Methods and systems for preparing and analyzing samples for extracellular vesicle biomarkers associated with brain-related conditions or disorders

Abstract

Disclosed herein are embodiments related to analyzing brain-derived extracellular vesicles (EVs). Some methods described herein comprise: (a) enriching a biological sample for brain-derived EVs, the biological sample being from a subject having a brain-related condition or disorder and comprising the brain-derived EVs; (b) subjecting the enriched sample to EV disruption to produce an analytic sample; (c) purifying the analytic sample to capture EV material after the EV disruption; and (d) analyzing the captured EV material to determine a differential amount of the captured EV material relative to a control. The method allows for improving 10 outcomes for patients suffering from brain-related conditions or disorders.

Description

ATTORNEY DOCKET NO. 223711-700001 / PCTMETHODS AND SYSTEMS FOR PREPARING AND ANALYZING SAMPLES FOR EXTRACELLULAR VESICLE BIOMARKERS ASSOCIATED WITH BRAIN- RELATED CONDITIONS OR DISORDERSCROSS REFERENCE

[0001] This application claims the benefit under 35 U. S. C. § 119(e) of U. S. Provisional Application No. 63 / 743,329 filed January 9, 2025, with the entire disclosure incorporated herein by reference m its entirety'.BACKGROUND

[0002] The blood-brain barrier (BBB) is generally known to inhibit material in the blood from easily passing into the brain, and likewise inhibits material m the brain, such as extracellular (EVs), from reaching the bloodstream. Analyzing biomarkers in the brain is conventionally performed using a biopsy of the brain, which can be complex and risk adverse effects on the subject due to removal of brain tissue or interference with bram function from the biopsy. There is a need for less burdensome methods of analyzing biomarkers in the brain for evaluating brain conditions.SUMMARY

[0003] In some aspects, the techniques described herein relate to a method including: (a) enriching a biological sample for bram-derived extracellular vesicles (EVs), the biological sample being from a subject having a brain-related condition or disorder and including the brain-derived EVs; (b) subjecting the enriched sample to EV disruption to produce an analytic sample; (c) purifying the analytic sample to capture EV material after the EV disruption; and (d) analyzing the captured EV material to determine differential amounts of the captured polypeptides relative to a control. In some aspects, the techniques described herein relate to a method, wherein the analyzing includes: generating polypeptides from the purified EV material; eluting the polypeptides from the purified analytic sample and loading the eluted polypeptides onto an analytical column; separating the polypeptides on the analytical column; and, detecting separated polypeptides with a detector. In some aspects, the techniques described herein relate to a method, wherein the biological sample includes blood, serum, plasma, saliva, cerebrospinal fluid, amniotic fluid, or urine. In some aspects, the techniques described herein relate to a method, wherein the brain-related condition or disorder is selected from traumatic brain injury, neurodegenerative disease, metabolic diseasesACTIVE 717682885v4 IATTORNEY DOCKET NO. 223711-700001 / PCTinvolving the nervous system, neurotoxicity, diabetes, spontaneous, birth-related neurological deficits, or post-natal, trauma-related neurological deficits. In some aspects, the techniques described herein relate to a method, wherein the biological sample is enriched for EVs by one or more of size exclusion chromatography, ultrafiltration, magnetic beads, magnetic beads coupled to protein capture agents, reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, or polystyrene-divinylbenzene chromatography. In some aspects, the techniques described herein relate to a method, wherein the brain-derived EVs include exosomes. In some aspects, the techniques described herein relate to a method, wherein the EV disruption includes exposure to one or more of urea, a reducing agent, or iodoacetamide. In some aspects, the techniques described herein relate to a method, wherein the reducing agent is DTT, DTE, mercaptoethanol, tris (2-carboxyethyl) phosphine, or glutathione. In some aspects, the techniques described herein relate to a method, wherein the analytic sample is subjected to proteolytic digestion, wherein the proteolytic digestion includes exposing the sample to cyanogen bromide (CnBr) or to one or more proteases. In some aspects, the techniques described herein relate to a method, wherein the one or more proteases include an aminopeptidase, a carboxypeptidase, trypsin, chymotrypsin, bromelian, papain, pronase, or proteinase k. In some aspects, the techniques described herein relate to a method, wherein the detecting includes adding stable isotope standard peptides for detecting specific protein markers to the analytical sample. In some aspects, the techniques described herein relate to a method, wherein the purifying includes performing chromatography selected from reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, or polystyrene-divinlybenzene chromatography. In some aspects, the techniques described herein relate to a method, wherein a purification column includes a high-performance liquid chromatography (HPLC) column. In some aspects, the techniques described herein relate to a method, wherein the high-performance liquid chromatography (HPLC) column includes a reverse phase material selected from Cl 8 or C6. In some aspects, the techniques described herein relate to a method, the uncaptured material includes material selected from salts and macromolecules or complexes having a size greater than 5.5 kD or the macromolecule is a polypeptide greater than about 50 amino acids long. In some aspects, the techniques described herein relate to a method, wherein the analytical column includes a high- performance liquid chromatography (HPLC) column. In some aspects, the techniques described herein relate to a method, wherein the HPLC column includes a reverse-phase material selectedACTIVE 717682885v4ATTORNEY DOCKET NO. 223711-700001 / PCTfrom Cl 8 or C6. In some aspects, the techniques described herein relate to a method, wherein the detecting includes providing separated analytes to the detector through a fluid connection between the analytical column and the detector. In some aspects, the techniques described herein relate to a method, wherein the detector detects analytes by mass spectrometry. In some aspects, the techniques described herein relate to a method, wherein the detector includes a mass spectrometer selected from quadrupole mass spectrometer or time-of-flight mass spectrometer. In some aspects, the techniques described herein relate to a method, wherein the detector includes a tandem mass spectrometer. In some aspects, the techniques described herein relate to a method, wherein the detector includes a triple quadrupole mass spectrometer. In some aspects, the techniques described herein relate to a method, wherein the detector includes a triple quadrupole mass spectrometer and detection includes multiple reaction monitoring. In some aspects, the techniques described herein relate to a method, wherein the detector includes a linear ion trap mass spectrometer and detection includes full-scan Synchronous Precursor Selection (SPS) MS3acquisition.

[0004] In some aspects, the techniques described herein relate to a system including: (a) an extracellular vesicle (EV) purification column, the EV purification column being configured to purify one or more of proteins or peptides from circulating brain-derived EVs in a biological sample, the EV purification column including (a) a first solution for enriching the biological sample for the brain-derived EVs and (b) a second solution for subjecting the enriched biological sample to EV disruption to produce an analytic sample; (b) a multi-port valve connected through a first fluid conduit to a column containing the EV purification column; and (c) an analytical column connected through a second fluid conduit to the multi-port valve, the analytical column being configured to determine a differential amount of captured EV material from the analytic sample relative to a control sample. In some aspects, the techniques described herein relate to a system, wherein the EV purification column further includes stable isotope standard polypeptides. In some aspects, the techniques described herein relate to a system, further including an outlet fluid conduit connecting an outlet port to the multi-port valve. In some aspects, the techniques described herein relate to a system, further including a detector fluid conduit connecting a detector to the analytical column. In some aspects, the techniques described herein relate to a system, further including a pump fluid conduit connecting a pump connected to the EV purification column. In some aspects, the techniques described herein relate to a system, wherein the EV purification column is selected from a reverse-phase chromatography column, an anion exchangeACTIVE 717682885v4 3ATTORNEY DOCKET NO. 223711-700001 / PCTchromatography column, a cation exchange chromatography column, a polystyrene-divinylbenzene column or a size exclusion column. In some aspects, the techniques described herein relate to a system, wherein the EV purification column includes a high-performance liquid chromatography (HPLC) column. In some aspects, the techniques described herein relate to a system, wherein the HPLC column includes a reverse-phase material selected from Cl 8 or C6. In some aspects, the techniques described herein relate to a system, wherein the analytical column includes a high- performance liquid chromatography (HPLC) column. In some aspects, the techniques described herein relate to a system, wherein the detector includes a mass spectrometer. In some aspects, the techniques described herein relate to a system, wherein the mass spectrometer is selected from a quadrupole mass spectrometer or a time-of-flight mass spectrometer. In some aspects, the techniques described herein relate to a system, wherein the detector includes a tandem mass spectrometer. In some aspects, the techniques described herein relate to a system, wherein the detector includes a triple quadrupole mass spectrometer. In some aspects, the techniques described herein relate to a system, wherein the detector includes a linear ion trap mass spectrometer. In some aspects, the techniques described herein relate to a system, wherein the EVs include exosomes.

