Xudong Lou

Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.

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.

Study of Cell Antigens and Intracellular DNA by Identification of Element-Containing Labels and Metallointercalators Using Inductively Coupled Plasma Mass Spectrometry

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.

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.

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.