Abstract
Human Proteome Project (HPP) presents a systematic characterization of the protein landscape under different conditions using several complementary-omic techniques (LC-MS/MS proteomics, affinity proteomics, transcriptomics, etc.). In the present study, using a B-cell lymphoma cell line as a model, comprehensive integration of RNA-Seq transcriptomics, MS/MS, and antibody-based affinity proteomics (combined with size-exclusion chromatography) (SEC-MAP) were performed to uncover correlations that could provide insights into protein dynamics at the intracellular level. Here, 5672 unique proteins were systematically identified by MS/MS analysis and subcellular protein extraction strategies (neXtProt release 2020-21, MS/MS data are available via ProteomeXchange with identifier PXD003939). Moreover, RNA deep sequencing analysis of this lymphoma B-cell line identified 19,518 expressed genes and 5707 protein coding genes (mapped to neXtProt). Among these data sets, 162 relevant proteins (targeted by 206 antibodies) were systematically analyzed by the SEC-MAP approach, providing information about PTMs, isoforms, protein complexes, and subcellular localization. Finally, a bioinformatic pipeline has been designed and developed for orthogonal integration of these high-content proteomics and transcriptomics datasets, which might be useful for comprehensive and global characterization of intracellular protein profiles.
Highlights
Increased understanding of the events taking place during intracellular signaling has revealed a highly dynamic protein landscape for eukaryotic cells, especially in pathological settings [1,2,3]
Knowing and exploring the different cell lysis strategies is fundamental to know the compatibility of the different proteomics methodologies and the feasibility to perform an integration of multi-pronged proteomic strategies (LC-MS/MS and size-exclusion chromatography (SEC)-MAP, for example)
Multi-pronged proteomics characterization is highly dependent on protein extraction procedures, being a key step for functional outcomes from detection of proteins/multiprotein complexes in samples
Summary
Increased understanding of the events taking place during intracellular signaling has revealed a highly dynamic protein landscape for eukaryotic cells, especially in pathological settings (such as cancer, neurodegenerative, auto-immune diseases, etc.,) [1,2,3]. Proteins are shuttling between different subcellular localizations to execute the expected and/or programmed biological processes according to the needs and requirements of the cell These fluctuations could produce re-wiring of signaling networks for enabling phenotypic changes required for the adaptation to microenvironmental or external perturbations and/or stimuli [4,5,6]. Given these temporal and spatial variabilities, high-throughput biochemical methods are required to deepen the knowledge on the interacting biomolecules across subcellular localizations during the response to external stimulus, drug administration, and drug resistance mechanisms, etc., [1,2,4,6,7]. Given these temporal and spatial variabilities, high-throughput biochemical methods are required to deepen the knowledge on the interacting biomolecules across subcellular localizations during the response to external stimulus, drug administration, and drug resistance mechanisms, etc., [1,2,4,6,7]. 4.0/).
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