006 Jun 16.

The Buck Institute for Age Research, Novato, California 94945, USA.

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.
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http://dx.doi.org/10.1074/jbc.M513507200DOI Listing
August 2006

Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria.

Biochim Biophys Acta 2006 Feb 3;1762(2):213-22. Epub 2005 Aug 3.

Buck Institute for Age Research, Novato, CA 94945, USA.

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.
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http://dx.doi.org/10.1016/j.bbadis.2005.07.003DOI Listing
February 2006

Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line.

Mol Cell Proteomics 2005 Jan 10;4(1):84-96. Epub 2004 Dec 10.

Buck Institute for Age Research, Novato, CA 94945, USA.

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.
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http://dx.doi.org/10.1074/mcp.M400143-MCP200DOI Listing
January 2005

Exercise-induced ischemia initiates the second window of protection in humans independent of collateral recruitment.

J Am Coll Cardiol 2003 Apr;41(7):1174-82

Department of Cardiology, King's College London, The Rayne Institute, St. Thomas' Hospital, London, United Kingdom.

Objectives: This study was designed to examine if exercise-induced ischemia initiated late preconditioning in humans that becomes manifest during subsequent exercise and serial balloon occlusion of the left anterior descending coronary artery (LAD).

Background: The existence of late preconditioning in humans is controversial. We therefore compared myocardial responses to exercise-induced and intracoronary balloon inflation-induced ischemia in two groups of patients subjected to different temporal patterns of ischemia.

Methods: Thirty patients with stable angina secondary to single-vessel LAD disease underwent percutaneous coronary intervention (PCI) after two separate exercise tolerance test (ETT) protocols designed to investigate isolated early preconditioning (IEP) alone or the second window of protection (SWOP). The IEP subjects underwent three sequential ETTs at least two weeks before PCI. The SWOP subjects underwent five sequential ETTs commencing 24 h before PCI.

Results: During PCI there was no significant difference in intracoronary pressure-derived collateral flow index (CFI) between groups (IEP = 0.15 +/- 0.13, SWOP = 0.19 +/- 0.15). In SWOP patients, compared with the initial ETT, the ETT performed 24 h later had a 40% (p < 0.001) increase in time to 0.1-mV ST depression and a 60% (p < 0.05) decrease in ventricular ectopic frequency. During the first balloon inflation, peak ST elevation was reduced by 49% (p < 0.05) in the SWOP versus the IEP group, and the dependence on CFI observed in the IEP group was abolished (analysis of covariance, p < 0.05). The significant attenuation of ST elevation (47%, p < 0.005) seen at the time of the second inflation in the IEP patients was not seen in the SWOP patients.

Conclusions: Exercise-induced ischemia triggers late preconditioning in humans, which becomes manifest during exercise and PCI. This is the first evidence that ischemia induced by coronary occlusion is attenuated in humans by a late preconditioning effect induced by exercise.
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http://dx.doi.org/10.1016/s0735-1097(03)00055-xDOI Listing
April 2003

Systemic inflammation in unstable angina is the result of myocardial necrosis.

J Am Coll Cardiol 2002 Jun;39(12):1917-23

Department of Cardiology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom.

Objectives: We investigated whether the source of the acute phase response in unstable angina (UA) lay within the culprit coronary plaque or distal myocardium.

Background: An inflammatory response is an important component of the acute coronary syndromes. However, its origin and mechanism remain unclear.

Methods: In 94 stable patients undergoing coronary angiography, the relationship between systemic levels of tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6) and C-reactive protein (CRP) and extent of atherosclerosis was studied. The temporal relationship between these markers and troponin T (TnT) was determined in 91 patients with UA. Cytokine levels were measured in the aortic root and coronary sinus of 36 unstable patients.

Results: There was no relationship found between stable coronary atherosclerosis and inflammatory marker levels. Compared with this group, admission levels of IL-6 (3.6 +/- 0.3 ng/ml vs. 10.7 +/- 1.7 ng/ml, p < 0.05) and CRP (2.3 +/- 0.1 mg/l vs. 4.6 +/- 0.6 mg/l, p < 0.05) were elevated in patients with UA. In this group, IL-6 and CRP remained elevated in those who subsequently experienced major adverse cardiac events. This inflammatory response occurred in parallel to the appearance of TnT. Both TNF-alpha (19.2 +/- 3.4 ng/ml vs. 17.1 +/- 3.3 ng/ml, p < 0.001) and IL-6 (10.3 +/- 1.4 ng/ml vs. 7.7 +/- 1.1 ng/ml, p < 0.01) were elevated in the coronary sinus compared with aortic root in patients with UA. This was principally observed in those who were TnT positive. There was no cytokine gradient across the culprit plaque.

Conclusions: There is an intracardiac inflammatory response in UA that appears to be the result of low-grade myocardial necrosis. The ruptured plaque does not appear to contribute to the acute phase response.
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http://dx.doi.org/10.1016/s0735-1097(02)01899-5DOI Listing
June 2002