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Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived “red cell storage lesions.” The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers.
2',7'-dichlorfluorescein-diacetate (DCFH-DA); 4-hydroxynonenal (4-HNE); adenosine triphosphate (ATP); carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP); cytosolic calcium concentration ([Ca 2+ ]c); dihydroethidium probe (DHE); Dulbecco's Modified Eagle's Medium (DMEM); extracellular acidification rates (ECAR); fetal bovine serum (FBS); gene of interest (GOI); horse serum (HS); housekeeping gene (HKG); lipid peroxidation (LPO); oxygen consumption rates (OCR); polyunsaturated fatty acids (PUFA); reactive oxygen species (ROS); relative fluorescence units (RFU); threshold cycle (CT) Summary Background: Skeletal muscle cells continuously generate reactive oxygen species (ROS). Excessive ROS can affect lipids resulting in lipid peroxidation (LPO). Here we investigated the effects of myotube contraction on the LPO induction and the impact of LPO-product 4-hydroxynonenal (4-HNE) on physiology/pathology of myotubes using C2C12 myoblasts. Methods: C2C12 myoblasts were differentiated into myotubes, stimulated with caffeine and analyzed for the induction of LPO and formation of 4-HNE protein adducts. Further effects of 4-HNE on mitochondrial bioenergetics, NADH level, mitochondrial density and expression of mitochondrial metabolism genes were determined. Results: Short and long-term caffeine stimulation of myotubes promoted superoxide production, LPO and formation of 4-HNE protein adducts. Furthermore, low 4-HNE concentrations had no effect on myotube viability and cellular redox homeostasis, while concentrations from 10µM and above reduced myotube viability and significantly disrupted homeostasis. A time and dose-dependent 4-HNE effect on superoxide production and mitochondrial NADH-autofluorescence was observed. Finally, 4-HNE had strong impact on maximal respiration, spare respiratory capacity, ATP production, coupling efficiency of mitochondria and mitochondrial density. Conclusion: Data presented in this work make evident for the first time that pathological 4-HNE levels elicit damaging effects on skeletal muscle cells while acute exposure to physiological 4-HNE induces transient adaptation. General significance: This work suggests an important role of 4-HNE on the regulation of myotube's mitochondrial metabolism and cellular energy production. It further signifies the importance of skeletal muscle cells hormesis in response to acute stress in order to maintain essential biological functions.
The accurate processing of complex liquid chromatography coupled to tandem mass spectrometry (LC− MS/MS) data from biological samples is a major challenge for metabolomics, proteomics, and related approaches. Here, we present the pipelines and systems for threshold-avoiding quantification (PASTAQ) LC−MS/MS preprocessing toolset, which allows highly accurate quantification of data-dependent acquisition LC−MS/MS datasets. PASTAQ performs compound quantification using single-stage (MS1) data and implements novel algorithms for high-performance and accurate quantification, retention time alignment, feature detection, and linking annotations from multiple identification engines. PASTAQ offers straightforward parameterization and automatic generation of quality control plots for data and preprocessing assessment. This design results in smaller variance when analyzing replicates of proteomes mixed with known ratios and allows the detection of peptides over a larger dynamic concentration range compared to widely used proteomics preprocessing tools. The performance of the pipeline is also demonstrated in a biological human serum dataset for the identification of gender-related proteins.
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