Combination of size‐exclusion chromatography and ion exchange adsorption for improving the proteomic analysis of plasma‐derived extracellular vesicles

Wang, Yaojie; Zhang, Ying; Li, Zhi; Wei, Sisi; Chi, Xiuping; Yan, Xi; Lv, Huilai; Zhao, Libo; Zhao, Lianmei

doi:10.1002/pmic.202200364

Cited by 8 publications

(4 citation statements)

References 62 publications

(60 reference statements)

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“…Exosomes were isolated from plasma and supernatant as previously reported [ 19 ]. Briefly, samples were first filtered through a 0.22 μM filter (SLGPR33RB, Millipore, USA).…”

Section: Methodsmentioning

confidence: 99%

LncRNA CCAT1 facilitates the progression of gastric cancer via PTBP1-mediated glycolysis enhancement

Zhang,

Wang,

Liu

et al. 2023

J Exp Clin Cancer Res

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Background Gastric cancer (GC) is one of the most prevalent malignant tumors of the digestive system. As a hallmark of cancer, energy-related metabolic reprogramming is manipulated by multiple factors, including long non-coding RNAs (lncRNAs). Notably, lncRNA CCAT1 has been identified as a crucial regulator in tumor progression. Nevertheless, the precise molecular mechanisms underlying the involvement of CCAT1 in metabolic reprogramming of GC remain unclear. Methods Gain- and loss-of-function experiments were performed to evaluate the roles of CCAT1 in tumorigenesis and glycolysis of GC. Bioinformatics analyses and mechanistic experiments, such as mass spectrometry (MS), RNA-pulldown, and RNA immunoprecipitation (RIP), were employed to reveal the potential interacting protein of CCAT1 and elucidate the regulatory mechanism of CCAT1 in GC glycolysis. Moreover, the nude mice xenograft assay was used to evaluate the effect of CCAT1 on GC cells in vivo. Results In this study, we identified that CCAT1 expression was significantly elevated in the tissues and plasma exosomes of GC patients, as well as GC cell lines. Functional experiments showed that the knockdown of CCAT1 resulted in a substantial decrease in the proliferation, migration and invasion of GC cells both in vitro and in vivo through decreasing the expression of glycolytic enzymes and glycolytic rate. Conversely, overexpression of CCAT1 exhibited contrasting effects. Mechanistically, CCAT1 interacted with PTBP1 and effectively maintained its stability by inhibiting the ubiquitin-mediated degradation process. As a critical splicing factor, PTBP1 facilitated the transition from PKM1 to PKM2, thereby augmenting the glycolytic activity of GC cells and ultimately fostering the progression of GC. Conclusions Our findings demonstrate that CCAT1 plays a significant role in promoting the proliferation, migration, and invasion of GC cells through the PTBP1/PKM2/glycolysis pathway, thus suggesting CCAT1’s potential as a biomarker and therapeutic target for GC.

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“…Exosomes were isolated from plasma and supernatant as previously reported [ 19 ]. Briefly, samples were first filtered through a 0.22 μM filter (SLGPR33RB, Millipore, USA).…”

Section: Methodsmentioning

confidence: 99%

LncRNA CCAT1 facilitates the progression of gastric cancer via PTBP1-mediated glycolysis enhancement

Zhang,

Wang,

Liu

et al. 2023

J Exp Clin Cancer Res

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“…Proteomic analysis was performed on SGC-7901 cells with stably knocked down CCAT1 and corresponding control cells using an HPLC Easy-nLC1200 system (Thermo Fisher Scienti c, USA) and a Q Exactive HF mass spectrometer (Thermo Fisher Scienti c, USA) as previously described [19], with three biologic replicates per group. Cells were sampled, and 3552 proteins were quanti ed.…”

