Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.

This article provides a review of the routine methods currently utilized for total naphthenic acid analyses. There is a growing need to develop chemical methods that can selectively distinguish compounds found within industrially derived oil sands process affected waters (OSPW) from those derived from the natural weathering of oil sands deposits. Attention is thus given to the characterization of other OSPW components such as oil sands polar organic compounds, PAHs, and heavy metals along with characterization of chemical additives such as polyacrylamide polymers and trace levels of boron species. Environmental samples discussed cover the following matrices: OSPW containments, on-lease interceptor well systems, on- and off-lease groundwater, and river and lake surface waters. There are diverse ranges of methods available for analyses of total naphthenic acids. However, there is a need for inter-laboratory studies to compare their accuracy and precision for routine analyses. Recent advances in high- and medium-resolution mass spectrometry, concomitant with comprehensive mass spectrometry techniques following multi-dimensional chromatography or ion-mobility separations, have allowed for the speciation of monocarboxylic naphthenic acids along with a wide range of other species including humics. The distributions of oil sands polar organic compounds, particularly the sulphur containing species (i.e., OxS and OxS2) may allow for distinguishing sources of OSPW. The ratios of oxygen- (i.e., Ox) and nitrogen-containing species (i.e., NOx, and N2Ox) are useful for differentiating organic components derived from OSPW from natural components found within receiving waters. Synchronous fluorescence spectroscopy also provides a powerful screening technique capable of quickly detecting the presence of aromatic organic acids contained within oil sands naphthenic acid mixtures. Synchronous fluorescence spectroscopy provides diagnostic profiles for OSPW and potentially impacted groundwater that can be compared against reference groundwater and surface water samples. Novel applications of X-ray absorption near edge spectroscopy (XANES) are emerging for speciation of sulphur-containing species (both organic and inorganic components) as well as industrially derived boron-containing species. There is strong potential for an environmental forensics application of XANES for chemical fingerprinting of weathered sulphur-containing species and industrial additives in OSPW.

Sample preparation remains a challenge in untargeted metabolomics studies and no method currently results in complete extraction of all metabolite classes in human plasma. Because a large variety of molecules, with vast differences in dynamic range, could be involved in human disease, there is an urgent need to develop analytical techniques that result in comprehensive coverage of metabolites. Furthermore, analysis of more focused molecular classes could be necessary to more fully interrogate markers of human disease. However, such techniques, which generally involve multiple steps, often result in high variability. We have optimized a combined liquid-liquid and solid phase extraction method for plasma and have compared that to traditional methanol precipitation using spiked internal standards as controls. This method, based largely on previously published methods, results in 5 separate fractions enriched for aqueous species, phospholipids, fatty acids, neutral lipids, and hydrophobic lipids. Using liquid chromatography mass spectrometry as the analytical method, we detect over 3,806 metabolites using the new method versus 1,851 metabolites using methanol alone. Qualitative analysis and quantitative analysis of both internal standards (ISTDs) and endogenous metabolites demonstrate excellent reproducibility with CV’s below 15% for the combined method compared to 30% using the methanol method. While both methods have applications for clinical metabolomics, fractionation resulted in greater overall coverage and can be used for initial classification of molecular species.

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reversephase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/ MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.

Currently, no reliable markers are available to evaluate the epileptogenic potential of a brain injury. The electroencephalogram is the standard method of diagnosis of epilepsy; however, it is not used to predict the risk of developing epilepsy. Biomarkers that indicate an individual’s risk to develop epilepsy, especially those measurable in the periphery are urgently needed. Temporal lobe epilepsy (TLE), the most common form of acquired epilepsy, is characterized by spontaneous recurrent seizures following brain injury and a seizure-free “latent” period. Elucidation of mechanisms at play during epilepsy development (epileptogenesis) in animal models of TLE could enable the identification of predictive biomarkers. Our pilot study using liquid chromatography-mass spectrometry metabolomics analysis revealed changes (p-value ≤ 0.05, ≥1.5-fold change) in lipid, purine, and sterol metabolism in rat plasma and hippocampus during epileptogenesis and chronic epilepsy in the kainic acid model of TLE. Notably, disease development was associated with dysregulation of vitamin D3 metabolism at all stages and plasma 25-hydroxyvitamin D3 depletion in the acute and latent phase of injury-induced epileptogenesis. These data suggest that plasma VD3 metabolites reflect the severity of an epileptogenic insult and that a panel of plasma VD3 metabolites may be able to serve as a marker of epileptogenesis.

Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of "candidate biomarkers" specific to each of five body fluids (i.e., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time-of-flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single-source samples of these human body fluids were accurately identified by the detection of one or more high-specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2-component mixtures of human body fluids, the multiplex assay accurately identified both components in a single-pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins.

DNA profiling has transformed the field of forensic biology by making it possible to individualize biological stains. The identification of the stain itself, however, continues to present forensic serologists with significant challenges. Current antibody- and enzyme activity-based assays yield only presumptive results as detection in nontarget body fluids or cross-reactivity with nonhuman sources have both been well documented. For other critical body fluids such as vaginal and menstrual fluids, there are no commercial tests at all. Using a three-pronged, comparative proteomic strategy based on proteome fractionation by HPLC followed by MS, a panel of 29 candidate protein biomarkers have been proposed as highly specific indicators of human saliva, urine, seminal fluid, vaginal fluid, peripheral blood, and menstrual fluid. The combination of consistent identification by multiple strategies in the current study; confirmation in independently compiled proteomic databases; and information on tissue expression and/or functionality from the proteomic literature all support the proposition that these proteins will have utility as reliable biomarkers of their target body fluids. The identification of candidate high-specificity protein biomarkers for human body fluids encountered in forensic investigations lays the foundation for the development of faster and more reliable approaches to the serological analysis of evidentiary stains.

A new analytical method suitable for high throughput measurements of LTE 4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/ tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/mL in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE 4 from healthy adults and children are reported.