Analysis of hexafluoropropylene oxide-dimer acid (HFPO-DA) by Liquid Chromatography-Mass Spectrometry (LC-MS): Review of Current Approaches and Environmental Levels.

Emerging per- and polyfluorinated compounds (PFAS) compounds are of increasing interest for environmental monitoring, one being hexafluoropropylene oxide-dimer acid (HFPO-DA), commonly referred to as GenX. The following review describes existing liquid chromatography-mass spectrometry (LC-MS) methods used to analyze HFPO-DA, including sample preparation and method sensitivity relative to other PFAS. Analytical challenges are also described, in particular the significant formation of in-source fragmentation, dimer and dimer adducts which detract from [M-H]- signal. Lastly, detected levels of HFPO-DA in environmental and biological samples are compared across the limited number of available field exposure studies, which found several µg/L concentrations in water samples taken near fluorochemical plant discharges.

Reducing overestimated intentions and expectations for physical activity: The effect of a corrective entreaty.

We assessed whether intentions and expectations formed in a hypothetical physical activity situation are different from those formed in a real situation; and whether the intentions and expectations of participants who are hypothetically given a free pass to attend a fitness class to better match their behaviour if they are administered a corrective entreaty (CE), than if they are not. In two separate studies, undergraduate university students were randomised into three groups: (a) Hypothetical (H); (b) Hypothetical with CE (HE) and (c) Real (R), and were asked to rate their intention and expectation to use a fitness pass. As hypothesised, significantly more participants expected that they would use the free fitness pass in the H group compared to the R group in both studies. Significantly fewer participants in the HE condition expected to use their free pass compared to the H group in Study 2. Also, significantly more corresponding expectation-behaviour relationships were found in the HE and R groups compared to the H group in both studies. Administering a CE influenced expectations formed in a hypothetical situation making them more similar to expectations formed in a real situation, and increased the specificity of tests of correspondence between expectation and behaviour.

Proteomic analysis of a podocyte vesicle-enriched fraction from human normal and pathological urine samples.

Podocytes (glomerular visceral epithelial cells) release vesicles into urine. Podocyte vesicle-enriched fractions from normal and pathological human urine samples were prepared for proteomic analysis. An immunoadsorption method was applied and enrichment of podocyte vesicles was assessed. We identified 76 unique proteins. One protein, serum paraoxonase/arylesterase 1 (PON-1), was newly identified in normal human urine sample. We confirmed this result and showed PON-1 expression in normal human kidney. These results demonstrated the potential for using the urine samples enriched in podocyte vesicles as a starting material in studies aimed at discovery of biomarkers for diseases.

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid.

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.

Deciphering the human nucleolar proteome.

Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states.

PARK7 and nucleoside diphosphate kinase A as plasma markers for the early diagnosis of stroke.

BACKGROUND: Plasma markers for stroke could be useful in diagnosis and prognosis and in prediction of response of stroke patients to therapy. PARK7 and nucleoside diphosphate kinase A (NDKA) are increased in human postmortem cerebrospinal fluid (CSF), a model of global brain insult, suggesting that measurement in CSF and, more importantly, in plasma may be useful as a biomarker of stroke. METHODS: We used ELISA to measure PARK7 and NDKA in plasma in 3 independent European and North American retrospective studies encompassing a total of 622 stroke patients and 165 control individuals. RESULTS: Increases in both biomarkers were highly significant, with sensitivities of 54%-91% for PARK7 and 70%-90% for NDKA and specificities of 80%-97% for PARK7 and 90%-97% for NDKA. The concentrations of both biomarkers increased within 3 h of stroke onset. CONCLUSIONS: PARK7 and NDKA may be useful plasma biomarkers for the early diagnosis of stroke. In addition, this study demonstrated the utility of analysis of postmortem CSF proteins as a first step in the discovery of plasma markers of ischemic brain injury.

Identification of post-mortem cerebrospinal fluid proteins as potential biomarkers of ischemia and neurodegeneration.

Only few biological markers are currently available for the routine diagnosis of brain damage-related disorders including cerebrovascular, dementia, and other neurodegenerative diseases. In this study, post-mortem cerebrospinal fluid samples were used as a model of massive brain insult to identify new markers potentially relevant for neurodegeneration. The protein pattern of this sample was compared to the one of cerebrospinal fluid from healthy subjects by two-dimensional gel electrophoresis. Using gel imaging, N-terminal microsequencing, mass spectrometry, and immunodetection techniques, we identified 13 differentially expressed proteins. Most of these proteins have been previously reported to be somehow associated with brain destruction or with the molecular mechanisms underlying certain neurodegenerative conditions. These data indicate that the identified proteins indeed represent potential biomarkers of brain damage. We recently showed that H-FABP, a protein highly homologous to E-FABP and A-FABP identified in this study, is a potential marker of Creutzfeldt-Jakob disease and stroke.

Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis.

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins.

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.

Mass spectrometry analysis of CD4-associating proteins using affinity chromatography and affinity tag-mediated purification of tryptic peptides.

The study of protein interactions using mass spectrometry (MS) for identification of the components of purified protein complexes is leading to the description of increasingly valuable data on protein function. Commonly proteins in a given complex are identified via MS analysis of in-gel digests of gel electrophoretically separated proteins. In this study, we have evaluated the use of an approach employing the digest of the whole protein complex to identify directly the proteins present in a purification of the CD4 receptor complex. We used a cysteinyl affinity capture method to reduce the complexity of the peptide mixture that was obtained from the tryptic digest of the whole protein complex to the rather limited mixture of only cysteine-containing peptides. Here we report the use of this approach with MS for identification of the CD4 receptor complex components CD4 and p56lck, along with several other proteins present in the detergent-solubilized fractions from the purification. We have been able to identify these proteins using peptide sequence data obtained from cysteine-containing peptides. With appropriate control experiments, we have demonstrated the specific nature of the CD4-p56lck interaction. In contrast, the other proteins identified are shown to arise from nonspecific interactions during the affinity chromatography purification suggesting a possible loss of specific interactions during the chromatography procedure. We found that the complexity of the mixture was reduced such that only 10% of the peptides derived from tryptic digest of the identified proteins were detected. This represents only one-third of the cysteine-containing peptides, however, suggesting that this approach does not enable detection of all individual proteins.