[show abstract][hide abstract] ABSTRACT: Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.
Cancer Immunology and Immunotherapy 04/2013; · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: This article describes a systematic study of ion release, carried out on lanthanide-encoded polystyrene-co-methacrylic acid (P(S-MAA)) and polystyrene-co-acrylic acid (P(S-AA)) copolymer particles, which were synthesized by two-stage or three-stage dispersion polymerizations. These particles with different levels of lanthanide (Ln) ion content and containing several different types of Ln ions were dispersed in normal buffer media, namely 2-(N-morpholino)ethanesulfonic acid (MES), phosphate buffered saline solution (PBS), ammonium acetate (AmAc) and buffers containing strong chelating molecules like ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Traditional inductively coupled plasma mass spectrometry (ICP-MS) was employed to follow the loss of ions into the aqueous medium as a function of time. The release behavior of the Ln ions were compared. In MES and PBS buffers at neutral pH, and AmAc at pH 9.0, there was essentially no significant loss of Ln ions from the particles to the buffer. When the chelating agents EDTA or DTPA were present in the buffer, the loss of Ln ions was more prominent, but less than 15% after 8 weeks under stirring. The differences among the different samples were small. Considering the ability of EDTA and DTPA to remove Ln ions from the particles and the fact of minimal ion release in the presence of PBS buffer, we infer that the ion loss is a more active process involving the EDTA or DTPA molecules. The main conclusion is that in the absence of strong chelating agents, these particles are stable against ion leakage, even upon prolonged storage and stirring. This is of great importance for their application in bead-array biological assays based on mass cytometry detection.
[show abstract][hide abstract] ABSTRACT: Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.
[show abstract][hide abstract] ABSTRACT: Lanthanide-encoded polystyrene particles synthesized by dispersion polymerization are excellent candidates for mass cytometry based immunoassays, however they have previously lacked the ability to conjugate biomolecules to the particle surface. We present here three approaches to post-functionalize these particles, enabling the covalent attachment of proteins. Our first approach used partially hydrolyzed poly(N-vinylpyrrolidone) as a dispersion polymerization stabilizer to synthesize particles with high concentration of -COOH groups on the particle surface. In an alternative strategy to provide -COOH functionality to the lanthanide-encoded particles, we employed seeded emulsion polymerization to graft poly(methacrylic acid) (PMAA) chains onto the surface of these particles. However, these two approaches gave little to no improvement in the extent of bioconjugation. In our third approach, seeded emulsion polymerization was subsequently used as a method to grow a functional polymer shell (in this case, poly(glycidyl methacrylate) (PGMA)) onto the surface of these particles, which proved highly successful. The epoxide-rich PGMA shell permitted extensive surface bioconjugation of NeutrAvidin, as probed by an Lu-labeled biotin reporter (ca. 7 × 10(5) binding events per particle with a very low amount of non-specific binding) and analyzed by mass cytometry. It was shown that coupling agents such as EDC were not needed, such was the reactivity of the particle surface. These particles were stable and the addition of a polymeric shell was shown did not affect the narrow lanthanide ion distribution within the particle interior as analyzed by mass cytometry. These particles represent the most promising candidates for the development of a highly multiplexed bioassay based on lanthanide-labeled particles to date.
[show abstract][hide abstract] ABSTRACT: This article describes the synthesis and characterization of two series of functional polyelectrolyte copolymer microgels intended for bioassays based upon mass cytometry, a technique that detects metals by inductively coupled plasma mass spectrometry (ICP-MS). The microgels were loaded with Eu(III) ions, which were then converted in situ to EuF(3) nanoparticles (NPs). Both types of microgels are based upon copolymers of N-isopropylacrylamide (NIPAm) and methacrylic acid (MAA), poly(NIPAm/VCL/MAA) (VCL = N-vinylcaprolactam, V series), and poly(NIPAm/MAA/PEGMA) (PEGMA = poly(ethylene glycol)methacrylate, PG series). Very specific conditions (full neutralization of the MAA groups) were required to confine the EuF(3) NPs to the core of the microgels. We used mass cytometry to measure the number and the particle-to-particle variation of Eu ions per microgel. By controlling the amount of EuCl(3) added to the neutralized microgels. we could vary the atomic content of individual microgels from ca. 10(6) to 10(7) Eu atoms, either in the form of Eu(3+) ions or EuF(3) NPs. Leaching profiles of Eu ions from the hybrid microgels were measured by traditional ICP-MS.