[0005] In some aspects, the techniques described herein relate to a method including: (a) providing a system including: (i) an extracellular vesicle (EV) purification column; (ii) a multi¬ port valve connected through a first fluid conduit to the EV purification column; (iii) an analytical column connected through a second fluid conduit to the multi-port valve; (iv) an outlet port connected to the multi-port valve through outlet fluid conduit; and (v) a detector connected through a detector fluid conduit to the analytical column; (b) loading the EV purification column with a biological sample, the biological sample including circulating brain-derived EVs, the circulating brain-derived EVs including one or a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, and complex lipids, (c) flowing at least a first solution and a second solution through the biological sample to produce an analytic sample such that the analytic sample passes through the first fluid conduit, the multi-port valve, and a third fluid conduit, wherein the first solution includes a first composition for enriching the biological sample for the brain-derived EVs, and wherein the second solution includes for subjecting the enriched biological sample to EV disruption; (d) washing the EV purification column such that a wash passes through the first fluid conduit, the multi-port valve, and the third fluid conduit; (e) eluting the analytical columnACTIVE 717682885v4 4ATTORNEY DOCKET NO. 223711-700001 / PCTsuch that an eluate passes through the first fluid conduit, the multi-port valve and the second fluid conduit onto the analytical column; (f) separating the eluate on the analytical column; (g) passing the separated eluate through the detector fluid conduit to the detector; and (h) detecting one or more components of the separated eluate to determine a differential amount of the one or more components from the analytic sample relative to a control sample.

[0006] In some aspects, the techniques described herein relate to a method including: loading an extracellular vesicle (EV) purification column with a biological sample, the biological sample comprising circulating brain-derived EVs, the circulating brain-derived EVs comprising one or a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, and complex lipids, flowing at least a first solution and a second solution through the biological sample to produce an analytic sample such that the analytic sample passes through a multi-port valve connected through a first fluid conduit to the EV purification column, wherein the first solution comprises a first composition for enriching the biological sample for the brain-derived EVs, and wherein the second solution comprises a second composition for subjecting the enriched biological sample to EV disruption; washing the EV purification column such that a wash passes through the first fluid conduit, the multi-port valve, and the third fluid conduit; eluting an analytical column connected through a second fluid conduit to the multi-port valve such that an eluate passes through the first fluid conduit, the multi-port valve and the second fluid conduit onto the analytical column; separating the eluate on the analytical column; passing the separated eluate through a detector fluid conduit to a detector, wherein the detector is connected through the detector fluid conduit to the analytical column; and detecting one or more components of the separated eluate to determine a differential amount of the one or more components from the analytic sample relative to a control sample. In some aspects, the techniques described herein relate to a method, wherein detecting includes mass spectrometry. In some aspects, the techniques described herein relate to a method, wherein mass spectrometry includes multiple reaction monitoring mass spectrometry. In some aspects, the techniques described herein relate to a method, wherein mass spectrometry includes full-scan Synchronous Precursor Selection (SPS) MS3acquisition. In some aspects, the techniques described herein relate to a method, wherein the analytical sample of step (a) includes between about 1 pg and about 200 pg of protein or peptide. In some aspects, the techniques described herein relate to a method, wherein the analytical sample includes disrupted exosomes / extracellular vesicles that have been subjected to a proteolytic treatment. In some aspects, the techniquesACTIVE 717682885v4 5ATTORNEY DOCKET NO. 223711-700001 / PCTdescribed herein relate to a method, wherein the analytical sample includes a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, lipids, complex lipids, salts, or metals. In some aspects, the techniques described herein relate to a method, wherein the sample includes a homogenate of cells or a tissue homogenate.

[0007] Methods and systems of the presently disclosed embodiments were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages will be apparent from the detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A-1D illustrate exemplary optical quantification and protein measurements from extracellular vesicles (EVs) and plasma / serum proteins fractionated using size exclusion chromatography SEC) columns for common elution reagents (FIG. 1A) relative to 5 mM TEAA buffer (FIG. IB), 25 mM TEAA buffer (FIG. 1C), or 50 mM TEAA buffer (FIG. ID);

[0009] FIG. 2 is a flowchart of an example method for analyzing brain-derived extracellular vesicles (EVs) using MS; and

[0010] FIGS.3A-3B illustrate an exemplary process for a lateral flow (LF) system (FIG. 3A) and embodiment of an LF device (FIG. 3B) for measuring target analytes.

[0011] While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.DETAILED DESCRIPTION

[0012] Disclosed are various embodiments of techniques for analyzing a subject having a brain-related condition. In some embodiments, a brain-related condition is related to trauma or the like, such as, for example, a traumatic brain injury (TBI), neuroinflammation, or a related condition. In some embodiments, a bram-related condition is not linked to trauma, e.g., a neurode enerative disease, metabolic diseases involving the nervous system, neurotoxicity, diabetes, birth-related neurological deficits, or post-natal, trauma-related neurological deficits. In some embodiments, a method includes enriching a biological sample for brain-derived extracellularACTIVE 717682885v4 6ATTORNEY DOCKET NO. 223711-700001 / PCTvesicles (EVs), the biological sample being from a subject having a brain-related condition or disorder and comprising the brain-derived EVs. In some embodiments, the enriched sample may be subjected to EV disruption to produce an analytic sample, which is then purified to capture EV material after the EV disruption. In some embodiments, the captured EV material are then analyzed to determine an elevated or a differential amount of the captured EV material relative to a control. In some embodiments, the EV material comprises proteins, polypeptides, peptides, or the like.

[0013] EVs are small spheres of membranes pinched off or extruded from cells and tissues of the body that are generated by all cells of the body and carry biological molecules such as nucleic acids, proteins, lipids, and metabolites. EVs can be divided according to their size in exosomes (about 1-150 nm), microvesicles (about 100-1000 nm), and apoptotic bodies (about 1- 10 pm). As used herein, the term "exosome" refers to a cell-derived vesicle comprising a membrane enclosing an internal space. Using their internal space, exosomes are little message carriers of communication between nearby and long-distance cells and affect various aspects of health and disease throughout the body.

[0014] An advantage of the existence of EVs such as exosomes is their ability to enter various biological fluids, including blood, urine, saliva, or the like, thus allowing for their capture in diagnostic purposes. However, the history of the isolation of exosomes from biological fluids includes the use of large, expensive centrifuges and procedures that can take hours and even days. The analysis of exosomes using such burdensome methods is mostly for biology research. However, others have used exosomes for clinical purposes, e.g., for prenatal diagnostics and for prostate cancer, as well as in human clinical trials for the development of therapeutics for conditions like arthritis, chronic pain, and regenerative medicine. Others have developed topical therapies for wound healing that currently use exosomes, including post-cosmetic surgery to reduce scarring, while others are developing exosome-based therapeutic agents for TBI with interest in a diagnostic for ongoing brain injury that might indicate improvements during therapy with their agents.

[0015] Conventional systems for protein analysis rely on affinity reagents or capture agents, such as antibodies or aptamers, and their interactions with targets on the exosomes — membrane- associated and integral membrane proteins — and then breaking the exosomes open (e.g., via lysis) while they are attached to the affinity molecules. The affinity reagents may be attached to a solid support beforehand, say a column (i.e., an affinity column), and the biological sample is incubatedACTIVE 717682885v4 7ATTORNEY DOCKET NO. 223711-700001 / PCTwith or passed over the immobilized affinity reagents on the solid support, such as through liquid chromatography. Different biological samples (e.g., blood, urine, etc.) may be used for such a process.

[0016] While there are some advantages to a binding interaction between an exosome membrane marker and an interacting affinity reagent, such an approach is biased in that the population of EV s captured d epends on a previous decision of what population to remove with the reagents. Such an approach makes new discovery dependent on preconceived notions of what populations of EVs for which the sample should be enriched. The information gained from selecting a limited sub-population of EVs is thus biased to those preconceived notions.

[0017] While this bias is known, it is conventionally recognized as a necessary aspect of analysis of EVs, as a biological sample including EVs would typically include a massive and diverse set of EVs, with only a very minute amount of any particular type of EV in the sample. The scale of EVs and scale of diversity of EVs means that, conventionally, a choice needed to be made of which EVs may be relevant and should be searched for in a sample and analyzed if found.

[0018] This conventional approach to EV analysis also made for limitations on what EVs were searched for and how EVs were used in analysis. While a peripheral blood / tissue sample (e.g., a sample from an extremity of a subject) could be taken and analyzed, such a peripheral blood / tissue sample would often include EVs from that periphery or nearby parts of the subject. EVs from more distant parts would not be present in the sample, or would be present in even fewer numbers than the small numbers of EVs from nearby parts. An EV analysis may therefore focus on EVs from parts of the body that could be reliably analyzed with a peripheral blood / tissue sample.

[0019] These limitations impeded analysis of brain conditions or disorders using EVs. The blood-brain barrier (BBB) inhibits material in the blood from easily passing into the brain, and likewise inhibits material in the brain, including EVs, from reaching the bloodstream. This means brain EVs in the bloodstream number even lower than other types of EVs. A peripheral blood sample might be said to include a vanishingly small number of brain EVs, which deterred brain EV analysis. As a result, conventionally, EVs were not used to analyze brain conditions or disorders. This was particularly the case when the sample was taken as a peripheral blood sample. If a researcher were even interested in analyzing brain EVs, they naturally would have taken a biopsy of the brain. Brain biopsies are, however, generally avoided given the complexity ofACTIVE 717682885v4 8ATTORNEY DOCKET NO. 223711-700001 / PCTreaching the brain through the skull, and the risk of adverse effects on the subject due to removal of brain tissue or interference with brain function from the biopsy.

[0020] The inventors have recognized and appreciated, however, that brain EV s can be reliably analyzed for diagnostic purposes. Brain conditions or disorders can be identified and / or monitored through analysis of brain EVs in a sample, including a peripheral sample such as a peripheral tissue or blood sample. The amounts of EVs present in the sample, including absolute amounts of EVs or relative amounts of EVs, or other manner of characterizing brain EVs present in the sample can indicate a brain condition or disorder, such as traumatic brain injury.

