Tantalum Oxide Nanoparticle-Based Mass Tag for Mass Cytometry.

Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k-N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k-goat antimouse, TaO2-PEG2k-CD25, TaO2-PEG2k-CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k-goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k-CD25 and TaO2-PEG2k-CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.

Automated Data Cleanup for Mass Cytometry.

Mass cytometry is an emerging technology capable of 40 or more correlated measurements on a single cell. The complexity and volume of data generated by this platform have accelerated the creation of novel methods for high-dimensional data analysis and visualization. A key step in any high-level data analysis is the removal of unwanted events, a process often referred to as data cleanup. Data cleanup as applied to mass cytometry typically focuses on elimination of dead cells, debris, normalization beads, true aggregates, and coincident ion clouds from raw data. We describe a probability state modeling (PSM) method that automatically identifies and removes these elements, resulting in FCS files that contain mostly live and intact events. This approach not only leverages QC measurements such as DNA, live/dead, and event length but also four additional pulse-processing parameters that are available on Fluidigm Helios™ and CyTOF® (Fluidigm, Markham, Canada) 2 instruments with software versions of 6.3 or higher. These extra Gaussian-derived parameters are valuable for detecting well-formed pulses and eliminating coincident positive ion clouds. The automated nature of this new routine avoids the subjectivity of other gating methods and results in unbiased elimination of unwanted events. © 2019 International Society for Advancement of Cytometry.

Enabling Indium Channels for Mass Cytometry by Using Reinforced Cyclam-Based Chelating Polylysine.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In(cb-te2pa)]+ chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a fluorescein isothiocyanate/neutravidin receptor. High quality imaging mass cytometry trials, based on 115In detection were performed to highlight the behavior of the material. Anti-CD20 antibody was labeled by the so-obtained In(III)-modified polylysine using the biotin/neutravidin interaction. Ramos (CD20[+]) and HL-60 (CD20[-]) cell lines were costained with the In(III)-modified bioconjugate by finding the best staining conditions. Both immunofluorescence microscopy (IF-M) and mass cytometry analyses confirmed the specific binding of anti-CD20 onto Ramos cells. CyTOF histograms constructed on the 115In detection allowed us to define and to separate, with a good signal-to-noise ratio, two populations (Ramos and HL-60). The inertness of In(III)-MCP-NAv over a three-month storage period was proved by performing new functionality tests involving Jurkat cells (CD20[-]) and multiparametric trials involving the 115In channel. The results ensure a promising future use of the previously announced [In(cb-te2pa)]+ complex-based polymers for mass cytometry.

Highly Stable and Inert Complexation of Indium(III) by Reinforced Cyclam Dipicolinate and a Bifunctional Derivative for Bead Encoding in Mass Cytometry.

H2 cb-te2pa, a cross-bridged cyclam functionalized by two picolinate arms, was used for the formation of an incredible inert InIII chelate. The inertness of the complex was evaluated by UV/Vis experiments in several competitive media and was highlighted by the comparison with [In(dota)]- and [In(dtpa)]2- (H4 dota = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, H5 dtpa = diethylenetriamine pentaacetic acid), which are currently used in biological applications. For the first time, a bifunctional analogue of H2 cb-te2pa was prepared by C-functionalization to keep its coordination properties intact. However, this strategy leads to the formation of two diastereoisomers as evidenced and studied by NMR experiments and DFT calculations. Kinetic studies proved nevertheless that both isomers of the complex are equally inert. They were therefore used without distinction for their covalent grafting on polystyrene beads. The so-called metal-encoded beads were tested for imaging mass cytometry. The detection of 115 In allows the generation of images with high quality, proving the great potential of the bifunctional [In(cb-te2pa)]+ derivatives for single-cell analysis by mass cytometry.

Opposing Roles of Dendritic Cell Subsets in Experimental GN.