[show abstract][hide abstract] ABSTRACT: Lanthanide-encoded polystyrene microspheres with methacrylic acid (MAA) as a co-monomer and with diameters on the order of 2 μm and a very narrow size distribution were synthesized by two-stage dispersion polymerization (2-DisP). These microspheres were designed as a platform for mass cytometry-based bioassays. Different lanthanides (Ln) were loaded into these microspheres during the synthesis, through the addition of LnCl3 salts and excess MAA to the reaction after about 10% conversion of styrene, i.e., well after the microsphere nucleation stage was complete. Different levels of MAA were employed to investigate the relationship between the number of carboxyl group on the particle surface and the amount of MAA used. The reaction remained well controlled with both 2 and 4 wt % MAA. As monitored by inductively coupled plasma (ICP) mass spectrometry, we found high incorporation efficiency (>95%) of Ln ions into the particles when the total amount of LnCl3 salts in the reaction mixture was sufficiently small. The Ln incorporation efficiency decreased with the increasing amount of LnCl3 salts. Mass cytometry analysis shows that individual microspheres contain ca. 105–108 chelated lanthanide ions, either a single element or a mixture of elements. This method of incorporating lanthanide into P(S-MAA) particles through the second stage of two-stage dispersion polymerization yields microspheres suitable for the highly multiplexed detection of biomolecules.
[show abstract][hide abstract] ABSTRACT: Inductively coupled plasma-mass spectrometry (ICP-MS)-based assays lend themselves to multiplexing due to the high resolution between mass channels, the sensitivity, and the reliability of the technique. Here the potential of ICP-MS-based protease assays is demonstrated with a quadruplex assay of cysteine proteases and metalloproteases. Four orthogonal peptide substrates were synthesized for the proteases calpain-1, caspase-3, matrix metalloprotease-9 (MMP-9), and a disintegrin and metalloprotease-10 (ADAM10). Each substrate carries a biotin tag at the C terminus and a diethylenetriaminepentaacetic acid (DTPA)-based lanthanide complex at the N terminus. The results demonstrate that this is a simple and reproducible analysis technique with excellent correlation between the single and multiplex assay formats.
[show abstract][hide abstract] ABSTRACT: This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.
Journal of immunological methods 09/2010; 361(1-2):1-20. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.
[show abstract][hide abstract] ABSTRACT: We present the synthesis and characterization of monodisperse, sub-micron poly(styrene) (PS) particles loaded with up to and including 10(7) lanthanide (Ln) ions per particle. These particles have been synthesized by seeded emulsion polymerization with a mixture of monomer and a pre-formed Ln complex, and analyzed on a particle-by-particle basis by a unique inductively coupled plasma mass cytometer. Seed particles were prepared by surfactant-free emulsion polymerization (SFEP) to obtain large particle sizes in aqueous media. Extensive surface acid functionality was introduced using the acid-functional initiator ACVA, either during seed latex synthesis or in the second stage of polymerization. The loading of particles with three different Ln ions (Eu, Tb, and Ho) has proven to be close to 100 % efficient on an individual and combined basis. Covalent attachment of metal-tagged peptides and proteins such as Neutravidin to the particle surface was shown to be successful and the number of bound species can be readily determined. We believe these particles can serve as precursors for multiplexed, bead-based bio-assays utilizing mass cytometric detection.