[0021] More particularly, the inventors recognized and appreciated the utility of enriching EVs (which may include brain EVs) using an unbiased approach of isolating vesicles based on size, rather than limiting enrichment based on expression of particular molecules. The inventors have further recognized how to adapt such an enrichment approach for various technologies, including mass spectrometry (MS) or lateral flow (LF), discussed in more detail below. MS, for example, allows for greater specificity for measuring biomarkers of interest as it is a protein sequencespecific approach, not a binding interaction to detect a protein like enzyme-linked immunosorbent assays (ELISAs). Thus, MS is one of the most specific methods for detecting a molecule, more-so than affinity agents, and it results in absolute quantification of the markers with high precision. Alternatively, while LF depends on affinity agents for the final detection, the inventors have recognized its use in preliminary screening for a diagnostic target of interest that benefits from a size-based enrichment process.

[0022] Using enrichment methods and compositions described herein, the inventors have demonstrated that they can rapidly detect brain injuries such as TBI, especially those injuries not severe enough to be easily noticed but can affect performance in various contexts, e.g., on a battlefield. In particular, by using such an enrichment scheme, there is little to no need for affinity agents to purify a targeted population of EVs (e.g. EVs sourced from cardiac cells, hepatic cells, or others) because this enrichment allows for sensitive enough analysis to measure molecules in a sub-population of EVs within an entire sample. Further, such enrichment avoids loss of populations of EVs that might express non-target surface markers that would be lost using a biased enrichment approach, allowing for broader discovery for new targets.

[0023] Accordingly, some of the examples described herein include techniques that may be useful in identifying EVs at lower concentrations in samples than those previously attainable inACTIVE 717682885v4 9ATTORNEY DOCKET NO. 223711-700001 / PCTvarious biological samples. For example, due to the tight connections between cells that form the blood-brain-barrier (BBB), biological targets sourced from brain cells are only present in low concentrations in circulation. Using the enrichment techniques herein, EVs small enough to cross the BBB and enter circulation can thus be concentrated in test samples, and their contents can be analyzed for identifying brain-related injuries. As indicated above, some embodiments for analysis may include a) MS-based methods for identifying precise markers, and b) LF-based systems and devices for portable and rapid diagnostics. These specific analytical schemes may be selected based on the target application and their context for use.I. EV Enrichment Methods and Compositions

[0024] Provided herein are examples of methods and compositions for enriching a biological sample for brain-derived EVs. In some embodiments, a biological sample is from a subject having a brain-related condition or disorder and including the brain-derived EVs.

[0025] In some embodiments, the biological sample includes blood, serum, plasma, saliva, cerebrospinal fluid, amniotic fluid, or urine. In some embodiments, the biological sample is from a subject suffering from a brain-related condition or disorder. In some embodiments, the brain-related condition or disorder is selected from traumatic brain injury, neurodegenerative disease, metabolic diseases involving the nervous system, neurotoxicity, diabetes, birth-related neurological deficits, post-natal, trauma-related neurological deficits, or any other condition described herein.

[0026] In some embodiments, the biological sample comprises at least one EV. In some embodiments, the biological sample comprises at least one exosome. In some embodiments, the biological sample comprises at least one target analyte, described in more detail below.

[0027] In some embodiments, biological samples are enriched for EVs through size selection. In some embodiments, EVs are directly captured. In some embodiments, cells are captured and EVs are collected from the remaining sample. In some embodiments, the biological sample is enriched for EVs by one or more of size exclusion chromatography, ultrafiltration, magnetic beads, magnetic beads coupled to protein capture agents, or reverse-phase and other chromatography. In some embodiments, the brain-derived EV s include exosomes.ACTIVE 717682885v4 10ATTORNEY DOCKET NO. 223711-700001 / PCT

[0028] In some embodiments, a composition for enriching brain-derived EVs includes a buffer. In some embodiments, compositions comprise one or more buffers. Non-limiting examples of buffer includes phosphate buffered saline (PBS); triethylammonium acetic acid (TEAA); 4-(2-hydroxyethyl)-! -piperazineethanesulfonic acid (HEPES); 2-(N-Morpholino)-ethanesulfonic acid (MES); piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES); 3-(N-morpholino)propanesulfonic acid (MOPS); 2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-l-sulfonic acid (TES); N, N-Bis(2-hydroxyethyl)glycine (Bicine); 3-[4-(2-hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acid (HEPPS or EPPS); N-[l,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]glycine (Tricine); and 2-amino-2-(hydroxymethyl)propane-l,3 -diol (Tris).

[0029] In some embodiments compositions include buffers comprising mixed ion pairing agents, which allow for achieving a neutral system for isolation of proteins. A neutral pH allows for enhanced fractionation of sialic acid containing peptides, proteins, and / or phosphopeptides. In some embodiments, compositions include buffers that are adjusted for minimal ion pairing of proteins. Such enrichment allows for effective detection of low concentrations of analytes in a biological sample.

[0030] In an exemplary embodiment, TEAA is used as a buffer reagent for enriching a biological sample. TEAA is effective for anion exchange while minimizing cation exchange. TEAA is conventionally used m oligonucleotide purification but not for protein isolation. TEAA can be evaporated away easily, allowing for leaving a relatively pure preparation of EVs in a biological sample or analytical sample for downstream processing, such as for MS- or LF-based diagnostics. In some embodiments, a TEAA buffer reagent may be used with size exclusion columns (SEC), which separates the EVs by size and removes serum / plasma proteins (soluble proteins), circulating “free” nucleic acids, and small molecules, like metabolites, and salts from the EVs, resulting in a population of EVs suspended in TEAA. The TEAA can then be evaporated, leaving behind a relatively pure population of EV, often in the form of a powder. In some embodiments, the EV s are then dissolved in solutions and buffers suitable for either MS or LF analysis.

[0031] FIGS. 1A-1D illustrate exemplary optical quantification and protein measurements from extracellular vesicles (EVs) and plasma / serum proteins fractionated using size exclusion chromatography SEC) columns for common elution reagents (FIG. 1 A) relative to 5 mM TEAAACTIVE 717682885v4 11ATTORNEY DOCKET NO. 223711-700001 / PCTbuffer (FIG. IB), 25 mM TEAA buffer (FIG. 1C), or 50 ruM TEAA buffer (FIG ID). A unique separation between the A320 peak (EVs) and BCA peak (plasma / serum proteins) is seen, represented by a “bridge”. This “bridge” that appears between the vesicular and soluble protein peaks suggests a population of EVs that offers a purer form of exosomes.II. Mass Spectrometry-based analysis

[0032] Provided herein are examples of methods and systems for analyzing brain-derived extracellular vesicles (EVs) using mass spectrometry (MS).

[0033] In some embodiments, a method for analyzing brain-derived EVs using MS includes subjecting an enriched sample to one or more of EV disruption and proteolytic digestion to produce an analytic sample. In some embodiments, EV disruption includes exposure to one or more of urea, a reducing agent, or iodoacetamide. In some embodiments, the reducing agent is DTT, DTE, mercaptoethanol, tris (2-carboxyethyl) phosphine or glutathione. In some embodiments, proteolytic digestion includes exposing the sample to cyanogen bromide (CnBr) or to one or more proteases. In some embodiments, the one or more proteases include an aminopeptidase, a carboxypeptidase, trypsin, chymotrypsin, bromelian, papain, pronase, or proteinase k.

[0034] In some embodiments, a method includes purifying the analytic sample to capture EV material (e.g., proteins, polypeptides, peptides, or the like) after the EV disruption. In some embodiments, the purifying includes performing chromatography selected from reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, or polystyrene-divinlybenzene chromatography. In some embodiments, the purifying is performed using a high-performance liquid chromatography (HPLC) column. In some embodiments, the HPLC column includes a reverse-phase material selected from Cl 8 or C6.

[0035] In some embodiments, a method includes analyzing the captured EV material to determine an elevated or a differential amount of the captured EV material relative to a control. In some embodiments, a control may include a sample from a subject not having a brain- related condition or disorder. In some embodiments, a control may include a sample not including brain- derived EVs.

[0036] In some embodiments, the analyzing includes removing uncaptured EV material from the purified analytic sample. In some embodiments, the uncaptured material includes materialACTIVE 717682885v4 12ATTORNEY DOCKET NO. 223711-700001 / PCTselected from salts and macromolecules or complexes having a size greater than 5.5 kD or the macromolecule is a polypeptide greater than about 50 amino acids long.

[0037] In some embodiments, the analyzing includes eluting the captured EV material from the purified analytic sample and loading the eluted EV material onto an analytical column. In some embodiments, the analytical column includes a high- performance liquid chromatography (HPLC) column. In some embodiments, the high-performance liquid chromatography (HPLC) column includes a reverse-phase material selected from Cl 8 or C6.

