132 research outputs found
How shall we use the proteomics toolbox for biomarker discovery?
Biomarker discovery for clinical purposes is one of the major areas in which
proteomics is used. However, despite considerable effort, the successes have
been relatively scarce. In this perspective paper, we try to highlight and
analyze the main causes for this limited success, and to suggest alternate
strategies, which will avoid them, without eluding the foreseeable weak points
of these strategies. Two major strategies are analyzed, namely, the switch from
body fluids to cell and tissues for the initial biomarker discovery step or, if
body fluids must be analyzed, the implementation of highly selective protein
selection strategies
The proteomic to biology inference, a frequently overlooked concern in the interpretation of proteomic data: A plea for functional validation
Proteomics will celebrate its 20th year in 2014. In this relatively short
period of time, it has invaded most areas of biology and its use will probably
continue to spread in the future. These two decades have seen a considerable
increase in the speed and sensitivity of protein identification and
characterization, even from complex samples. Indeed, what was a challenge
twenty years ago is now little more than a daily routine. Although not
completely over, the technological challenge now makes room to another
challenge, which is the best possible appraisal and exploitation of proteomic
data to draw the best possible conclusions from a biological point of view. The
point developed in this paper is that proteomic data are almost always
fragmentary. This means in turn that although better than an mRNA level, a
protein level is often insufficient to draw a valid conclusion from a
biological point of view, especially in a world where PTMs play such an
important role. This means in turn that transformation of proteomic data into
biological data requires an important intermediate layer of functional
validation, i.e. not merely the confirmation of protein abundance changes by
other methods, but a functional appraisal of the biological consequences of the
protein level changes highlighted by the proteomic screens
Organelle proteomics.
International audienceThis unit describes strategies for studying the proteomes of organelles, which is one example of targeted proteomics. It relies heavily on previously published units dealing with organelle preparation, protein solubilization, and proteomics techniques. A specific commentary for organelle proteomics is provided. Specific protocols for the isolation of nuclei from various sources (cell cultures, tissues) are also provided
Organelle proteomics
This unit describes strategies for studying the proteomes of organelles,
which is one example of targeted proteomics. It relies heavily on previously
published units dealing with organelle preparation, protein solubilization, and
proteomics techniques. A specific commentary for organelle proteomics is
provided. Specific protocols for the isolation of nuclei from various sources
(cell cultures, tissues) are also provided
Progress in the definition of a reference human mitochondrial proteome
Owing to the complexity of higher eukaryotic cells, a complete proteome is
likely to be very difficult to achieve. However, advantage can be taken of the
cell compartmentalization to build organelle proteomes, which can moreover be
viewed as specialized tools to study specifically the biology and "physiology"
of the target organelle. Within this frame, we report here the construction of
the human mitochondrial proteome, using placenta as the source tissue. Protein
identification was carried out mainly by peptide mass fingerprinting. The
optimization steps in two-dimensional electrophoresis needed for proteome
research are discussed. However, the relative paucity of data concerning
mitochondrial proteins is still the major limiting factor in building the
corresponding proteome, which should be a useful tool for researchers working
on human mitochondria and their deficiencies.Comment: website publisher http://www.interscience.wiley.co
Electron Transfer Dissociation Mass Spectrometry of Hemoglobin on Clinical Samples
A mass spectrometry-based assay combining the specificity of selected reaction monitoring and the protein ion activation capabilities of electron transfer dissociation was developed and employed for the rapid identification of hemoglobin variants from whole blood without previous proteolytic cleavage. The analysis was performed in a robust ion trap mass spectrometer operating at nominal mass accuracy and resolution. Subtle differences in globin sequences, resulting with mass shifts of about one Da, can be unambiguously identified. These results suggest that mass spectrometry analysis of entire proteins using electron transfer dissociation can be employed on clinical samples in a workflow compatible with diagnostic application
LacaScore: a novel plasma sample quality control tool based on ascorbic acid and lactic acid levels
Introduction
Metabolome analysis is complicated by the continuous dynamic changes of metabolites in vivo and ex vivo. One of the main challenges in metabolomics is the robustness and reproducibility of results, partially driven by pre-analytical variations.
Objectives
The objective of this study was to analyse the impact of pre-centrifugation time and temperature, and to determine a quality control marker in plasma samples.
Methods
Plasma metabolites were measured by gas chromatography-mass spectrometry (GC–MS) and analysed with the MetaboliteDetector software. The metabolites, which were the most labile to pre-analytical variations, were further measured by enzymatic assays. A score was calculated for their use as quality control markers.
