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MB Sample ID: SA092152
| Local Sample ID: | 17Dec22_24exo |
| Subject ID: | SU001337 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001337 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 17Dec22_24exo | SA092152 | FL013207 | Early | Cancer Stage |
Collection:
| Collection ID: | CO001331 |
| Collection Summary: | Ten mL samples of blood were drawn into a purple top vacutainer containing K2-EDTA (Becton-Dickson), inverted twice to ensure dissolution of the EDTA, and kept on ice immediately after blood draw. The whole blood was separated into packed red cells, buffy coat, and plasma within 30 min of collection by centrifuging at 3500 g for 15 min at 4 C in a swing out rotor. All blood processing procedures were performed in a class II biosafety cabinet housed in a BSL category 2 laboratory. Plasma (0.7 mL) was aliquotted into 1.5 mL screw cap vials, flash frozen in liq. N2, and stored at-80 C until exosomal isolation. These collection and processing procedures were designed to minimize variations in plasma and exosome quality. |
| Sample Type: | Blood (whole) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001352 |
| Treatment Summary: | No Treatment |
Sample Preparation:
| Sampleprep ID: | SP001345 |
| Sampleprep Summary: | Exosomes were isolated from plasma by differential ultracentrifugation adapted from Refs. [47,48]. 0.7 mL cleared plasma (see above) were placed in 5 41 mm polyallomer ultraclear ultracentrifuge tubes on ice, and centrifuged for 1 h at 70,000 g at 4 C in a SWTi55 swing out rotor (Beckman). The supernatant was recentrifuged at 100,000 g for 1 h at 4 C, and the pellet was drained and resuspended in 0.7 mL cold PBS, and recentrifuged at 100,000 g for 1 h at 4 C. The washed exosomal pellets were resuspended in 100 mL nanopure water, vortexed for 30 s and transferred to a fresh microcentrifuge tube. The ultracentrifuge tube was washed with another 100 mL of nanopure water, vortexed for 30 s and the wash was transferred into same microcentrifuge tube, using the same pipet tip. The combined exosome suspensions were then lyophilized except for a small portion that was used for characterization by particle size distribution analysis (see below). These nanoparticles are operationally defined as exosomes. The lyophilized EXO preparations were extracted for lipidic metabolites using a solvent partitioning method with CH3CN:H2O:CHCl3 (2:1.5:1, v/v) as described previously [49]. The resulting lipid extracts were vacuum-dried in a vacuum centrifuge (Eppendorf), redissolved in 200 mL CHCl3:CH3OH (2:1) with 1 mM butylated hydroxytoluene, which was further diluted 1:20 in isopropanol/CH3OH/CHCl3 (4:2:1) with 20 mM ammonium formate for UHR-FTMS analysis. |
| Sampleprep Protocol Comments: | A small fraction (<1%) of each exosome preparation was characterized by size distribution analysis using a Nanosight 300 (Malvern Instruments), which provided the distribution of the Stokes' radius (mean 60e66 nm) and the number density of the particles. A typical analysis is shown in Fig. S1. The method eliminates very small particles, and provides a strongly peaked, narrow distribution at the expected size for exosomes (40e100nm, observed mode of 60e65 nm for the main peaks in Figs. S1A and B). |
Combined analysis:
| Analysis ID | AN002109 |
|---|---|
| Analysis type | MS |
| Chromatography type | None (Direct infusion) |
| Chromatography system | Thermo Orbitrap Fusion |
| Column | none |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Fusion Orbitrap |
| Ion Mode | POSITIVE |
| Units | Ion Intensity |
Chromatography:
| Chromatography ID: | CH001539 |
| Instrument Name: | Thermo Orbitrap Fusion |
| Column Name: | none |
| Chromatography Type: | None (Direct infusion) |
MS:
| MS ID: | MS001960 |
| Analysis ID: | AN002109 |
| Instrument Name: | Thermo Fusion Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | High sample throughput ( 16 min total cycle time per sample, <7 min for MS1 portion) was achieved using the nanoelectrospray TriVersa NanoMate (Advion Biosciences, Ithaca, NY, USA) with 1.5 kV electrospray voltage and 0.4 psi head pressure. UHR-FTMS data were acquired from an Orbitrap Fusion Tribrid (Thermo Scientific, San Jose, CA, USA) set at a resolving power of 450,000 (at 200 m/z) for MS1 full scans using 10 microscans per scan in the m/z range of 150e1,600, achieving sub ppm mass accuracy through <1200 m/z in positive mode. AGC (Automatic Gain Control) target was set to 1e5 and maximal injection time was set to 100 ms. During the MS1 run, the top 500 most intense monoisotopic precursor ions were isolated via quadrupole using 1m/z isolation window and HCD (Higher Energy Collisional Dissociation) set at 25% collision energy was performed in positive mode for datadependent MS2 at a resolving power of 120,000 (at 200 m/z) to obtain fragments for acyl chain assignment and neutral loss of specific head groups. The AGC target was set to 5e4 with maximal injection time of 500 ms. MS2 does not distinguish the sn1 and sn2 acyl positions of glycerolipids, nor the position of unsaturations in acyl chains and acyl branching. Representative full scan MS along with an example MS2 spectrum are shown in Fig. S2. The UHR-FTMS raw data were assigned by our (CESB) in-house software PREMISE (PRecalculated Exact Mass Isotopologue Search Engine) that compares UHR-FTMS m/z data against our metabolite m/z library (calculated with mass accuracy to the 5th decimal point) to discern all known lipid MF and their 13C isotopologues, including hypothetical lipids, while simultaneously taking into account all of the major adducts (here Hþ, Naþ, Kþ and NHþ4 ) [50,51]. An in-house developed natural abundance (NA) correction algorithm [52,53] was applied to simultaneously examine the distribution of naturally occurring 13C isotopologues of the unlabeled lipids to help verify the assigned molecular formulae, and to eliminate non- monoisotopic 13C isotopologues from further analysis. For statistical classification, we used only high accuracy monoisotopic m/z values that mapped to lipid molecular formulae, and multiple adducts of each were tracked throughout to avoid redundancy. Below, such m/z values are referred to as “lipid features”, and neither molecular formulae nor lipid names were directly used. The number of assigned lipid features in each sample varied from 1 to 70. After combining all samples into a master file, the data set had a total of 430 such lipid features. Prior to multivariate statistical analyses, MS1 peaks arising from solvent blanks and known contaminants were removed from the lipid feature lists. As absolute intensities vary from sample to sample, the lipid features must be normalized. The intensities of the lipid features in each sample were thus normalized to the summed intensities of all mass peaks that were non-zero in 20%, 50%, 75%, 97%, 100% of all samples. This is equivalent to estimating the mole fraction of each lipid feature present, and therefore can be used for determining relative changes in composition. We found that normalization using the summed intensities of lipid features that were non-zero in 20% of all samples provided the best statistical outcome according to the ROC analysis. |
| Ion Mode: | POSITIVE |
