Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes

Biological Fluids ◽

Protein Detection ◽

Cancer Diagnostics ◽

Small Sample ◽

Surface Proteins ◽

Protein Profiling ◽

Size Exclusion ◽

Pcr Method

Abstract Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq™ antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using live cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.

Multiplex protein profiling method for extracellular vesicle protein detection

Scientific Reports ◽

10.1038/s41598-021-92012-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Li Sun ◽

David G. Meckes

Keyword(s):

Biological Fluids ◽

Protein Detection ◽

Cancer Diagnostics ◽

Small Sample ◽

Surface Proteins ◽

Protein Profiling ◽

Size Exclusion ◽

Serum Samples ◽

Chromatography Method ◽

Pcr Method

AbstractExtracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

International Journal of Molecular Sciences ◽

10.3390/ijms21186466 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6466 ◽

Cited By ~ 6

Author(s):

Karim Sidhom ◽

Patience O. Obi ◽

Ayesha Saleem

Keyword(s):

Culture Media ◽

Biological Fluids ◽

Vital Role ◽

Size Exclusion ◽

Isolation Techniques ◽

Specialized Equipment ◽

Pros And Cons ◽

Poly Ethylene

Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.

Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

Electrophoresis ◽

10.1002/elps.200600037 ◽

2006 ◽

Vol 27 (13) ◽

pp. 2722-2733 ◽

Cited By ~ 42

Author(s):

David C. Simpson ◽

Seonghee Ahn ◽

Ljiljana Pasa-Tolic ◽

Bogdan Bogdanov ◽

Heather M. Mottaz ◽

...

Keyword(s):

Protein Profiling ◽

Size Exclusion ◽

Two Dimensional ◽

Critical study of the speciation of aluminum in biological fluids by size-exclusion chromatography and electrothermal atomic absorption spectrometry

Analytica Chimica Acta ◽

10.1016/s0003-2670(00)83075-5 ◽

1987 ◽

Vol 196 ◽

pp. 103-114 ◽

Cited By ~ 20

Author(s):

H. Keirsse ◽

J. Smeyers-Verbeke ◽

D. Verbeelen ◽

D.L. Massart

Keyword(s):

Atomic Absorption Spectrometry ◽

Atomic Absorption ◽

Biological Fluids ◽

Electrothermal Atomic Absorption Spectrometry ◽

Absorption Spectrometry ◽

Size Exclusion ◽

Critical Study ◽

Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed

International Journal of Molecular Sciences ◽

10.3390/ijms222011144 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11144

Author(s):

Mikel Azkargorta ◽

Ibon Iloro ◽

Iraide Escobes ◽

Diana Cabrera ◽

Juan M. Falcon-Perez ◽

...

Keyword(s):

Human Serum ◽

Extracellular Vesicles ◽

Membrane Attack Complex ◽

Protein Detection ◽

Size Exclusion ◽

Blood Proteins ◽

Proteomic Profiling ◽

Serum Samples ◽

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

10.20944/preprints202007.0485.v2 ◽

2020 ◽

Author(s):

Karim Sidhom ◽

Patience O. Obi ◽

Ayesha Saleem

Keyword(s):

Culture Media ◽

Biological Fluids ◽

Vital Role ◽

Size Exclusion ◽

Isolation Techniques ◽

Specialized Equipment ◽

Pros And Cons ◽

Poly Ethylene

Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media among others. While differential ultracentrifugation (dUC), has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics and size exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise.

COMPARISONS OF NANOPARTICLE PROTEIN CORONA COMPLEXES ISOLATED WITH DIFFERENT METHODS

Nano LIFE ◽

10.1142/s1793984413430046 ◽

2013 ◽

Vol 03 (04) ◽

pp. 1343004 ◽

Cited By ~ 10

Author(s):

MARCO P. MONOPOLI ◽

SHA WAN ◽

FRANCESCA BALDELLI BOMBELLI ◽

EUGENE MAHON ◽

KENNETH A. DAWSON

Keyword(s):

Residence Time ◽

Biological Fluids ◽

Protein Composition ◽

Protein Corona ◽

Membrane Receptors ◽

Size Exclusion ◽

Dispersion Properties ◽

Nanoparticle Surface ◽

Nanoparticles, after incubation in biological fluids, adsorb several kinds of biomolecules like lipids, sugars and mainly proteins with high affinities for the nanoparticle surface and with long residence time, forming the so-called hard corona. The biological machinery, such as cellular barriers and membrane receptors can directly engage with the protein corona while the pristine surface may remain inaccessible. Here we isolate nanoparticles associated with strongly bound biomolecules from the unbound and loosely bound ones, by different approaches: centrifugation, size exclusion chromatography and magnetic isolation. The different separation methodologies, despite requiring diverse time and operating mechanisms, gave nanoparticle-hard corona complexes which were found to be remarkably similar in both dispersion properties and protein composition thus proving to be equally valid.

SEC-SANS: size exclusion chromatography combinedin situwith small-angle neutron scattering

Journal of Applied Crystallography ◽

10.1107/s1600576716016514 ◽

2016 ◽

Vol 49 (6) ◽

pp. 2015-2020 ◽

Cited By ~ 29

Author(s):

Ashley Jordan ◽

Mark Jacques ◽

Catherine Merrick ◽

Juliette Devos ◽

V. Trevor Forsyth ◽

...

Keyword(s):

Neutron Scattering ◽

Small Angle ◽

Small Angle Neutron Scattering ◽

Small Sample ◽

Size Exclusion ◽

Biological Macromolecules ◽

Sec System

The first implementation and use of anin situsize exclusion chromatography (SEC) system on a small-angle neutron scattering instrument (SANS) is described. The possibility of deploying such a system for biological solution scattering at the Institut Laue–Langevin (ILL) has arisen from the fact that current day SANS instruments at ILL now allow datasets to be acquired using small sample volumes with exposure times that are often shorter than a minute. This capability is of particular importance for the study of unstable biological macromolecules where aggregation or denaturation issues are a major problem. The first use of SEC-SANS on ILL's instrument D22 is described for a variety of proteins including one particularly aggregation-prone system.