Project description:Nanoflow liquid chromatography-mass spectrometry is key to enabling in-depth proteome profiling of trace samples such as single cells, but these separations can lack robustness due to the use of narrow-bore columns that are susceptible to clogging. In the case of single-cell proteomics, offline cleanup steps are generally omitted to avoid losses to additional surfaces, and online solid-phase extraction/trap columns frequently provide the only opportunity to remove salts and insoluble debris before the sample is introduced to the analytical column. Trap columns are traditionally short, packed columns used to load and concentrate analytes at flow rates greater than those employed in analytical columns, and since these first encounter the uncleaned sample mixture, trap columns are also susceptible to clogging. We hypothesized that clogging could be avoided by using large-bore porous layer open tubular trap columns (PLOTrap). The low back pressure ensured that the PLOTraps could also serve as the sample loop, thus allowing sample cleanup and injection with a single 6-port valve. We found that PLOTraps could effectively remove debris to avoid column clogging. We also evaluated multiple stationary phases and PLOTrap diameters to optimize performance in terms of peak widths and sample loading capacities. Optimized PLOTraps were compared to conventional packed trap columns operated in forward and backflush modes, and were found to have similar chromatographic performance of backflushed traps while providing improved debris removal for robust analyses of trace samples.
Project description:Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in adenine nucleotide translocase 1 (Ant1), and its yeast homolog Aac2, cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis and cell viability independent of Ant1’s nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/Ant1 cause severe clogging primarily at the Translocase of the Outer Membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger Ant1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy Ant1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.
2023-03-17 | GSE227295 | GEO
Project description:Metagenomic sequencing of bio-clogging
| PRJNA633717 | ENA
Project description:Microbial community associated with iron clogging
Project description:Ischemic stroke is one of the leading causes of disability and mortality worldwide, recognizing aging as a prominent risk factor and determinant of dismal outcome. Aging is known to lead to overall frailty due to multifactorial changes, but pathogenic mechanisms underlying poor outcome remain unclear. Here we show that deterioration in elderly stroke is preceded by neutrophil accumulation and clogging in the ischemic brain microcirculation leading to a worse no-reflow phenomenon. With high-dimensional single-cell profiling over time of the brain's, blood's, and bone marrow's immune response, we could delineate after stroke four main neutrophil clusters in the blood whose quantitative and temporal dynamic of release is deranged in the bone marrow of the old. In the elderly, stroke triggers an early surge in the blood of the CD62Llo neutrophil subset characterized by a signature of bone marrow proximity, senescence, and oxidative stress. Functionally, transfer of this neutrophil subset displaying prominent thrombogenic features in young stroke mice leads to increased clogging of the ischemic brain microcirculation, worse no-reflow and outcome. Interrogating the blood leukocyte landscape of a large human stroke cohort with extensive single-cell proteome analyses, we confirmed that older stroke patients display a similar precocious accumulation of blood CD62Llo neutrophil subset, worse reperfusion and outcome. Our results demonstrate how age-related alterations in the process of neutrophil differentiation and release from the bone marrow have a relevant pathogenic role in the major cerebrovascular disorder affecting the world population, that unleash emergency granulopoiesis.