[0038] In some embodiments, the analyzing includes separating the polypeptides on the analytical column; and detecting separated polypeptides with a detector. In some embodiments, the detecting includes adding stable isotope standard peptides for detecting specific protein markers to the analytical sample. In some embodiments, the detecting includes providing separated analytes to the detector through a fluid connection between the analytical column and the detector. In some embodiments, the detector detects analytes by MS, flow cytometry, electron or atomic force microscopy or affinity-based assays. In some embodiments, the detector includes a mass spectrometer selected from quadrupole mass spectrometer or time-of-flight mass spectrometer. In some embodiments, the detector is selected from a tandem mass spectrometer, a triple quadrupole mass spectrometer, or a linear ion trap mass spectrometer. In some embodiments, the detector includes a triple quadrupole mass spectrometer and detection includes multiple reaction monitoring. In some embodiments, the detector includes a linear ion trap mass spectrometer and detection includes full-scan Synchronous Precursor Selection (SPS) MS3acquisition.

[0039] In some embodiments, a system for analyzing brain-derived EVs using MS includes an EV purification module, the EV purification module being configured to purify one or more of proteins or peptides from circulating brain-derived EVs in a biological sample, the EV purification module including (a) a first solution for enriching the biological sample for the brain-derived EVs and (b) a second solution for subjecting the enriched biological sample to one or more of EV disruption or proteolytic digestion to produce an analytic sample.

[0040] In some embodiments, the EV purification module is selected from a reverse-phase chromatography column, an anion exchange chromatography column, a cation exchange chromatography column, a polystyrene-divinylbenzene column or a size exclusion column. In some embodiments, the EV purification module includes a high-performance liquid chromatography (HPLC) column. In some embodiments, the HPLC column includes a reverse-ACTIVE 717682885v4 13ATTORNEY DOCKET NO. 223711-700001 / PCTphase material selected from Cl 8 or C6. In some embodiments, the EV purification module further includes stable isotope standard polypeptides.

[0041] In some embodiments, a system includes a multi-port valve connected through a first fluid conduit to a column containing the EV purification module. In some embodiments, a system further includes an outlet fluid conduit connecting an outlet port to the multi-port valve. In some embodiments, a system further includes a pump fluid conduit connecting a pump connected to the EV purification module. In some embodiments, a system includes an analytical column connected through a second fluid conduit to the multi-port valve, the analytical column being configured to determine an elevated or a differential amount of captured EV material from the analytic sample relative to a control sample. In some embodiments, the analytical column includes a high-performance liquid chromatography (HPLC) column,

[0042] In some embodiments, a system further includes a detector fluid conduit connecting a detector to the analytical column. In some embodiments, the detector is selected from a mass spectrometer, a flow cytometer, an electron microscope, or an atomic force microscope. In some embodiments, the detector includes a mass spectrometer selected from quadrupole mass spectrometer or time-of-flight mass spectrometer. In some embodiments, the detector is selected from a tandem mass spectrometer, a triple quadrupole mass spectrometer, or a linear ion trap mass spectrometer. In some embodiments, the detector includes a triple quadrupole mass spectrometer and detection includes multiple reaction monitoring. In some embodiments, the detector includes a linear ion trap mass spectrometer and detection includes full-scan Synchronous Precursor Selection (SPS) MS3acquisition.

[0043] Further provided herein are methods of operating systems for analyzing brain-derived EVs. In some embodiments, a system includes: (i) an extracellular vesicle (EV) purification module; (ii) a multi-port valve connected through a first fluid conduit to the EV purification module; (hi) an analytical column connected through a second fluid conduit to the multi-port valve; (iv) an outlet port connected to the multi-port valve through outlet fluid conduit; and (v) a detector connected through a detector fluid conduit to the analytical column.

[0044] In some embodiments, a method includes loading the EV purification module with a biological sample, the biological sample including circulating brain-derived EVs, the circulating brain-derived EV s including one or a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, and complex lipids.ACTIVE 717682885v4 14ATTORNEY DOCKET NO. 223711-700001 / PCT

[0045] In some embodiments, a method includes flowing at least a first solution and a second solution through the biological sample to produce an analytic sample such that the analytic sample passes through the first fluid conduit, the multi-port valve, and a third fluid conduit, wherein the first solution includes a first composition for enriching the biological sample for the brain-derived EVs, and wherein the second solution includes for subjecting the enriched biological sample to one or more of EV disruption and proteolytic digestion. In some embodiments, the analytical sample includes between about 1 pg and about 200 pg of protein or peptide. In some embodiments, the analytical sample includes disrupted EVs or exosomes that have been subjected to a proteolytic treatment. In some embodiments, the analytical sample includes a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, lipids, complex lipids, salts, or metals. In some embodiments, the sample includes a homogenate of cells or a tissue homogenate.

[0046] In some embodiments, a method includes washing the EV purification module such that a wash passes through the first fluid conduit, the multi-port valve, and the third fluid conduit. In some embodiments, a method includes eluting the analytical column such that an eluate passes through the first fluid conduit, the multi-port valve and the second fluid conduit onto the analytical column. In some embodiments, a method includes separating the eluate on the analytical column. In some embodiments, a method includes passing the separated eluate through the detector fluid conduit to the detector.

[0047] In some embodiments, a method includes detecting one or more components of the separated eluate to determine an elevated or a differential amount of the one or more components from the analytic sample relative to a control sample. In some embodiments, detecting includes mass spectrometry. In some embodiments, mass spectrometry includes multiple reaction monitoring mass spectrometry. In some embodiments, mass spectrometry includes full-scan Synchronous Precursor Selection (SI’S) MSdacquisition.

[0048] FIG. 2 illustrates a flowchart of an example method for analyzing brain-derived EVs using MS. The process 1000 begins at step 110, which includes enriching a biological sample for brain-derived extracellular vesicles (EVs), the biological sample being from a subject having a brain-related condition or disorder and comprising the brain-derived EVs. At step 120, the enriched sample is subjected to one or more of EV disruption and proteolytic digestion to produce an analytic sample. At step 130, the analytic sample is purified to capture EV material after the EVACTIVE 717682885v4 15ATTORNEY DOCKET NO. 223711-700001 / PCTdisruption. Finally, at step 140, the captured EV material are analyzed to determine an elevated or a differential amount of the captured EV material relative to a control.DI. Lateral flow-based analysis

[0049] Provided herein are examples of LF-based systems and methods thereof for analyzing brain-derived extracellular vesicles (EVs).

[0050] Lateral flow (LF) devices have emerged in portable diagnostic tools as an extension of paper chromatography, a simple way to physically separate chemicals from each other as they tra veled through paper materials. Paper chromatography was developed in 1943, but modifications have led to lateral flow immunochromatographic assays, or rapid tests that use antibodies to recognize and bind to molecules, lake proteins, antibodies are used by the immune system to tightly bind up microorganisms, such as bacteria, to target them for destruction and removal; they were commandeered for detecting chemicals in research settings and, later, in medical settings. The presence of a target molecule recognized by the antibody often was signaled as a colored line on a flat, elongated strip of paper-like material or other flexible polymer as thin as paper. Colored particles (e.g., nanoparticles) bound to the antibodies allow for visualizing the antibody binding. Any colored particle could be used, but blue-colored latex or nanometer-sized gold particles that produce a red color are commonly used. In some embodiments, instead of colored particles, small enzymes can be linked to the antibodies that chemically produce a colored substance. In all cases, a simple line appears when binding to a target molecule occurs, and a lack of a line signals none or limited amounts of the target molecule being present.

[0051] LF membranes may be inserted into cassettes, often made of a plastic, that became simple devices intended to detect the presence of a target analyte in a liquid sample, like blood, without the need for sophisticated and costly equipment. Because the compactness and simplicity of the devices were so practical, the basic designs have stood the test of time. Many innovations, however, have come from materials used for the strips and chemistries used for detection. For example, chemicals that produce light, or special imaging systems, may be substituted to generate lines when the targets for disease were present. LF may also act as a filter, trapping debris and leftover cells from a biological sample in the paper-like membrane, while fluids from the biological sample and buffers mixed with the sample flow through the membrane via capillary flow (i.e., capillary action), leaving debris and larger biological components behind. EVs may be purifiedACTIVE 717682885v4 16ATTORNEY DOCKET NO. 223711-700001 / PCTaway from the debris and any cells or other large matter, and various compositions, such as, for example, a detergent-buffer combination, may disrupt the EVs and break them open to release their contents. Membranes may further clean up the EVs in the sample to allow for any additional chemistries in the system to act.

[0052] In some embodiments, provided herein is a system including: (1) an EV isolation device (herein “isolation column” or “capture column”) for extracting EVs such as exosomes from a biological sample of a subject; and (2) an LF device for measuring one or more target analytes. In some embodiments, a capture column is selected from a reverse-phase chromatography column, an anion exchange chromatography column, a cation exchange chromatography column, a polystyrene-divinylbenzene column or a size exclusion column. As discussed above, an LF device allows for rapid and easy readout, using lines that may appear in minutes, of the presence of target analytes for diagnosis,

[0053] Exosomes are small (e.g., often in sub-micrometer and even sub-nanometer diameter ranges) pieces of organs and tissue that are shed regularly, as well as in response to disease and injury, such as TBI. / Xs discussed above, they are ubiquitous in biological solutions and their contents can have substantial biological information. In particular, those sourced from the brain carry proteins, including target analytes used herein as biomarkers, and other molecules from the brain’s cells (including the neurons and their glial cell cushions). For example, after a brain injury, increased amounts of exosomes are released from brain cells and flow into circulation. By capturing brain (and other) exosomes from blood, it is possible to indirectly obtain biopsy-like information from the brain, which normally requires sticking a needle into the brain or using a lumbar puncture to obtain cerebrospinal fluid (brain and spinal cord fluid).