Dendritic cells (DCs) are thought to form a dendritic network across barrier surfaces and throughout organs, including the kidney, to perform an important sentinel function. However, previous studies of DC function used markers, such as CD11c or CX3CR1, that are not unique to DCs. Here, we evaluated the role of DCs in renal inflammation using a CD11c reporter mouse line and two mouse lines with DC-specific reporters, Zbtb46-GFP and Snx22-GFP. Multiphoton microscopy of kidney sections confirmed that most of the dendritically shaped CD11c+ cells forming a network throughout the renal interstitium expressed macrophage-specific markers. In contrast, DCs marked by Zbtb46-GFP or Snx22-GFP were less abundant, concentrated around blood vessels, and round in shape. We confirmed this pattern of localization using imaging mass cytometry. Motility measurements showed that resident macrophages were sessile, whereas DCs were motile before and after inflammation. Although uninflamed glomeruli rarely contained DCs, injury with nephrotoxic antibodies resulted in accumulation of ZBTB46 + cells in the periglomerular region. ZBTB46 identifies all classic DCs, which can be categorized into two functional subsets that express either CD103 or CD11b. Depletion of ZBTB46 + cells attenuated the antibody-induced kidney injury, whereas deficiency of the CD103+ subset accelerated injury through a mechanism that involved increased neutrophil infiltration. RNA sequencing 7 days after nephrotoxic antibody injection showed that CD11b+ DCs expressed the neutrophil-attracting cytokine CXCL2, whereas CD103+ DCs expressed high levels of several anti-inflammatory genes. These results provide new insights into the distinct functions of the two major DC subsets in glomerular inflammation.

Clickable and High-Sensitivity Metal-Containing Tags for Mass Cytometry.

Mass cytometry is a highly multiplexed single-cell analysis platform that uses metal-tagged reagents to identify multiple cellular biomarkers. The current metal-tagged reagent preparation employs thiol-maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues have been challenging to label using this method. This inherent limitation led us to develop a new conjugation strategy for labeling a wide range of biomolecules and affinity reagents. In this report, we present a metal tagging approach using a new class of azide- or transcyclooctene-terminated MCPs with copper(I)-free strain-promoted alkyne-azide cycloaddition or tetrazine-alkene click chemistry reactions, in which biomolecules with -NH2 functional groups are selectively activated with a dibenzocyclooctyne or tetrazine moiety, respectively. This approach enabled us to generate highly sensitive and specific metal-tagged IgGs, IgMs, small peptides, and lectins for applications in immunophenotyping and glycobiology. We also created dual-tagged reagents for simultaneous detection of markers by immunofluorescence, mass cytometry, and imaging mass cytometry using a two-step conjugation process. The Helios mass cytometer was used to test the functionality of reagents on suspension human leukemia cell lines and primary cells. The dual-tagged Abs, metal-tagged lectins, and phalloidin staining reagent were used to visualize target proteins and glycans on adherent cell lines and frozen/FFPE tissue sections using the Hyperion Imaging System. In some instances, reagents produced by click conjugation showed superior sensitivity and specificity compared to those of reagents produced by thiol-maleimide chemistry. In general, the click chemistry-based conjugation with new MCPs could be instrumental in developing a wide range of highly sensitive metal-containing reagents for proteomics and glycomics applications.

Aptamer-facilitated mass cytometry.

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.

Simultaneous Detection of Protein and mRNA in Jurkat and KG-1a Cells by Mass Cytometry.

Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope® platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells. © 2017 International Society for Advancement of Cytometry.

Imaging Mass Cytometry.

Imaging Mass Cytometry (IMC) is an expansion of mass cytometry, but rather than analyzing single cells in suspension, it uses laser ablation to generate plumes of particles that are carried to the mass cytometer by a stream of inert gas. Images reconstructed from tissue sections scanned by IMC have a resolution comparable to light microscopy, with the high content of mass cytometry enabled through the use of isotopically labeled probes and ICP-MS detection. Importantly, IMC can be performed on paraffin-embedded tissue sections, so can be applied to the retrospective analysis of patient cohorts whose outcome is known, and eventually to personalized medicine. Since the original description in 2014, IMC has evolved rapidly into a commercial instrument of unprecedented power for the analysis of histological sections. In this Review, we discuss the underlying principles of this new technology, and outline emerging applications of IMC in the analysis of normal and pathological tissues. © 2017 International Society for Advancement of Cytometry.

Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry.

Although of fundamental importance to the treatment of cancer patients, the quantitative study of drug distribution and action in vivo at the single cell level is challenging. We used the recently-developed technique of mass cytometry to measure cisplatin uptake into individual tumor cells (Pt atoms/cell), combined with measurement of the rate of IdU incorporation into DNA (I(127) atoms/cell/min) and tumor hypoxia identified by the 2-nitroimidazole EF5 in cisplatin-treated BxPC-3 and ME-180 xenografts. Pt levels of 10(5) to 10(6) atoms/cell were obtained following a single cisplatin treatment using clinically relevant doses. Cisplatin caused cell cycle arrest in a dose- and time-dependent manner that paralleled effects in vitro, and it readily penetrated into hypoxic tumor regions. Similar levels of Pt/cell were found in xenografts treated with oxaliplatin. Mass cytometry offers the unique capability to study the cellular uptake and anticancer effects of platinum-containing drugs at the single cell level in animal models, and it has the potential for application to samples obtained from cancer patients during treatment.

