Dataset Information

QCeltis Case Study 2: Native Plasma QC Dataset

ABSTRACT: A dataset containing native plasma samples used as quality control digestion replicates. Samples were automatically processed on i7 automated liquid handling system (Beckman Coulter). Briefly, proteins were extracted and denatured with a buffer composed of TFE (2,2,2-Trifluoroethanol), DTT and ammonium bicarbonate prior being alkylated, reduced and digested with trypsin. Obtained tryptic peptides were desalted by SPE (Solid Phase Extraction) on C18 stationary phase. Pooled whole blood and plasma samples were used as digestion control replicates (DRs). 5 DRs were added to each digestion plate to be digested with the cohort data. DIA-MS analysis was performed on an Orbitrap Exploris 480 (Thermo Fisher) instrument using MS-MS settings with tryptic peptides separated by EVOSEP One LC System (Evosep) over a 60-minute gradient. During LC-MS acquisition, the 5 DRs are run at the beginning of each batch.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jennifer Van Eyk

PROVIDER: MSV000095744 | MassIVE |

SECONDARY ACCESSION(S): PXD055483

REPOSITORIES: MassIVE

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Publications

<u>Enhancing Proteomics Quality Control: Insights from the Visualization Tool QCeltis</u>.

Vegesna Manasa M Sundararaman Niveda N Bharadwaj Ajay A Washington Kirstin K Pandey Rakhi R Haghani Ali A Chazarin Blandine B Binek Aleksandra A Fu Qin Q Cheng Susan S Herrington David D Van Eyk Jennifer E JE

Journal of proteome research 20250224 3

Large-scale mass-spectrometry-based proteomics experiments are complex and prone to analytical variability, requiring rigorous quality checks across each step in the workflow: sample preparation, chromatography, mass spectrometry, and the bioinformatics stages. This includes quality control (QC) measures that address biological and technical variation. Most QC approaches involve detecting sample outliers and monitoring parameters related to sample preparation and mass spectrometer performance. E ...[more]

PMID: 39992359

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Project description:Multiple sclerosis (MS) begins as an inflammatory/demyelinating disease but axon injury and neurodegeneration underlie chronic progression. Extracellular matrix (ECM) heparan sulfate proteoglycans (HSPG) are catabolized in acute inflammatory MS lesions and HSPG catabolic products may contribute to failure of neuron repair. Neurite outgrowth and axon elongation of PC12 cells are inhibited when these cells are grown on heparanase-digested HSPG. This inhibition of differentiation is counteracted by nerve growth factor (NGF) but failure to completely reverse inhibition may be due to loss of other growth factors affected by HSPG breakdown. To understand effects of the digested ECM in this model, expression of genes that control differentiation in PC12 cells grown on digested and undigested HSPG will be analyzed. This will permit identification of new potential therapeutic targets for specifically counteracting inhibitory effects of ECM breakdown on neuron regrowth/regeneration in MS. We will compare gene expression patterns of PC12 cells grown on undigested and digested HSPG in the presence and in the absence of NGF over a two week time course to identify the alterations induced by the catabolized HSPG on expression of genes involved in their acquisition of a neuronal phenotype and to determine which genes are and are not targeted by NGF. We are studying ECM HSPG breakdown in human and experimental demyelinating diseases and have developed an in vitro model of ECM catabolism-induced inhibition of neuronal differentiation. Our data indicate that: 1) the numbers of PC12 cells that exhibit neurite outgrowth are decreased when the ECM contains digested, as opposed to intact HSPG; and 2) in the presence of NGF, neurite outgrowth inhibition is counteracted, but complete axonal growth/elongation is not achieved. We will use gene microarray analysis to identify the specific gene expression alterations that digested ECM HSPG induces in PC12 cells in this model of inhibition of neural differentiation. We hypothesize that the genes that are critical for PC12 cell differentiation will be altered by the pathologically altered ECM. Rat pheochromocytoma PC12 cell lines are widely used for analyzing neuronal differentiation. Microarray analyses have identified gene expression patterns associated with their differentiation and response to growth factors. We developed a model of catabolized HSPG inhibition of PC12 cell differentiation that mimics the ECM in MS lesions. HSPG coated on tissue culture dishes is digested with heparanases prior to plating of the PC12 cells. Cells grown on digested ECM without NGF show inhibition of neuronal differentiation (decreased numbers with neurite outgrowth) from days 7 to 14. NGF can partially, but not completely counteract the inhibition, suggesting that signaling pathways in addition to those affected by NGF may be affected by the ECM HSPG digestion. Therefore, we will compare gene expression patterns in samples of cells grown on digested and undigested HSPG ECM both in the presence and absence of NGF. The cells are plated on washed, undigested or digested HSPG-coated 100 mm Petri dishes and grown with or without NGF. Total RNA will be extracted from cells grown in the 4 conditions (w/without digestion; w/without NGF) on 100mm Petri dishes using the RNeasy extraction kit (Qiagen). We propose: 2 duplicate samples/culture condition (8 samples) per time point X 5 time points (days 1, 5, 7, 10, and 14 of culture) = 40 samples/experiment. The RNA will be sent to the Microarray Consortium center for QC check and microarray analysis. Previous analyses of PC12 cells have used the NIA Mouse 15K cDNA clone set scanned with the Agilent Oligo array platform (Vaudry, 2002; Grumolato, 2003). Therefore, this methodology will be useful for comparisons to published studies. (Number of experiments to be determined). Vaudry D, et al, 2002. J Neurochem 83:1272-1284 Grumolato L, et al, 2003. Endocrinology 144:2368-2379 Keywords: time-course

Dataset Information

QCeltis Case Study 2: Native Plasma QC Dataset

Dataset's files

Publications

<u>Enhancing Proteomics Quality Control: Insights from the Visualization Tool QCeltis</u>.

Similar Datasets

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Dataset Information

QCeltis Case Study 2: Native Plasma QC Dataset

Dataset's files

Publications

&lt;u&gt;Enhancing Proteomics Quality Control: Insights from the Visualization Tool QCeltis&lt;/u&gt;.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

<u>Enhancing Proteomics Quality Control: Insights from the Visualization Tool QCeltis</u>.