[0054] In some embodiments, LF devices herein use nanoparticles to reveal the presence of various markers, e.g., proteins for TBI, as sensitive indicators of damage when coupled to EV capture using an LF membrane. EVs concentrate such markers in a small volume (e.g., 1 millionth of a liter). A clear advantage of LF devices is the lack of need of electronics for EV capture from blood and for measuring the markers on a scale of a positive or negative result. A capture column may be the size of a ballpoint pen and the LF membrane may fit into a cassette that, in some embodiments, may be shorter than a tongue depressor while being about 3 times as thick. Both a capture column and an LF device disclosed herein are compact, portable, and durable. The capture column uses gravity and what is called “capillary action,” or the natural flow through the paper-ACTIVE 717682885v4 17ATTORNEY DOCKET NO. 223711-700001 / PCTlike membrane, are necessary to make the whole thing work. Gravity and the seeping of fluid through capillary action are two properties that are fairly reliable. There are no batteries required, no plugs to plug in, and no sensitive electronic components to break loose or split apart. Generally, the two components may withstand swings in temperature, reasonable pressure, and jostling around. They are compact, allowing them to be tucked away in a medical backpack, and numerous devices can be shipped in small containers.

[0055] FIGS. 3A-3B illustrates exemplary process for an LF system (FIG 3A) and embodiment of an Id device (FIG. 3B) for measuring proteins related to TBI using LF, In particular, the I d;system may be used for molecule isolation and detection of proteins related to TBI, In some embodiments, the LF system may be used as a rule-out test for TBI, i.e., a negative test indicates no or minimal damage. For example, in a battlefield context, a soldier would be able to return to the team without a trip to a field hospital or an overseas medical complex. The LF system for sampling exosomes m a biological fluid allows for a reduction in total testing time to about 30 minutes, with each of the two devices, i.e,, the exosome isolation column and the LF device, being the size of a ballpoint pen (exosome capture column) and a tongue depressor (LF device). The positive readout (i.e., TBI present) of the LV device is a thm line, easily visualized by eye, on a membrane the size of a glucose monitoring strip placed within the tongue depressor¬ sized, plastic shelled device. If necessary for higher precision grading of the measurement, and for storing the data, a “reader” may be used, the reader including one or more imaging devices such as a camera, which may be adapted for communication using a dedicated software.

[0056] As illustrated in FIG. 3A, step (a) includes obtaining a blood plasma collection tube and transfer pipette, the collection tube containing the biological sample, i.e., blood sample, from a subject. Step (b) includes contacting the biological sample in the collection tube with a capture column, the capture column being a size exclusion column. The capture column may be kept upright, pointing towards the ground, to allow for gravity flow, and allows for the sample to isolate exosomes by size to produce an enriched sample. The capture columns for exosome isolation can be run in about 2 to about 5 minutes. Step (c) includes adding a lysis buffer to the enriched sample to dissolve the exosome membranes and disrupt the exosomes in the enriched sample, releasing their contents and producing an analytical sample. Finally, step (d) includes adding the analytical sample to the LF device by contacting a portion of an LF membrane in the LF device and allowing the analytical sample to flow through the LF membrane via capillary flow, causing potentialACTIVE 717682885v4 18ATTORNEY DOCKET NO. 223711-700001 / PCTbinding of the target analytes (i.e., proteins related to TBI) to a corresponding set of nanoparticles on the LF membrane. After a short period of time, a result is visually present on the LF membrane. As illustrated in FIG. 3B, an LF device may include a compact footprint that accommodates 4 specific membranes for targeted molecules.

[0057] In some embodiments, an electronics module may be incorporated into an LF system. In some embodiments, an electronics module may be configured to rapidly convert a whole blood to plasma, i.e., blood plasma generator. Such a generator could be used for plasma testing in a medical tent and would not require a heavy, mechanically sensitive centrifuge to spin the blood before testing. In some embodiments, an electronics module may include a mechanical stand configured to automate and / or increase a rate of EV isolation from the capture column. In some embodiments, an electronics module is configured to control flow of the sample through the capture column against gravity. In some embodiments, an electronics module may include an adaptor for connecting to one or more auxiliary devices, such as for power or data communications, documentation, and / or increasing a sensitivity or quantification ability of the LF system. In some embodiments, an auxiliary device includes a mobile device. In some embodiments, an auxiliary device includes an imaging device.

[0058] In some embodiments, an LF device provided herein includes one or more LF membranes and one or more internal controls for assessing a status of the sample or the rest of the LF device. In some embodiments, an LF device includes one or more reaction chamber for receiving a biological sample and one or more reagents. In some embodiments, a device may comprise more than one lateral flow membrane. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow membranes. In some embodiments, each of the one or more LF membranes may be downstream of one or more reaction chamber. In some embodiments, each reaction chamber may be interfaced with a lateral flow membrane for multiplexing. In some embodiments, one or more lateral flow membranes are configured to detect a control analyte instead of or in addition to a target analyte. For example, a device comprising six lateral flow membranes may comprise five lateral flow membranes configured to detect one or more target analytes (e.g., five different target analytes) and one lateral flow membrane configured to detect a control analyte.

[0059] In some embodiments, results may be ready m less than about 1 minute after the sample begins the capillary' process across the lateral flow membrane. In some embodiments, results mayACTIVE 717682885v4 19ATTORNEY DOCKET NO. 223711-700001 / PCTbe ready in less than about 2 minutes after the sample begins the capillary' process across the lateral flow membrane. In some embodiments, results may be ready in less than about 3 minutes after the sample begins the capillary process across the lateral flow membrane. In some embodiments, results may be ready in less than about 5 minutes after the sample begins the capillary' process across the lateral flow membrane. In some embodiments, results may be ready in less than about 10 minutes after the sample begins the capillary process across the lateral flow membrane. In some embodiments, results may be ready in less than about 20 minutes after the sample begins the capillary process across the lateral flow membrane. In some embodiments, results may be ready in less than about 30 minutes after the sample begins the capillary process across the lateral flow membrane. In some embodiments, results may be ready in less than about 60 minutes after the sample begins the capillary' process across the lateral flow membrane,IV, Target analytes

[0060] In some embodiments, a target analyte includes a protein. In some embodiments, a protein comprises a polypeptide chain of greater than 10 amino acids, greater than 20 amino acids, greater than 50 amino acids, greater than 100 ammo acids, greater than 500 amino acids, greater than 1000 ammo acids, greater than 1500 ammo acids, greater than 2000 ammo acids, or more. In some embodiments, a protein comprises a polypeptide chain of at least 10 ammo acids, at least than 20 ammo acids, at least than 50 ammo acids, at least than 100 ammo acids, at least than 500 amino acids, at least than 1000 amino acids, at least 1500 amino acids, at least 2000 ammo acids, or more. In some embodiments, a protein comprises a polypeptide chain has a molecular weight of greater than 1 kDa, greater than 2 kDa, greater than 5 kDa, greater than 10 kDa, greater than 20 kDa, greater than 30 kDa, greater than 40 kDa, greater than 50 kDa, or more. In some embodiments, a protein comprises a polypeptide chain has a molecular weight of at least 1 kDa, at least 2 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 100 kDa, at least 150 kDa, or more. In some embodiments, a protein comprises an antibody, an antigen, an enzyme, a hormone, or a functional fragment thereof. In some embodiments, an antibody is a monoclonal antibody or antibody-drug conjugate. In some embodiments, a protein comprises a membrane protein. In some embodiments, an enzyme comprises Ubiquitin Carboxyl-Terminal Hydrolase LI (UCH-L1).ACTIVE 717682885v4 20ATTORNEY DOCKET NO. 223711-700001 / PCT

[0061] In some embodiments, a target analyte includes a nucleic acid. In some embodiments, a nucleic acid is a double stranded nucleic acid (e.g., DNA). In some embodiments, a nucleic acid is a single stranded nucleic acid (e.g., a RNA, wherein the RNA comprises a mRNA, a rRNA, a tRNA, a non-coding RNA, a long non-coding RNA, a microRNA (miRNA), a small interfering RNA (siRNA), and a single-stranded RNA (ssRNA)).

[0062] In some embodiments, a target analyte includes a small molecule. In some embodiments, a small molecule comprises a molecular weight of up to 1000 Da. In some embodiments, a small molecule comprises a molecular weight of less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da, less than 500 Da, less than 400 Da, less than 300 Da, less than 200 Da, or less than 100 Da. In some embodiments, a small molecule comprises a molecular weight in a range of from 10 Da to 1000 Da, from 10 Da to 800 Da, from 10 Da to 500 Da, from 10 Da to 300 Da, from 50 Da to 1000 Da, from 50 Da to 800 Da, from 50 Da to 500 Da, from 50 Da to 300 Da, from 100 Da to 1000 Da, from 100 Da to 800 Da, from 100 Da to 500 Da, from 100 Da to 300 Da, from 300 Da to 1000 Da, from 300 Da to 800 Da, from 300 Da to 500 Da, from 500 Da to 1000 Da, from 500 Da to 800 Da, or from 800 Da to 1000 Da.