Section: Proteomic Analysismentioning

confidence: 99%

LncRNA CCAT1 facilitates the progress of gastric cancer via the PTBP1/glycolysis axis

wang

Zhao

et al. 2023

Preprint

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Background: Gastric cancer (GC) is one of the most common malignant tumors of the digestive system. As a hallmark of cancer, energy-related metabolic reprogramming was manipulated by various factors, including lncRNAs. It has been shown that lncRNA CCAT1 is a key regulator involved in tumor development. Nevertheless, the exact underlying molecular mechanisms by which lncRNA CCAT1 acts in GC metabolic reprogramming are yet to be elucidated. Methods: The expression of CCAT1 in GC tissues, serum, and exosome that was isolated from plasma and GC cell lines were detected by qRT-PCR. The gain and loss-function assays were performed to explore the role of CCAT1 on GC cells. Xenograft tumor formation models in nude mice were performed to estimate the proliferation of GC cells with CCAT1 stably knocking down in vivo. The proteins interacting with CCAT1 were first analyzed by online databases and further confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assays. The expression of glycolytic signaling pathway-related proteins were probed using western blotting and immunohistochemistry (IHC). Results: In this study, we identified that CCAT1 was remarkably enhanced in the tissues, serum, and plasma exosomes of GC patients as well as in GC cell lines. Functional experiments showed that knockdown of CCAT1 significantly reduced the proliferation, migration and invasion of GC cells in vitro and in vivo, and also decreased glycolytic rate and the expression of glycolytic enzymes in GC cells, whereas overexpression of CCAT1 had opposing effects. Mechanically, CCAT1 interacted with PTBP1 and maintained its stability by inhibiting the ubiquitin-mediated degradation. As a critical splicing factor, PTBP1 induced a switch from PKM1 to PKM2, leading to an increase in the glycolysis of GC cells and ultimately promoting GC progression. Conclusions: Our study exhibited that CCAT1 contributed to GC proliferation, migration and invasion via PTBP1 / glycolysis axis, making it a potential biomarker and therapeutic target in GC patients.

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“…Size‐exclusion chromatography has issues with sample loss and size overlap (Lozano‐Ramos et al., 2015 ). The application of charge‐based techniques is limited by the presence of complex charged biomolecules (Wang et al., 2023 ). Therefore, alternative methods such as immunoaffinity magnetic beads (MBs) that specifically target specific EV surface biomarkers have emerged as a viable approach.…”

Section: Introductionmentioning

confidence: 99%

Isolation and quantification of L1CAM‐positive extracellular vesicles on a chip as a potential biomarker for Parkinson's Disease

Li,

Zou,

Huang

et al. 2024

J of Extracellular Vesicle

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Extracellular vesicles (EVs) carry disease‐specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated biochip platform, termed EVID‐biochip (EVs identification and detection biochip), which integrates in situ electrochemical protein detection with on‐chip antifouling‐immunomagnetic beads modified with CD81 antibodies and zwitterion molecules, enabling efficient isolation and detection of neuronal EVs. The capability of the EVID‐biochip to isolate common EVs and detect neuronal EVs associated with Parkinson's disease in human serum is successfully demonstrated, using the transmembrane protein L1‐cell adhesion molecule (L1CAM) as a target biomarker. The EVID‐biochip exhibited high efficiency and specificity for the detection of L1CAM with a sensitivity of 1 pg/mL. Based on the validation of 76 human serum samples, for the first time, this study discovered that the level of L1CAM/neuronal EV particles in serum could serve as a reliable indicator to distinguish Parkinson's disease from control groups with AUC = 0.973. EVID‐biochip represents a reliable and rapid liquid biopsy platform for the analysis of complex biofluids offering EVs isolation and detection in a single chip, requiring a small sample volume (300 µL) and an assay time of 1.5 h. This approach has the potential to advance the diagnosis and biomarker discovery of various neurological disorders and other diseases.

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Combination of size‐exclusion chromatography and ion exchange adsorption for improving the proteomic analysis of plasma‐derived extracellular vesicles

Cited by 8 publications

References 62 publications

LncRNA CCAT1 facilitates the progression of gastric cancer via PTBP1-mediated glycolysis enhancement

LncRNA CCAT1 facilitates the progression of gastric cancer via PTBP1-mediated glycolysis enhancement

LncRNA CCAT1 facilitates the progress of gastric cancer via the PTBP1/glycolysis axis

Isolation and quantification of L1CAM‐positive extracellular vesicles on a chip as a potential biomarker for Parkinson's Disease

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