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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210109	2/3	Homo sapiens	Blood plasma	Asymmetric flow field-flow fractionation	Cai, Tanxi	2021	75%
Study summary Full title LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles All authors Tanxi Cai, Qing Zhang, Bowen Wu, Jifeng Wang, Na Li, Tingting Zhang, Zhipeng Wang, Jianjun Luo, Xiaojing Guo, Xiang Ding, Zhensheng Xie, Lili Niu, Weihai Ning, Zhen Fan, Xiaowei Chen, Xiangqian Guo, Runsheng Chen, Hongwei Zhang, Fuquan Yang Journal J Extracell Vesicles Abstract Advancements in omics-based technologies over the past few years have led to the discovery of numero (show more...)Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Asymmetric flow field-flow fractionation Protein markers EV: TSG101/ CD81/ HSP90/ CD63/ CD9 non-EV: APOB/ APOA1 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Asymmetric flow field-flow fractionation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ HSP90/ TSG101/ CD81 Not detected contaminants APOA1/ APOB Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 144.2+/-2.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.00E+10 EM EM-type Transmission-EM Image type Wide-field

	EV210109	3/3	Homo sapiens	Blood plasma	Asymmetric flow field-flow fractionation	Cai, Tanxi	2021	75%
Study summary Full title LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles All authors Tanxi Cai, Qing Zhang, Bowen Wu, Jifeng Wang, Na Li, Tingting Zhang, Zhipeng Wang, Jianjun Luo, Xiaojing Guo, Xiang Ding, Zhensheng Xie, Lili Niu, Weihai Ning, Zhen Fan, Xiaowei Chen, Xiangqian Guo, Runsheng Chen, Hongwei Zhang, Fuquan Yang Journal J Extracell Vesicles Abstract Advancements in omics-based technologies over the past few years have led to the discovery of numero (show more...)Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Giloma cancer patients Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Asymmetric flow field-flow fractionation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Asymmetric flow field-flow fractionation Characterization: Protein analysis Protein Concentration Method BCA Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210109	1/3	Homo sapiens	Glioma cells	(d)(U)C	Cai, Tanxi	2021	67%
Study summary Full title LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles All authors Tanxi Cai, Qing Zhang, Bowen Wu, Jifeng Wang, Na Li, Tingting Zhang, Zhipeng Wang, Jianjun Luo, Xiaojing Guo, Xiang Ding, Zhensheng Xie, Lili Niu, Weihai Ning, Zhen Fan, Xiaowei Chen, Xiangqian Guo, Runsheng Chen, Hongwei Zhang, Fuquan Yang Journal J Extracell Vesicles Abstract Advancements in omics-based technologies over the past few years have led to the discovery of numero (show more...)Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81/ CD63/ CD9/ Annexin A1 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioma cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Cell count 4.00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 70 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Annexin A1/ Alix/ CD81 Not detected contaminants Calnexin Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124.9+/-7.9 EV concentration Yes Particle yield Yes, as number of particles per million cells 4.00E+08 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV210109
species	Homo sapiens
sample type	Blood plasma	Blood plasma	Cell culture
cell type	NA	NA	Glioma cells
medium	NA	NA	EV-depleted medium
condition	Control condition	Giloma cancer patients	Control condition
separation protocol	Asymmetric flow field-flow fractionation	Asymmetric flow field-flow fractionation	(d)(U)C
Exp. nr.	2	3	1
EV-METRIC %	75	75	67