[0063] In some embodiments, a target analyte is specific to a target region, i.e., region-specific. In some embodiments, a target region comprises a region in the brain. In some embodiments, a region in the brain includes an occipital lobe, parietal lobe, frontal lobe, temporal lobes, amygdala, basal ganglia, hippocampus, cerebral cortex, corpus callosum, entorhinal cortex, a brain stem, or any other related or adjacent region or a combination thereof.

[0064] In some embodiments, a target analyte comprises a secretory protein, a neurotrophic factor, a growth factor, a cytokine, a chemokme, a pre-toxic molecule, a toxic molecule, or a combination thereof. In some embodiments, a protein comprises a Neurofilament Heavy Chain polypeptide (NF-H). In some embodiments, a neuronal polypeptide comprises S100B. In some embodiments, a target analyte is a surface marker of an EV. In some embodiments, a surface marker comprises EGFR, TREM2, VEGFR2, or the like or a combination thereof. In some embodiments, a target analyte is specific to a microglia or other brain inflammatory cell or is a microglial activator. In some embodiments, a target analyte comprises TREM2, Ionized calcium- binding adapter molecule 1 (IB Al), or the like or a combination thereof. In some embodiments, a target analyte is an inflammatory marker. In some embodiments, an inflammatory marker comprises an inflammatory cytokine (e g., IL-6, IL-1 (3, IL-34, IL-12B, FasL, tumor necrosisACTIVE 717682885v4 21ATTORNEY DOCKET NO. 223711-700001 / PCTfactor-alpha (TNFa), or the like). In some embodiments, a target analyte is a combination of a plurality of target analytes. In some embodiments, a combination of a plurality of target analytes comprises IBA1, IL-6, IL-ip, TNFa, GFAP, UCH-L1, SIOOB, NF-H, or a combination thereof.

[0065] In some embodiments, a target analyte comprises a member of the cytokine Family (e.g., Interleukin-6 Family), ciliary Neurotrophic Factor (CNTF), leukemia Inhibitory Factor (LIF), fibroblast growth factor (FGF) family (e.g., fibroblast growth factor-2 (FGF-2, bFGF), and fibroblast growth factor-8 (FGF-8), and other growth factors, including insulin-like growth factor- 1 (IGF-1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-p (TGF-[3), epidermal growth factor (EGF), or the like or a combination thereof. In some embodiments, a target analyte is a cell-specific marker. In some embodiments, a cell-specific marker comprises a neuron-specific protein (e.g,, synaptosome associated protein 25 (SNAP25), a Purkinje cell-specific protein (e.g., Purkinje cell protein 2 (PCP2 (L7), Purkinje cell protein 4 (PCP4), Zebrin-II, Purkinje cell-specific phosphoprotein 260 kDA (PCPP-260), cerebellin-1, or the like), a neurogramn (NRGN), tau, phosphorylated tau, 0 -42, aP-40, synaptophysin, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and neureglin-1 (NRG1)), an astrocyte-specific protein (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter I (EAATl )), a microglia-specific protein (CD1 lb), an oligodendrocyte-specific protein (e.g., myelin basic protein (MBP), an oligodendrocyte myelin glycoprotein (OMG), a cytosolic protein (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL), a chemokine (CX3CL1, CCLs, CXCLs), cytokine (TNFa or the like, interleukins or the like, such as IL-1 P, IL-34, IL-12B or FasL, etc.). In some embodiments, a cell-specific marker is specific to a glial cell, a neuron, a nerve cell, an astrocyte, a microglia, an oligodendrocyte, a granule cell, a Purkinje cell, a cerebellar Purkinje cell, a cerebral cortex cell (e.g., deep layers), a hippocampus cell (e.g., CA2), an olfactory bulb cell, a thalamus cell, a basal ganglia cell (e.g., putamen, globus pallidus, caudate nucleus or the like), amygdala cell, a granule cell, or the like or a combination thereof.

[0066] In some embodiments, a target analyte is specific to a condition. In some embodiments, a condition is related to a trauma or traumatic injury. In some embodiments, a condition is not related to a trauma or traumatic injury. In some embodiments, a trauma is due to one or more injuryACTIVE 717682885v4 22ATTORNEY DOCKET NO. 223711-700001 / PCTmechanisms — e.g., explosions, blunt force, penetrating lesions, rotational forces, etc. — the intensity of the mechanisms, and several other factors including genetic predispositions and cellular defense mechanisms, including cellular resistance to shearing forces, anoxia, oxidative damage, and inflammation initiation or control (and others). In some embodiments, the traumatic injury can cause diffuse changes, such as diffuse axonal injury’ (DAI), or be concentrated in localized areas, such as the cerebellum or frontal or occipital lobes. In some embodiments, a condition includes traumatic brain injury’ (TBI), neurodegenerative disease, metabolic diseases involving the nervous system, neuroinflammation and related conditions, neurotoxicity or related neurodegeneration induced by chemicals, drugs and toxins, diabetes, birth-related neurological deficits, post-natal, trauma-related neurological deficits, conditions related to shaken baby syndrome, conditions related to post-natal trauma, age-related memory impairment, agyrophilic gram dementia, Parkinson’s disease, neurological effects of auto-immune conditions (e.g. Guillain-Barre syndrome, Lupus), Biswanger's disease, brain and spinal tumors (including neurofibromatosis), cerebral amyloid angiopathies, cerebral palsy, chronic fatigue syndrome, corticobasal degeneration, conditions due to developmental dysfunction of the CNS parenchyma, conditions due to developmental dysfunction of the cerebrovasculature, dementia, multi infarct, dementia or related conditions, neurofibrillary tangles with calcification, diseases of the eye, ear and vestibular systems involving neurodegeneration (e.g., macular degeneration and glaucoma), Down's syndrome, dyskinesias, essential tremor, Fahr's syndrome, frontotemporal lobar degeneration, frontal lobe dementia, hepatic encephalopathy, hereditary spastic paraplegia, hydrocephalus, conditions involving CSF dysfunction, Gaucher's disease, Hallervorden-Spatz disease, Korsakoffs syndrome, mild cognitive impairment, monomeric amyotrophy, motor neuron diseases, multiple system atrophy, multiple sclerosis and other related demyelinating conditions, neurological / cognitive effects of bacterial and / or virus infections, including but not restricted to enteroviruses, primary lateral sclerosis, prion diseases including Creutzfeldt- Jakob disease and variants thereof, kuru, sporadic fronto-temporal dementias, sulphite oxidase deficiency, Tay-Sach's disease, Tourette's syndrome, vascular dementia, Wilson disease, neurological manifestations of various diseases, or a combination thereof. In some embodiments, a target analyte is present in the biological sample in an amount that correlates with a severity of a condition.ACTIVE 717682885v4 23ATTORNEY DOCKET NO. 223711-700001 / PCTILLUSTRATIVE EMBODIMENTS

[0067] Embodiment!. A method comprising:

[0068] a. enriching a biological sample for brain-derived extracellular vesicles (EVs), the biological sample being from a subject having a brain-related condition or disorder and comprising the brain-derived EVs;

[0069] b. subjecting the enriched sample to EV disruption to produce an analytic sample;

[0070] c, purifying the analytic sample to capture EV material after the EV disruption; and

[0071] d. analyzing the captured EV material to determine a differential amount of the captured EV material relative to a control,

[0072] Embodiment 2, The method of Embodiment 1, wherein the analyzing compri ses:

[0073] generating polypeptides from the EV material;

[0074] eluting the polypeptides from the purified analytic sample and loading the eluted polypeptides onto an analytical column;

[0075] separating the polypeptides on the analytical column; and

[0076] detecting separated polypeptides with a detector.

[0077] Embodiment 3. The method of Embodiment 1 or 2, wherein the biological sample comprises blood, serum, plasma, saliva, cerebrospinal fluid, amniotic fluid, or urine.

[0078] Embodiment 4. The method of any one of Embodiments 1-3, wherein the brain- related condition or disorder is selected from traumatic brain injury, neurodegenerative disease, metabolic diseases involving the nervous system, neurotoxicity, diabetes, birth-related neurological deficits, or post-natal, trauma-related neurological deficits.

[0079] Embodiment 5. The method of any one of Embodiments 1-4, wherein the biological sample is enriched for EVs by one or more of size exclusion chromatography, ultrafiltration, magnetic beads, magnetic beads coupled to protein capture agents, reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, or polystyrene- divinylbenzene chromatography.

[0080] Embodiment 6. The method of any one of Embodiments 1-5, wherein the brain- derived EVs comprise exosomes.

[0081] Embodiment 7. The method of any one of Embodiments 1-6, wherein the EV disruption comprises exposure to one or more of urea, a reducing agent, or iodoacetamide.ACTIVE 717682885v4 24ATTORNEY DOCKET NO. 223711-700001 / PCT

[0082] Embodiment 8. The method of Embodiment 7, wherein the reducing agent is DTT, DTE, mercaptoethanol, tris (2- carboxy ethyl) phosphine or glutathione.

[0083] Embodiment 9. The method of any one of Embodiments 1-8, wherein the analytic sample is subjected to proteolytic digestion, wherein the proteolytic digestion comprises exposing the sample to cyanogen bromide (CnBr) or to one or more proteases.

