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Diagnosis and Pathobiology
Published in Franklyn De Silva, Jane Alcorn, The Elusive Road Towards Effective Cancer Prevention and Treatment, 2023
Franklyn De Silva, Jane Alcorn
Newer strategies for molecular characterization like epigenetic profiling, immunophenotyping, single-cell analyses (DNA, RNA, or protein), RNA data profiles, chromatin immunoprecipitation (ChIP), and transcriptomics could improve the precision of cancer medicine [220, 365, 517]. Furthermore, as robust complementary methods (with improved temporal and spatial resolution) to the classic affinity purification/mass spectrometry (AP/MS) of multiprotein complexes, proximity-based labeling has emerged for the mapping of additional molecular interactions and has led to innovations in protein-centric (protein molecule tagging to label adjacent proteins such as improved biotin-based proximity labeling approaches), RNA-centric (RNA molecule tagging to label RNA-binding proteins such as ribonucleoprotein complexes), and DNA-centric (tagging of a gene locus to label related protein complexes such as chromatin-associated protein complexes) assays [518–523]. One of the key barriers in modern biomedicine is studying the protein interaction networks of all proteins in an organism (i.e., interactomes) [524]. The advancements in our understanding on cellular and molecular pathways and the development of competent therapy for human disease intervention can surely benefit from such information regarding dynamic interactions [524]. Looking closely at the enormous diversity and complexity of the epigenome and the relationship between the genome and the epigenome, it makes sense that every angle of cancer heterogeneity is tightly meshed into the environment we live in through epigenetic modulations.
Ion Channel Conformational Coupling in Ischemic Neuronal Death
Published in Tian-Le Xu, Long-Jun Wu, Nonclassical Ion Channels in the Nervous System, 2021
From neuroprotection to neural injury, ion channels play a variety of important roles in different stages of ischemic stroke (Kahle et al., 2009; Lai et al., 2014; Weilinger et al., 2013; Xiong et al., 2004). However, for a very long period of time, the majority of researchers have stereotyped ion channels as just tunnels for ion exchange. Nearly all hypotheses regarding mechanisms of ion channel-mediated neurological damage were rooted from a simple idea: how ionic balance is disturbed by a certain channel during ischemic stroke. For example, numerous publications have shown that N-methyl-D-aspartate receptors (NMDARs) (Ge et al., 2020; Hoyte et al., 2004) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) (Akins and Atkinson, 2002) contribute to ischemic neuronal death by mediating Ca2+ influx that results in cytosolic Ca2+ overload (Table 14.1). There is no doubt that these are all important findings and they are very enlightening. The concern is that the dominating ion conducting-based hypotheses may narrow the scope of exploration. In living cells, proteins always form networks and function in groups (or modules). No protein can fulfill a physiological or pathological function solely on its own. Neither do ion channels. In fact, an increasing number of ion channel-binding proteins (ICBP) have been revealed by affinity purification–mass spectrometry (Dunham et al., 2012) and proximity labeling analysis (Branon et al., 2018). Some of the ICBPs serve as auxiliary subunits to support ion channel function, like protein interacting with C Kinase - 1 (PINK1) for AMPARs (Xia et al., 1999) and beta subunits of voltage-gated Na+, Ca2+ and K+ channels (Yu et al., 2005). Others are the substrates or downstream effectors, like receptor-interacting serine/threonine-protein kinase 1 (RIPK1) for acid-sensing ion channel 1a (ASIC1a) (Wang et al., 2015).
Proximity labeling and other novel mass spectrometric approaches for spatiotemporal protein dynamics
Published in Expert Review of Proteomics, 2021
Lindsay Pino, Birgit Schilling
One challenge to using proximity labeling is that ‘innocent bystander’ proteins nearby the proximity labeling enzyme may be labeled but are not true interactors. These contaminant proteins make it difficult to construct the true protein interaction network. To remove these confounders and gain higher-resolution spatial information, one creative solution is to use multiple proximity label fusion proteins that are specific to different subcellular localizations [19]. This approach enabled sub-minute resolution of G-protein-coupled receptor partners in HEK293T human cell culture by using a pair of APEX2 fusion constructs: beta-2 adrenergic receptor fused to APEX2 and delta opioid receptor fused to another APEX2 construct. If the same proximity-labeled proteins are present in each list of enriched proteins, then it is more likely that protein is a true ‘protein–protein interaction’ (PPI); if a protein is only found with one construct, then it is more likely that protein is an ‘innocent bystander.’
Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells
Published in Expert Review of Proteomics, 2021
Jacqueline S. Gerritsen, Forest M. White
Another challenge for phosphoproteomics is spatial analysis, due to the highly dynamic nature of this PTM. With the use of proximity labeling strategies, Liu et al. demonstrated the ability to monitor altered phosphorylation patterns due to ER stress in in vitro and in vivo systems [137]. We previously used phosphoproteomics to characterize the immediate-early signaling dynamics in the EGFR network and proximity ligation assays (PLA) to characterize dynamic recruitment of adaptor proteins to the membrane [82], or total internal reflection fluorescence (TIRF) microscopy to monitor in vivo SH2 binding dynamics and binding site kinetics [138]. Being able to directly quantify spatially resolved signaling networks by MS-based phosphoproteomic analysis has yet to be accomplished.
Proteotyping pluripotency with mass spectrometry
Published in Expert Review of Proteomics, 2019
Cristina Sayago, Ana Martinez-Val, Javier Munoz
Understanding the signaling pathways that safeguard pluripotency states is key. The vast complexity of post-translational modifications that participate in these pathways could be defined by exploiting the superior sensitivity and speed of modern mass spectrometers that enable the quantification of over 50,000 unique phosphorylation sites [77]. In addition, novel enrichment approaches are being developed, many of them based on the generation of highly pan- specific monoclonal antibodies directed towards certain modifications. Latest reports employing these immunoaffinity purification strategies have demonstrated their potential enabling the identification of 63,000 ubiquitination sites [78], 21,000 acetylation sites [79] and 8,000 arginine methylation sites [63]. Furthermore, novel approaches are available that allow to map the intricate physical interactions among thousands of proteins with high-throughput. Proximity-labeling is a promising technique that, by means of enzymatic reactions, allows the identification of proteins in the close vicinity of a candidate protein. Proximity-dependent biotin identification (BioID) relies on the fusion of the BirA biotin ligase to the protein of interest [80]. Promiscuous biotinylation of proximal proteins allows stringent purification and subsequent MS detection. Likewise, novel chemical cross-linking coupled to MS can provide thousands of endogenous interactions in protein complexes and assemblies directly from whole cellular lysates.