Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: Nanoparticle tracking analysis of EVs isolated from plasma using five different methods. ( a ) Representative particle size distribution from an EQ preparation showing counts versus diameter. ( b ) Mean particle diameter (nm) for each method (mean ± SD, n ≥ 3 independent preparations). No statistically significant differences were observed among methods by one-way ANOVA. ( c ) Particle yield is expressed as the number of particles isolated per mL of starting plasma (×10 10 particles per mL; mean ± SD, n ≥ 3 independent preparations). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test, and statistical significance is shown as *** p < 0.001, indicating that TI yielded significantly more particles than all other methods. EQ = ExoQuick, ES = ExoSpin, IZ = Izon qEV-35 nm column, TI = Total Exosome Isolation, T14 = Top14 abundant protein depletion kit, and PL = starting plasma.

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Isolation, Clinical Proteomics

Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: Comparative proteomic characterization of EV isolates vs. starting plasma. ( a ) MS1 image from each EV preparation was overlayed on image from PL (starting plasma) to identify shared features. Each spot on the image represents a peptide ion and has a cyan, red, or black color. The cyan color indicates the ion is predominantly present in the EV prep method, and the red color indicates the ion is predominantly present in the starting plasma PL, while the black color indicates the ion is common/overlaps between the EV prep method and PL. The numbers in parentheses indicate the percentage overlap with PL. ( b , c ) Total number of peptides ( b ) and their corresponding proteins ( c ) identified by each method, mean ± SD ( n ≥ 3 independent preparations). ( d , e ) Breakdown of peptides ( d ) and proteins ( e ) identified by each method into categories: “PL only” (detected in starting plasma only), “EV-only markers” (canonical EV marker proteins found only in EV isolates, not in plasma), “PL + EV markers” (canonical EV markers found in both plasma and EV isolates), and “Unique” (other proteins detected only in EV isolates and absent in plasma). Asterisks represent statistically significant differences (Kruskal–Wallis test, Dunn’s multiple-comparisons test, and adjusted p -values): * p < 0.05; ** p < 0.01; *** p < 0.001. EQ = ExoQuick, ES = ExoSpin, IZ = Izon qEV-35 nm column, TI = Total Exosome Isolation, T14 = Top14 abundant protein depletion kit, and PL = starting plasma.

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Clinical Proteomics, Marker, Isolation

Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: Relative levels of 26 abundant plasma proteins (including albumin and immunoglobulins) in the 5 EV preparations (EQ, ES, IZ, TI, and T14) and starting plasma PL. Tryptic digests (0.1 µg protein) of each EV preparation and starting PL were loaded onto nano-LC-MS/MS and analyzed by DDA. The peptide LFQ intensities for each indicated protein are plotted as log2 ± SD ( n ≥ 3 independent preparations) following mean normalization. Most of the proteins showed depletion depending on the EV preparation. Proteins in bold (from TF to APOA2) are supposed to be depleted by the T14 kit. EQ = ExoQuick, ES = ExoSpin, IZ = Izon qEV-35 nm column, TI = Total Exosome Isolation, T14 = Top14 abundant protein depletion kit, and PL = starting plasma.

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: Relative levels of 41 of the most frequently observed EV markers detected in the 5 EV preparations (EQ, ES, IZ, TI, and T14) and starting plasma PL. Tryptic digests (0.1 µg protein) of each EV preparation and starting PL were loaded onto nano-LC-MS/MS and analyzed by DDA. The peptide LFQ intensities for each indicated protein are plotted as log2 ± SD ( n ≥ 3 independent preparations) following mean normalization. Tetraspanins are shown in bold. EQ = ExoQuick, ES = ExoSpin, IZ = Izon qEV-35 nm column, TI = Total Exosome Isolation, T14 = Top14 abundant protein depletion kit, and PL = starting plasma.

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: Relative levels of 66 unique proteins detected in the 5 EV preparations (EQ, ES, IZ, TI, and T14) but absent in the starting plasma PL. Tryptic digests (0.1 µg protein) of each EV preparation and starting PL were loaded onto nano-LC-MS/MS and analyzed by DDA. The peptide LFQ intensities for each indicated protein are plotted as log2 ± SD ( n ≥ 3 independent preparations) following mean normalization. EQ = ExoQuick, ES = ExoSpin, IZ = Izon qEV-35 nm column, TI = Total Exosome Isolation, T14 = Top14 abundant protein depletion kit, and PL = starting plasma.

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: PRM analysis of positive and negative markers in 5 EV preparations (EQ, ES, IZ, TI, and T14), starting plasma PL, and positive control EV. Tryptic digests (0.1 µg protein) of each EV preparation and starting PL were analyzed by PRM for 50 peptides corresponding to the shown 24 proteins. The summed peptide intensities for each indicated proteins were mean normalized amongst the samples and plotted as the mean ± SD ( n ≥ 3 independent preps). Tetraspanins are shown in bold. EQ = ExoQuick, ES = ExoSpin, IZ = Izon qEV-35 nm column, TI = Total Exosome Isolation, T14 = Top14 abundant protein depletion kit, PL = starting plasma, and EV = EVs from A549 cells.

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Clinical Proteomics, Positive Control, Isolation

Journal: Proteomes

Article Title: Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery

doi: 10.3390/proteomes13030045

Figure Lengend Snippet: Assessment of EV enrichment for 5 EV preparations (EQ, ES, IZ, TI, and T14) relative to starting plasma PL and positive control EV. Relative levels of positive and negative EV markers (e.g., tetraspanins and albumin) from PRM results were used to calculate the ( a ) EV enrichment score and abundant protein contamination index, which were then used to calculate ( b ) the final composite score to rank the best EV isolation method for biomarker discovery. Results are plotted as the mean ± SD ( n ≥ 3 independent preps). EQ = ExoQuick (blue symbol and bar), ES = ExoSpin (red symbol and bar), IZ = Izon qEV-35 nm column (green symbol and bar), TI = Total Exosome Isolation (purple symbol and bar), T14 = Top14 abundant protein depletion kit (orange symbol and bar), PL = starting plasma (black symbol and bar), and EV = EVs from A549 cells (brown symbol and bar).

Article Snippet: The supernatant was used for the following isolation methods: Total Exosome Isolation Kit (Invitrogen, Burlington, ON, Canada), ExoQuick Ultra (System Biosciences, Palo Alto, CA, USA), ExoSpin (Cell Guidance Systems, St. Louis, MO, USA), High-SelectTM Top14 abundant protein depletion (ThermoFisher Scientific, San Jose, CA, USA), and qEV original 35 nM (Izon Science, Medford, MA, USA).

Techniques: Clinical Proteomics, Positive Control, Isolation, Biomarker Discovery