Nanoprojectile Secondary Ion Mass Spectrometry for Analysis of Extracellular Vesicles

Abstract

We describe a technique based on secondary ion mass spectrometry with nanoprojectiles (NP-SIMS) for determining the protein content of extracellular vesicles, EVs, via tagged antibodies. The technique uses individual gold nanoprojectiles (e.g., Au400 4+ and Au2800 8+), separated in time and space, to bombard a surface. For each projectile impact (10–20 nm in diameter), the co-emitted molecules are mass analyzed and recorded as an individual mass spectrum. Examining these individual mass spectra for co-localized species allows for nanoscale mass spectrometry to be performed. The high lateral resolution of this technique is well suited for analyzing nano-objects. SIMS is generally limited to analyzing small molecules (below ∼1500 Da); therefore, we evaluated three molecules (eosin, erythrosine, and BHHTEGST) as prospective mass spectrometry tags. We tested these on a model surface comprising a mixture of all three tags conjugated to antibodies and found that NP-SIMS could detect all three tags from a single projectile impact. Applying the method, we tagged two surface proteins common in urinary EVs, CD63 and CD81, with anti-CD63–erythrosine and anti-CD81–BHHTEGST. We found that NP-SIMS could determine the relative abundance of the two proteins and required only a few hundred or thousand EVs in the analysis region to detect the presence of the tagged antibodies.

Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

“…To test for co-localization, we used nanoprojectile secondary-ion mass spectrometry (NP-SIMS). NP-SIMS involves bombarding the surface with a suite of individual nanoprojectiles (e.g., Au 400 4+ ) separated in time and space, allowing for mass spectrometry measurements on the nanoscale . In our study, NP-SIMS was used to examine immunoprobes for co-localization of metal ions and Abs on the AuNPs.…”

“…NP-SIMS involves bombarding the surface with a suite of individual nanoprojectiles (e.g., Au 400 4+ ) separated in time and space, allowing for mass spectrometry measurements on the nanoscale. 37 In our study, NP-SIMS was used to examine immunoprobes for co-localization of metal ions and Abs on the AuNPs. Samples for NP-SIMS analysis were prepared on a clean silicon substrate using the Marangoni flow-assisted method which, resulted in a sparse distribution of NPs.…”

Using Electrochemical Immunoassay in a Novel Microtiter Plate to Detect Surface Markers of Preeclampsia on Urinary Extracellular Vesicles

“…We used three types of samples in this study: (1) commercial pregnancy urine that was used for immunoassay characterization, (2) media conditioned by hepatic cells (Huh7) for selectivity experiments, and (3) patient urine samples for assessing clinical utility of our immunoassay. EVs from commercial urine samples were isolated using a total EV isolation kit (Invitrogen, Carlsbad, CA) as described before. , Briefly, urine was mixed with an EV isolation reagent and incubated for 1 h. After, EVs were collected by centrifugation at 10,000 g and dispersed in 1× PBS. EVs from Huh7 cells were isolated by differential ultracentrifugation of conditioned media as previously described . EVs isolated at 100,000 g were resuspended 1× PBS. Clinical urine samples were obtained from normotensive (NT) and preeclamptic (PE) pregnant women at Mayo Clinic according to the IRB protocol 2104–05.…”

“…(1) EVs from commercial urine samples were isolated using a total EV isolation kit (Invitrogen, Carlsbad, CA) as described before. 15,31 Briefly, urine was mixed with an EV isolation reagent and incubated for 1 h. After, EVs were collected by centrifugation at 10,000g and dispersed in 1× PBS. (2) EVs from Huh7 cells were isolated by differential ultracentrifugation of conditioned media as previously described.…”

New Methodology for Accurate Determination of Molecular Co-localization at the Nanoscale

“…Neither super-resolution microscopy nor nano-SIMS, two of the most advanced technologies for biomolecule detection, allows us to quantify many distinct co-localization events. Nanoprojectile-SIMS (NP-SIMS) operating in the event-by-event bombardment mode, while lacking imaging capability, is well suited for quantifying molecular co-localization. − In this approach, the surface is sampled randomly with individual nanoprojectile impacts that act like nanoprobes. By recording the emitted secondary ions from each bombardment event separately, we can identify two or more different tagged moieties which are located within ∼20 nm of each other.…”