[show abstract][hide abstract] ABSTRACT: In this article we demonstrate that hybrid nanogels can be prepared by encapsulation of reactive nanoparticles (NPs) directly during nanogel synthesis. Nanogels investigated in present study consist of poly(N-vinylcaprolactam-co-(2-acetoacetoxyethyl) methacrylate) copolymer. The modification of the LaF3:Eunanoparticle surface with reactive double bonds allows effective incorporation of the NPs into the nanogel structure. This approach ensures effective encapsulation of varying amounts of NPs into the nanogel interior with a loading efficiency close to 95%. The NPs are covalently bound to the nanogel core, and no NP leakage occurs. Reactive NPs act as multifunctional cross-linking agents and increase the cross-linking degree of the nanogels. We demonstrate the possibility of the incorporation of LaF3 nanoparticles doped with different ions (Eu, Tb, Pr, Gd) or nanoparticle mixtures into nanogels. These nanogels exhibit temperature-sensitive properties and superior colloidal stability in water and other aqueous media.
Journal of Materials Chemistry 01/2010; 20(24). · 5.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic alpha-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr approximately Asp-Lys(biotin) and Lu-DTPA-betaAla-betaAla-betaAla-betaAla-Gly-Ser-Ala-Tyr approximately Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for alpha-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2pM could be readily detected, superior to the detection limit of an assay using the alpha-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.
[show abstract][hide abstract] ABSTRACT: A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200-300 micros duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard inductively coupled plasma (ICP) operating conditions (<3% cerium oxide ratio). At mass resolution (full width at half-maximum) M/DeltaM > 900 for m/z = 159, the sensitivity with a standard sample introduction system of >1.4 x 10(8) ion counts per second per mg L(-1) of Tb and an abundance sensitivity of (6 x 10(-4))-(1.4 x 10(-3)) (trailing and leading masses, respectively) are shown. The mass range (m/z = 125-215) and abundance sensitivity are sufficient for elemental immunoassay with up to 60 distinct available elemental tags. When <15 elemental tags are used, a higher sensitivity mode at lower resolution (M/DeltaM > 500) can be used, which provides >2.4 x 10(8) cps per mg L(-1) of Tb, at (1.5 x 10(-3))-(5.0 x 10(-3)) abundance sensitivity. The real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown. A real time single cell 20 antigen expression assay of model cell lines and leukemia patient samples immuno-labeled with lanthanide-tagged antibodies is presented.
[show abstract][hide abstract] ABSTRACT: We describe the preparation and thermal response of aqueous microgels based on poly(N-vinyl caprolactam) containing grafted poly(ethylene glycol) (PEG) chains. These microgels were synthesized by free radical
copolymerization of vinyl caprolactam and acetoacetoxyethyl methacrylate in the presence of methoxy-capped poly(ethylene glycol)methacrylate
macromonomers. We show that variation of the amount of PEG macromonomer or the length of the PEG chain provides effective
control of the microgel diameter in the range 60–220nm. The presence of the grafted PEG chains improves the colloidal stability
of the microgels. The incorporation of the PEG macromonomers into microgel structure decreases the swelling degree and induces
a shift of the volume phase transition to higher temperatures.
Colloid and Polymer Science 02/2009; 287(3):269-275. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lectins have been increasingly important in the study of glycoproteins. Here, we report a glycoprofiling method based on the covalent attachment of metal-chelating polymers to lectins for use in an ICP-MS-based assay. The labeled lectins are able to distinguish between glycoproteins covalently attached to a microtiter plate and their binding can be directly quantified by ICP-MS. Since each conjugate contains a different lanthanide, the assays can be conducted in a single or multiplex fashion, and may be readily elaborated to many different assay formats.
Journal of Proteome Research 01/2009; 8(2):443-9. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well.
Journal of Immunological Methods 08/2008; 336(1):56-63. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.