Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for Quantitative Proteomics

Hoedt, Esthelle; Zhang, Guoan; Neubert, Thomas A.

doi:10.1007/978-3-319-06068-2_5

Cited by 36 publications

(33 citation statements)

References 118 publications

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“…S1) were reprogrammed to hiPSCs via nonintegrating episomalmediated (42), lentivirus-mediated (1), or retrovirus-mediated (19) gene transfer, characterized (43), and differentiated to NPCs and cortical interneurons, as per our routine and as previously described (17)(18)(19)(20). Protein isolation, 2D-DIGE and SILAC, Western blotting, and coimmunoprecipitation were performed as described previously (17,28). Immunofluorescence was quantified by pixel number captured via image analysis software using unbiased stereology.…”

Section: Methodsmentioning

confidence: 99%

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis

Tobe

Crain

Winquist

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

133

121

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The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSCderived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active nonphosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2:CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the "lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknownmight reveal otherwise inscrutable intracellular pathogenic pathways. have proven valuable for studying the molecular pathology of monogenic diseases, one of the technique's greatest challenges has been to offer similar insights into the molecular pathogenesis of polygenic, multifactorial disorders for which the underlying pathophysiology is unknown. The struggle has been to go beyond phenotypic description to discerning underlying molecular mechanisms. Neuropsychiatric illnesses are a prototype for such complex conditions (1-3). They are difficult to model not only because of the likelihood of polygenic influences, but also because of the subjectivity with which these diseases must often be diagnosed, the empirical fashion with which drugs are prescribed, and the heterogeneity of patient response. Of such maladies, bipolar disorder

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Section: Methodsmentioning

confidence: 99%

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis

Tobe

Crain

Winquist

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

133

121

View full text Add to dashboard Cite

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“…Thus, for example, FUNCAT allows for the visualization of NSPs but does not resolve the identity of the NSPs. Methods based on metabolic labeling and mass spectroscopy, such as stable isotope labeling with amino acids in cell culture (SILAC) and MS (for a recent review see Hoedt et al, 2014) have provided unprecedented information on the turnover rates of major neuronal proteins, yet they tend to “miss” less abundant proteins, have poor temporal resolution and practically no spatial resolution. Genetic methods, such as TimeSTAMP (Butko et al, 2012) allow for the visualization of newly synthesized copies of particular proteins and for measurements of their degradations rates, but the physiological relevance of these data are sensitive to the degree to which the regulation of expression as well as the degradation rates of these exogenous fusion proteins mimic those of their endogenous counterparts.…”

Section: Technological Obstacles and Outlookmentioning

confidence: 99%

The roles of protein expression in synaptic plasticity and memory consolidation

Rosenberg¹,

Gal-Ben-Ari²,

Dieterich³

et al. 2014

Front. Mol. Neurosci.

136

103

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The amount and availability of proteins are regulated by their synthesis, degradation, and transport. These processes can specifically, locally, and temporally regulate a protein or a population of proteins, thus affecting numerous biological processes in health and disease states. Accordingly, malfunction in the processes of protein turnover and localization underlies different neuronal diseases. However, as early as a century ago, it was recognized that there is a specific need for normal macromolecular synthesis in a specific fragment of the learning process, memory consolidation, which takes place minutes to hours following acquisition. Memory consolidation is the process by which fragile short-term memory is converted into stable long-term memory. It is accepted today that synaptic plasticity is a cellular mechanism of learning and memory processes. Interestingly, similar molecular mechanisms subserve both memory and synaptic plasticity consolidation. In this review, we survey the current view on the connection between memory consolidation processes and proteostasis, i.e., maintaining the protein contents at the neuron and the synapse. In addition, we describe the technical obstacles and possible new methods to determine neuronal proteostasis of synaptic function and better explain the process of memory and synaptic plasticity consolidation.

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“…Proteomics has been widely utilized to profile, screen and identify specific phenotype-or genotype-associated biomarkers (36,37). In recent years, SILAC has stood out when distinguishing the proteomics from one group to another, such as in cancer biomarker discovery (11,13) and in the identification of chemoresistance-associated biomarkers (16). In the present study, two rounds of SILAC procedures were performed with paired groups of H-labeled A549R cells and L-labeled A549 cells, or with paired groups of L-labeled A549R cells and H-labeled A549 cells.…”

Section: Discussionmentioning

confidence: 99%

“…However, the relatively low sensitivity and poor lower thresholds of microarray detection reduce its accuracy; thus, follow-up quantitative methods are required to confirm the results. Stable isotope labeling by amino acids in cell culture (SILAC) is effective in distinguishing the protein profiling from one group to the other (11)(12)(13). Five passages would transform ~97% of 12 C-labeled amino acids in A549 cells into 13 C-labeled amino acids [1-(1/2) 5 = 97%] and thus, cells only contain 'heavy' proteins (11,13).…”

Section: Profiling Of Apoptosis-and Autophagy-associated Molecules Inmentioning

confidence: 99%

“…Stable isotope labeling by amino acids in cell culture (SILAC) is effective in distinguishing the protein profiling from one group to the other (11)(12)(13). Five passages would transform ~97% of 12 C-labeled amino acids in A549 cells into 13 C-labeled amino acids [1-(1/2) 5 = 97%] and thus, cells only contain 'heavy' proteins (11,13). The incorporation of stable isotopes facilitates the quantitative recognition of the differences in expression profiles by tandem mass spectrometry between the 12 C-and 13 C-labeled A549 cells.…”

Section: Profiling Of Apoptosis-and Autophagy-associated Molecules Inmentioning

confidence: 99%

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Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture

Wang

Jiang

2019

Int J Oncol

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Cis-diammine-dichloro-platinum II-based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non-small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin-treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin-treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20 passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post-cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy-related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15 proteins [glucose-regulated protein, 78 kDa (GRP78), heat shock protein 71, pre-mRNA processing factor 19, polypyrimidine tract binding protein 1, translationally controlled tumor protein, Cathepsin D, Cytochrome c, thioredoxin domain containing 5, MutS homolog (MSH) 6, Annexin A2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor 1A interacting protein, MSH2, protein phosphatase 2A 55 kDa regulatory subunit Bα, Rho glyceraldehyde-3-phosphate-dissociation inhibitor 1 and ANXA4] that were upregulated by >1.5-fold in heavy (H)-and light (L)-labeled A549R cells. In addition, 16 and 14 proteins were downregulated by >1.5-fold in the H-and L-labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin-resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin-resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti-apoptosis and/or autophagy promotion.

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Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for Quantitative Proteomics

Cited by 36 publications

References 118 publications

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis

The roles of protein expression in synaptic plasticity and memory consolidation

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture

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