An introduction to mass cytometry: fundamentals and applications.

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.

Dual-purpose polymer labels for fluorescent and mass cytometric affinity bioassays.

We describe the synthesis and characterization of a family of poly(N-alkylacrylamide) polymers carrying 2-6 fluorescent dye molecules, ∼70 pendant DTPA (diethylenetriaminepentaacetic acid) groups, and an orthogonal maleimide end-group for covalent attachment to an antibody (Ab). These dual-purpose labels were designed for use in multiplexed immunoassays based on both mass cytometry and fluorescent flow cytometry. A challenge in the polymer synthesis was finding conditions for attaching a sufficient number of dye molecules to each polymer chain. Although attachment of a terminal maleimide to the polymers was not as efficient as anticipated, the end-functional polymers were still effective in labeling Abs. Secondary goat antimouse IgG was labeled with the four dual-label polymers as well as a control polymer, and while the resultant antibody-polymer conjugates showed positive performance in mass cytometric and fluorescent assays, some trials showed problems such as low signal and nonspecific adsorption. Four primary antibody conjugates were prepared and used to stain cells in 4-plex assays. The results of both primary assays are bittersweet in that the CD3-FITC and CD45-DyLight 649 conjugates performed well, while the CD13-DyLight 405 and the CD38-DyLight 549 conjugates did not.

Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry.

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.

Metal-chelating polymers by anionic ring-opening polymerization and their use in quantitative mass cytometry.

Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined. Here we describe the synthesis of a new type of MCP based on anionic ring-opening polymerization of an activated cyclopropane (the diallyl ester of 1,1-cyclopropane dicarboxylic acid) using a combination of 2-furanmethanethiol and a phosphazene base as the initiator. This reaction takes place with rigorous control over molecular weight, yielding a polymer with a narrow molecular weight distribution, reactive pendant groups for introducing a metal chelator, and a functional end group with orthogonal reactivity for attaching the polymer to the mAbs. Following the ring-opening polymerization, a two-step transformation introduced diethylenetriaminepentaacetic acid (DTPA) chelating groups on each pendant group. The polymers were characterized by NMR, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The binding properties toward Gd(3+) as a prototypical lanthanide (Ln) ion were also studied by isothermal titration calorimetry (ITC). Attachment to a mAb involves a Diels-Alder reaction of the terminal furan with a bismaleimide, followed by a Michael addition of a thiol on the mAb, generated by mild reduction of a disulfide bond in the hinge region. Polymer samples with a number average degree of polymerization of 35, with a binding capacity of 49.5 ± 6 Ln(3+) ions per chain, were loaded with 10 different types of Ln ions and conjugated to 10 different mAbs. A suite of metal-tagged Abs was tested by mass cytometry in a 10-plex single cell analysis of human adult peripheral blood, allowing us to quantify the antibody binding capacity of 10 different cell surface antigens associated with specific cell types.

Multiplexed protease assays using element-tagged substrates.

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.

Development of mass cytometry methods for bacterial discrimination.

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.

Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.

We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis.

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Synthesis of a functional metal-chelating polymer and steps toward quantitative mass cytometry bioassays.

We describe the synthesis and characterization of metal-chelating polymers with a degree of polymerization of 67 and 79, high diethylenetriaminepentaacetic acid (DTPA) functionality, M(w)/M(n) ≤ 1.17, and a maleimide as an orthogonal functional group for conjugation to antibodies. The polymeric disulfide form of the DP(n) = 79 DTPA polymer was analyzed by thermogravimetric analysis to determine moisture and sodium-ion content and by isothermal titration calorimetry (ITC) to determine the Gd(3+) binding capacity. These results showed each chain binds 68 ± 7 Gd(3+) per chain. Secondary goat antimouse IgG was covalently labeled with the maleimide form of the DTPA polymer (DP(n) = 79) carrying (159)Tb. Conventional ICPMS analysis of this conjugate showed each antibody carried an average of 161 ± 4 (159)Tb atoms. This result was combined with the ITC result to show there are an average of 2.4 ± 0.3 polymer chains attached to each antibody. Eleven monoclonal primary antibodies were labeled with different lanthanide isotopes using the same labeling methodology. Single cell analysis of whole umbilical cord blood stained with a mixture of 11 metal-tagged antibodies was performed by mass cytometry.

Development of inductively coupled plasma-mass spectrometry-based protease assays.

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.