[0084] Embodiment 10. The method of Embodiment 9, wherein the one or more proteases comprise an aminopeptidase, a carboxypeptidase, trypsin, chymotrypsin, bromelian, papain, pronase, or proteinase k,

[0085] Embodiment 11. The method of any one of Embodiments 2-10, wherein the detecting comprises adding stable isotope standard peptides for detecting specific protein markers to the analytical sample.

[0086] Embodiment 12. The method of any one of Embodiments 1-11, wherein the purifying comprises performing chromatography selected from reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography or poly styrene-divinlybenzene chromatography.

[0087] Embodiment 13. The method of any one of Embodiments 1-12, wherein the purifying is performed using a high-performance liquid chromatography (HPLC) column.

[0088] Embodiment 14. The method of Embodiment 13, wherein the high-performance liquid chromatography (HPLC) column comprises a reverse phase material selected from Cl 8 or C6.

[0089] Embodiment 15. The method of any one of Embodiments 2-14, wherein uncaptured material comprises material selected from salts and macromolecules or complexes having a size greater than 5.5 kD or the macromolecule is a polypeptide greater than about 50 amino acids long.

[0090] Embodiment 16. The method of any one of Embodiments 2- 15, wherein the analytical column comprises a high- performance liquid chromatography (HPLC) column.

[0091] Embodiment 17. The method of Embodiment 16, wherein the HPLC column comprises a reverse- phase material selected from Cl 8 or C6.

[0092] Embodiment 18. The method of any one of Embodiments 2- 17, wherein the detecting comprises providing separated analytes to the detector through a fluid connection between the analytical column and the detector.ACTIVE 717682885v4 25ATTORNEY DOCKET NO. 223711-700001 / PCT

[0093] Embodiment 19. The method of any one of Embodiments 2-18, wherein the detector detects analytes by mass spectrometry’.

[0094] Embodiment 20. The method of any one of Embodiments 2-19, wherein the detector comprises a mass spectrometer selected from quadrupole mass spectrometer or time-of-flight mass spectrometer.

[0095] Embodiment 21. The method of any one of Embodiments 2-20, wherein the detector comprises a tandem mass spectrometer.

[0096] Embodiment 22. The method of any one of Embodiments 2-21, wherein the detector comprises a triple quadrupole mass spectrometer.

[0097] Embodiment 23. The method of any one of Embodiments 2-22, wherein the detector comprises a triple quadrupole mass spectrometer and detection comprises multiple reaction monitoring,

[0098] Embodiment 24, The method of any one of Embodiments 2-23, wherein the detector comprises a linear ion trap mass spectrometer and detection comprises full -scan Synchronous Precursor Selection (SPS) MS3acquisition.

[0099] Embodiment 25. A system comprising:

[0100] a. an extracellular vesicle (EV) purification column, the EV purification column being configured to purify one or more of proteins or peptides from circulating brain-derived EVs in a biological sample, the EV purification column comprising (a) a first solution for enriching the biological sample for the brain-derived EVs and (b) a second solution for subjecting the enriched biological sample to EV disruption to produce an analytic sample;

[0101] b. a multi-port valve connected through a first fluid conduit to a column containing the EV purification column; and

[0102] c. an analytical column connected through a second fluid conduit to the multi-port valve, the analytical column being configured to determine a differential amount of captured EV material from the analytic sample relative to a control sample.

[0103] Embodiment 26. The system of Embodiment 25, wherein the EV purification column further comprises stable isotope standard polypeptides.

[0104] Embodiment 27. The system of Embodiment 25 or 26, further comprising an outlet fluid conduit connecting an outlet port to the multi-port valve.ACTIVE 717682885v4 26ATTORNEY DOCKET NO. 223711-700001 / PCT

[0105] Embodiment 28. The system of any one of Embodiments 25-27, further comprising a detector fluid conduit connecting a detector to the analytical column.

[0106] Embodiment 29. The system of Embodiment 25, further comprising a pump fluid conduit connecting a pump connected to the EV purification column.

[0107] Embodiment 30. The system of any one of Embodiments 25-29, wherein the EV purification column is selected from a reverse-phase chromatography column, an anion exchange chromatography column, a cation exchange chromatography column, a polystyrene-divinylbenzene column or a size exclusion column.

[0108] Embodiment 31. The system of any one of Embodiments 25-30, w'herein the EV purification column comprises a high-performance liquid chromatography (HPLC) column.

[0109] Embodiment 32, The system of Embodiment 31, wherein the HPLC column comprises a reverse-phase material selected from Cl 8 or C6.

[0110] Embodiment 33. The system of any one of Embodiments 25-32, wherein the analytical column comprises a high- performance liquid chromatography (HPLC) column.

[0111] Embodiment 34. The system of Embodiment 28, wherein the detector comprises a mass spectrometer.

[0112] Embodiment 35. The system of Embodiment 34, wherein the mass spectrometer is selected from a quadrupole mass spectrometer or a time-of-flight mass spectrometer.

[0113] Embodiment 36. The system of any one of Embodiments 28-35, wherein the detector comprises a tandem mass spectrometer.

[0114] Embodiment 37. The system of any one of Embodiments 28-36, wherein the detector comprises a triple quadrupole mass spectrometer.

[0115] Embodiment 38. The system of any one of Embodiments 28-37, wherein the detector comprises a linear ion trap mass spectrometer.

[0116] Embodiment 39. The system of any one of Embodiments 25-38, wherein the EVs comprise exosomes.

[0117] Embodiment 40. A method comprising:

[0118] loading an extracellular vesicle (EV) purification column with a biological sample, the biological sample comprising circulating brain-derived EVs, the circulating brain-derived EVs comprising one or a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, and complex lipids,ACTIVE 717682885v4 27ATTORNEY DOCKET NO. 223711-700001 / PCT

[0119] flowing at least a first solution and a second solution through the biological sample to produce an analytic sample such that the analytic sample passes through a multi-port valve connected through a first fluid conduit to the EV purification column, wherein the first solution comprises a first composition for enriching the biological sample for the brain-derived EVs, and wherein the second solution comprises a second composition for subjecting the enriched biological sample to EV disruption;

[0120] washing the EV purification column such that a wash passes through the first fluid conduit, the multi-port valve, and the third fluid conduit;

[0121] eluting an analytical column connected through a second fluid conduit to the multi-port valve such that an eluate passes through the first fluid conduit, the multi-port valve and the second fluid conduit onto the analytical column;

[0122] separating the eluate on the analytical column;

[0123] passing the separated eluate through a detector fluid conduit to a detector, wherein the detector is connected through the detector fluid conduit to the analytical column; and

[0124] detecting one or more components of the separated eluate to determine a differential amount of the one or more components from the analytic sample relative to a control sample.

[0125] Embodiment 41. The method of Embodiment 40, wherein detecting comprises mass spectrometry.

[0126] Embodiment 42. The method of Embodiment 41, wherein mass spectrometry comprises multiple reaction monitoring mass spectrometry.

[0127] Embodiment 43. The method of Embodiment 41 or 42, wherein mass spectrometry comprises full-scan Synchronous Precursor Selection (SPS) MS3acquisition.

[0128] Embodiment 44. The method of any one of Embodiments 40-43, wherein the analytical sample of step (a) comprises between about 1 pg and about 200 pg of protein or peptide.

[0129] Embodiment 45. The method of any one of Embodiments 40-44, wherein the analytical sample comprises disrupted exosomes / extracellular vesicles that have been subjected to a proteolytic treatment.

[0130] Embodiment 46. The method of any one of Embodiments 40-45, wherein the analytical sample comprises a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, lipids, complex lipids, salts, or metals.ACTIVE 717682885v4 28ATTORNEY DOCKET NO. 223711-700001 / PCT

[0131] Embodiment 47. The method of any one of Embodiments 40-46, wherein the sample comprises a homogenate of cells or a tissue homogenate.OTHER EMBODIMENTS

[0132] From the foregoing description, it will be apparent that variations and modifications may be made to the embodiments described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0133] It will be understood by one skilled in the art that this disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the above description or illustrated in the drawings. The embodiments herein are capable of other embodiments, and capable of being practiced or carried out in various ways. Also, it will be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equi valents thereof as well as additional items. Unless limited otherwise, the terms “connected,” “coupled,” and “mounted,” and variations thereof herein are used broadly and encompass direct and indirect connections, couplings, and mountings. In addition, the terms “connected” and “coupled”, and variations thereof are not restricted to physical or mechanical connections or couplings. Further, terms such as up, down, bottom, and top are relative, and are employed to aid illustration, but are not limiting. Use of ordinal terms such as “first,” “second,” “third,” etc., m the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. The word “exemplary” is used herein to mean serving as an example, instance, or illustration. Any embodiment, implementation, process, feature, etc. described herein as exemplary should therefore be understood to be an illustrative example and should not be understood to be a preferred or advantageous example unless otherwise indicated.

[0134] The above-presented description and figures are intended by way of example only and are not intended to limit the illustrative embodiments in any way except as set forth in the following claims. It is particularly noted that persons skilled in the art can readily combine the variousACTIVE 717682885v4 29ATTORNEY DOCKET NO. 223711-700001 / PCTtechnical aspects of the various elements of the various illustrative embodiments that have been described above in numerous other ways, all of which are considered to be within the scope of the claims.

