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Evaluation of deep and dynamic proteomic screening strategies at sub-50Hz scan rate and without automation

ABSTRACT: In this study, we systematically evaluate sample preparation strategies, data acquisition modes and data-analysis approaches for both labeled and label-free proteomics, with the explicit goal of maximizing throughput and quantitative depth using Orbitrap-based instruments operating below 50Hz scan rates and without reliance on automation. Here we benchmarked DIA and TMT based acquistion of pSILAC data (2-SILAC and 3-SILAC)on Orbitrap Ascend, Lumos and Q-Exactive HF

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christian Münch

LAB HEAD: Christian Münch

PROVIDER: PXD073468 | Pride | 2026-03-27

REPOSITORIES: Pride

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			Action	DRS
	20250721_HF_LC2_BSP_20175_01_LH_D01.raw	Raw
	20250721_HF_LC2_BSP_20175_01_LH_D02.raw	Raw
	20250721_HF_LC2_BSP_20175_01_LH_D03.raw	Raw
	20250721_HF_LC2_BSP_20175_01_LH_D04.raw	Raw
	20250721_HF_LC2_BSP_20175_01_LH_T01.raw	Raw

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Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.

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Evaluation of deep and dynamic proteomic screening strategies at sub-50Hz scan rate and without automation

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