Results
The pre-centrifugation temperature was shown to be critical in the stability of plasma samples and had a significant impact on metabolite concentration profiles. In contrast, pre-centrifugation delay had only a minor impact. Based on the results of this study, whole blood should be kept on wet ice and centrifuged within maximum 3 h as a prerequisite for preparing EDTA plasma samples fit for the purpose of metabolome analysis.
Conclusions
We have established a novel blood sample quality control marker, the LacaScore, based on the ascorbic acid to lactic acid ratio in plasma, which can be used as an indicator of the blood pre-centrifugation conditions, and hence the suitability of the sample for metabolome analyses. This method can be applied in research institutes and biobanks, enabling assessment of the quality of their plasma sample collections
Type of mRNA COVID-19 vaccine and immunomodulatory treatment influence humoral immunogenicity in patients with inflammatory rheumatic diseases
Patients with inflammatory rheumatic diseases (IRD) are at increased risk for worse COVID-19 outcomes. Identifying whether mRNA vaccines differ in immunogenicity and examining the effects of immunomodulatory treatments may support COVID-19 vaccination strategies. We aimed to conduct a long-term, model-based comparison of the humoral immunogenicity following BNT162b2 and mRNA-1273 vaccination in a cohort of IRD patients. Patients from the Swiss IRD cohort (SCQM), who assented to mRNA COVID-19 vaccination were recruited between 3/2021-9/2021. Blood samples at baseline, 4, 12, and 24 weeks post second vaccine dose were tested for anti-SARS-CoV-2 spike IgG (anti-S1). We examined differences in antibody levels depending on the vaccine and treatment at baseline while adjusting for age, disease, and past SARS-CoV-2 infection. 565 IRD patients provided eligible samples. Among monotherapies, rituximab, abatacept, JAKi, and TNFi had the highest odds of reduced anti-S1 responses compared to no medication. Patients on specific combination therapies showed significantly lower antibody responses than those on monotherapy. Irrespective of the disease, treatment, and past SARS-CoV-2 infection, the odds of higher antibody levels at 4, 12, and 24 weeks post second vaccine dose were, respectively, 3.4, 3.8, and 3.8 times higher with mRNA-1273 versus BNT162b2 (p < 0.0001). With every year of age, the odds ratio of higher peak humoral immunogenicity following mRNA-1273 versus BNT162b2 increased by 5% (p < 0.001), indicating a particular benefit for elderly patients. Our results suggest that in IRD patients, two-dose vaccination with mRNA-1273 versus BNT162b2 results in higher anti-S1 levels, even more so in elderly patients
Type of mRNA COVID-19 vaccine and immunomodulatory treatment influence humoral immunogenicity in patients with inflammatory rheumatic diseases.
Patients with inflammatory rheumatic diseases (IRD) are at increased risk for worse COVID-19 outcomes. Identifying whether mRNA vaccines differ in immunogenicity and examining the effects of immunomodulatory treatments may support COVID-19 vaccination strategies. We aimed to conduct a long-term, model-based comparison of the humoral immunogenicity following BNT162b2 and mRNA-1273 vaccination in a cohort of IRD patients. Patients from the Swiss IRD cohort (SCQM), who assented to mRNA COVID-19 vaccination were recruited between 3/2021-9/2021. Blood samples at baseline, 4, 12, and 24 weeks post second vaccine dose were tested for anti-SARS-CoV-2 spike IgG (anti-S1). We examined differences in antibody levels depending on the vaccine and treatment at baseline while adjusting for age, disease, and past SARS-CoV-2 infection. 565 IRD patients provided eligible samples. Among monotherapies, rituximab, abatacept, JAKi, and TNFi had the highest odds of reduced anti-S1 responses compared to no medication. Patients on specific combination therapies showed significantly lower antibody responses than those on monotherapy. Irrespective of the disease, treatment, and past SARS-CoV-2 infection, the odds of higher antibody levels at 4, 12, and 24 weeks post second vaccine dose were, respectively, 3.4, 3.8, and 3.8 times higher with mRNA-1273 versus BNT162b2 (p < 0.0001). With every year of age, the odds ratio of higher peak humoral immunogenicity following mRNA-1273 versus BNT162b2 increased by 5% (p < 0.001), indicating a particular benefit for elderly patients. Our results suggest that in IRD patients, two-dose vaccination with mRNA-1273 versus BNT162b2 results in higher anti-S1 levels, even more so in elderly patients
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