[0135] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0136] While some embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein, or combinations of one or more of these embodiments or aspects described therein may be employed in practicing the present disclosure. It is intended that the fol lowing claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.ACTIVE 717682885v4 30

Claims

ATTORNEY DOCKET NO. 223711-700001 / PCTCLAIMSWhat is claimed is:

1. A method comprising:a. enriching a biological sample for brain-derived extracellular vesicles (EVs), the biological sample being from a subject having a brain-related condition or disorder and comprising the brain-derived EVs;b. subjecting the enriched sample to EV disruption to produce an analytic sample;c. purifying the analytic sample to capture EV material after the EV disruption; and d. analyzing the captured EV material to determine a differential amount of the captured EV material relative to a control,2. The method of claim 1, wherein the analyzing comprises:generating polypeptides from the EV material;eluting the polypeptides from the purified analytic sample and loading the eluted polypeptides onto an analytical column;separating the polypeptides on the analytical column; and detecting separated polypeptides with a detector.

3. The method of claim 1 or 2, wherein the biological sample comprises blood,serum, plasma, saliva, cerebrospinal fluid, amniotic fluid, or urine.

4. The method of any one of claims 1-3, wherein the brain-related condition or disorder is selected from traumatic brain injury, neurodegenerative disease, metabolic diseases involving the nervous system, neurotoxicity, diabetes, birth- related neurological deficits, or post-natal, trauma-related neurological deficits.

5. The method of any one of claims 1-4, wherein the biological sample is enrichedfor E Vs by one or more of size exclusion chromatography, ultrafiltration,magnetic beads, magnetic beads coupled to protein capture agents, reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, or polystyrene-divinylbenzene chromatography.

6. The method of any one of claims 1-5, wherein the brain-derived EVs comprise exosomes.ACTIVE 717682885v4 31ATTORNEY DOCKET NO. 223711-700001 / PCT7. The method of any one of claims 1-6, wherein the EV disruption comprises exposure to one or more of urea, a reducing agent, or iodoacetamide.

8. The method of claim 7, wherein the reducing agent is DTT, DTE, mercaptoethanol, tris (2-carboxyethyl) phosphine or glutathione.

9. The method of any one of claims 1-8, wherein the analytic sample is subjected to proteolytic digestion, wherein the proteolytic digestion comprises exposing the sample to cyanogen bromide (CnBr) or to one or more proteases.

10. The method of claim 9, wherein the one or more proteases comprise an aminopeptidase, a carboxypeptidase, trypsin, chymotrypsin, bromelian, papain, pronase, or proteinase k.

11. The method of any one of claims 2-10, wherein the detecting comprises addingstable isotope standard peptides for detecting specific protein markers to the analytical sample.

12. The method of any one of claims 1-11, wherein the purifying comprisesperforming chromatography selected from reverse-phase chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, or polystyrene-divinlybenzene chromatography.

13. The method of any one of claims 1-12, wherein the purifying is performed usinga high-performance liquid chromatography (HPLC) column.

14. The method of claim 13, wherein the high-performance liquid chromatography (HPLC) column comprises a reverse phase material selected from Cl 8 or C6.

15. The method of any one of claims 2-14, wherein uncaptured material comprises material selected from salts and macromolecules or complexes having a size greater than 5.5 kD or the macromolecule is a polypeptide greater than about 50 amino acids long.

16. The method of any one of claims 2-15, wherein the analytical column comprisesa high- performance liquid chromatography (HPLC) column.

17. The method of claim 16, wherein the HPLC column comprises a reverse-phase material selected from Cl 8 or C6.

18. The method of any one of claims 2-17, wherein the detecting comprisesproviding separated analytes to the detector through a fluid connection betweenACTIVE 717682885v4 32ATTORNEY DOCKET NO. 223711-700001 / PCTthe analytical column and the detector.

19. The method of any one of claims 2-18, wherein the detector detects analytes by mass spectrometry.

20. The method of any one of claims 2-19, wherein the detector comprises a mass spectrometer selected from quadrupole mass spectrometer or time-of-flight mass spectrometer.

21. The method of any one of claims 2-20, wherein the detector comprises a tandem mass spectrometer.

22. The method of any one of claims 2-21, wherein the detector comprises a triple quadrupole mass spectrometer.

23. The method of any one of claims 2-22, wherein the detector comprises a triple quadrupole mass spectrometer and detection comprises multiple reaction monitoring,24. The method of any one of claims 2-23, wherein the detector comprises a linearion trap mass spectrometer and detection comprises full-scan Synchronous Precursor Selection (SPS) MS3acquisition.

25. A system comprising:a. an extracellular vesicle (EV) purification column, the EV purification module being configured to purify one or more of proteins or peptides from circulating brain-derived EVs in a biological sample, the EV purification column comprising (a) a first solution for enriching the biological sample for the brain-derived EVs and (b) a second solution for subjecting the enriched biological sample to EV disruption to produce an analytic sample; b. a multi-port valve connected through a first fluid conduit to a column containing the EV purification column; andc. an analytical column connected through a second fluid conduit to the multiport valve, the analytical column being configured to determine a differential amount of captured EV material from the analytic sample relative to a control sample.

26. The system of claim 25, wherein the EV purification column further comprises stable isotope standard polypeptides.ACTIVE 717682885v4 33ATTORNEY DOCKET NO. 223711-700001 / PCT27. The system of claim 25 or 26, further comprising an outlet fluid conduit connecting an outlet port to the multi-port valve.

28. The system of any one of claims 25-27, further comprising a detector fluid conduit connecting a detector to the analytical column.

29. The system of claim 25, further comprising a pump fluid conduit connecting a pump connected to the EV purification column.

30. The system of any one of claims 25-29, wherein the EV purification column is selected from a reverse-phase chromatography column, an anion exchange chromatography column, a cation exchange chromatography column, apolystyrene-divinylbenzene column or a size exclusion column.

31. The system of any one of claims 25-30, wherein the EV purification column comprises a high-performance liquid chromatography (HPLC) column.

32. The system of claim 31, wherein the HPLC column comprises a reverse-phase material selected from Cl 8 or C6.

33. The system of any one of claims 25-32, wherein the analytical column comprises a high- performance liquid chromatography (HPLC) column.

34. The system of claim 28, wherein the detector comprises a mass spectrometer.

35. The system of claim 34, wherein the mass spectrometer is selected from a quadrupole mass spectrometer or a time-of-flight mass spectrometer.

36. The system of any one of claims 28-35, wherein the detector comprises a tandem mass spectrometer.

37. The system of any one of claims 28-36, wherein the detector comprises a triple quadrupole mass spectrometer.

38. The system of any one of claims 28-37, wherein the detector comprises a linear ion trap mass spectrometer.

39. The system of any one of claims 25-38, wherein the EVs comprise exosomes.

40. A method comprising:a. loading an extracellular vesicle (EV) purification column with a biological sample, the biological sample comprising circulating brain-derived EVs, the circulating brain-derived EVs comprising one or a plurality of molecules selected from polypeptides, complex carbohydrates, nucleic acids, and complex lipids,ACTIVE 717682885v4 34ATTORNEY DOCKET NO. 223711-700001 / PCTb. flowing at least a first solution and a second solution through the biological sample to produce an analytic sample such that the analytic sample passes through a multi-port valve connected through a first fluid conduit to the EV purification column, wherein the first solution comprises a first composition for enriching the biological sample for the brain-derived EVs, and wherein the second solution comprises a second composition for subjecting the enriched biological sample to EV disruption;c. washing the EV purification column such that a wash passes through the first fluid conduit, the multi-port valve, and the third fluid conduit;d. eluting an analytical column connected through a second fluid conduit to the multi-port valve such that an eluate passes through the first fluid conduit, the multi-port valve and the second fluid conduit onto the analytical column;e. separating the eluate on the analytical column;f. passing the separated eluate through a detector fluid conduit to a detector, wherein the detector is connected through the detector fluid conduit to the analytical column; andg. detecting one or more components of the separated eluate to determine a differential amount of the one or more components from the analytic sample relative to a control sample.

41. The method of claim 40, wherein detecting comprises mass spectrometry.

42. The method of claim 41, wherein mass spectrometry comprises multiple reaction monitoring mass spectrometry.

43. The method of claim 41 or 42, wherein mass spectrometry comprises full-scan Synchronous Precursor Selection (SPS) MS3acquisition.

44. The method of any one of claims 40-43, wherein the analytical sample of step (a) comprises between about 1 pg and about 200 pg of protein or peptide.

45. The method of any one of claims 40-44, wherein the analytical sample comprises disrupted exosomes / extracellular vesicles that have been subjected to a proteolytic treatment.

46. The method of any one of claims 40-45, wherein the analytical sample comprises a plurality’ of molecules selected from polypeptides, complex carbohydrates, nucleic acids,ACTIVE 717682885v4 35ATTORNEY DOCKET NO. 223711-700001 / PCTlipids, complex lipids, salts, or metals.

47. The method of any one of claims 40-46, wherein the sample comprises a homogenate of cells or a tissue homogenate.ACTIVE 717682885v4 36

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