Q : Isn't mass spectrometry an expensive, difficult, non-quantitative, low sensitivity method that is dependent on the specific chemical composition of the biomarker?

A : Use of the term "mass spectrometer" in the bioanalytical community almost invariably evokes recognition of organic (protein) mass spectrometry. This is quite distinct from the present technology. The mass spectrometer used in this application is an inorganic (elemental) mass spectrometer, specifically an ICP-MS. Conventional organic mass spectrometry attempts to recognize proteins or peptides through mass fingerprints with reference to massive libraries, and is encumbered by sample-dependent sensitivity (both the specific functionalization of the biomarker and ionization interference by the cell matrix). Accordingly, it has drawbacks inherent to the complexity of the instrument, sample preparation and data analysis.

ICP-MS provides virtually sample-matrix-independent sensitivity, absolute quantification, high dynamic range, excellent resolution of mass channels, and simple interpretation (only the elements of the periodic table). It has a reputation for great ruggedness and simplicity in the environmental analysis and metal-toxicology fields, but is new for the biosciences. It is not a simplification to say that the "mass spectrometer" in this instance is merely a detector that quantitatively informs on the elemental composition of the tags in the sample.

Q : What is an Inductively Coupled Plasma Mass Spectrometer?

A : ICP-MS uses an inductively coupled plasma ionization source linked to a mass spectrometer. It is the preferred means of determining the elemental composition, especially ultra-trace components, of materials. It has found acceptance in various applications including environmental (e.g., drinking, river, sea and waste water analyses), geological (e.g., trace element patterning), clinical (e.g., determination of trace metals in blood, serum and urine) and high purity materials (e.g., semiconductor reagents and components) analysis.

Briefly, a sample, most commonly an aerosol produced by nebulization, is injected into a high- temperature plasma obtained by heating a flowing argon gas stream with radio frequency (RF) energy. Under conditions approximating those at the surface of the sun, the sample is promptly vaporized, atomized and ionized as it flows through the plasma. High speed mass analysis provides a "mass fingerprint" that identifies the elements contained in the sample. The particular attributes of the method of note include: wide linear dynamic range (9 orders of magnitude), exceptional sensitivity (sub-part per trillion, or attomole/microlitre, detection), enormous abundance sensitivity (<10 -5 overlap between adjacent isotopes), counting-statistics-limited precision, absolute quantification, and tolerance of concomitant matrix. In its normal analytical application, ICP-MS is a simple push-button operated system, and often linked to an auto-sampler with data independently collected around the clock.

Q : Is compensation really not required?

A : The abundance sensitivity (overlap of one detection, or mass, channel on the neighbor channel) is less than one part in 10⁴ (10⁶ for solution assay) so that correction for overlapping signals is not required for relative biomarker concentrations less than 4 (or 6) orders of magnitude; virtually no overlap is possible when alternate mass channels are used. In practice, no corrections or compensations are required, because tag isotopes can be selected to be free of overlap or interference. At the current state of the art in sample preparation, the expected variability between sample replicates by far exceeds any uncertainty caused by variation of the natural or artificial isotope abundance or by trace amounts of polyatomic ions. Such "compensation", if ever needed, can be performed by software based on one-time calibration and does not require samples additional to the test and control sets.

Q : But isn't the real limitation to multiplex analysis the fact that antibodies are cross-reactive?

A : This clearly remains the fundamental limitation to wide dynamic range, massively multiplexed immunoassay. In recent years, antibody manufacturers have responded to the need for multiplexing by taking a responsible approach to verifying the independence of suites of antibodies for specific diagnostic challenges, and this is expected to continue so that multiplexing becomes increasingly viable. Nonetheless, element tagging is not subject to the challenges of autofluorescence (only isotopes that are absent in biological samples are employed), and multiplexing can be extended with the use of complementary affinity products (concomitant use of oligonucleotide probes, aptamers, intercalators and antibodies) that are labeled with metalopolymers as tags for ICP-MS detection. This clearly remains the fundamental limitation to wide dynamic range, massively multiplexed immunoassay. In recent years, antibody manufacturers have responded to the need for multiplexing by taking a responsible approach to verifying the independence of suites of antibodies for specific diagnostic challenges, and this is expected to continue so that multiplexing becomes increasingly viable. Antibody panels developed for protein arrays will be ready-qualified for mass cytometry. Importantly, element tagging is not subject to the challenges of autofluorescence (only isotopes that are absent in biological samples are employed), and multiplexing can be extended with the use of complementary affinity products (concomitant use of oligonucleotide probes, aptamers, intercalators and antibodies) that are labeled with metalopolymers as tags for ICP-MS detection.

Q : What do the products look like?

A : MAXPAR™ comprises a system of novel reagents designed specifically for massively multi-parametric biological assays. These may be considered the analog of fluorophores, where the fluorescent moiety is replaced by multiple copies of an element (stable, non-radioactive isotope), and for which analysis is provided by elemental mass spectrometry (e.g., ICP- MS). Another analog can be drawn to a radio-isotope immunoassay - with the critical advantage of the element-based immunoassay residing in the ability of the mass-spectrometry detector to quantify multiple different element/isotope tags simultaneously present in a sample. The core of the technology is a metalopolymeric tag that tightly binds (KD <10^-16) the reporter stable isotopes and is conjugated to the affinity reagent via a linker group or is included in the core of element bar-coded bead . The basic MAXPAR™ kit contains enough material to label 100 µg of antibody with a customer-specified element tag, as well as TBS, ammonium acetate buffers and instructions; not included are TCEP and 30K spin filters.

CyTOF™ is a high performance mass cytometer based on a novel elemental mass-spectrometry detection technology with multiple independent detection channels. The instrument combines a cell introduction system, operating at up to 1000 individual cells per second, with an elemental mass spectrometer (ICP-MS) detector that quantifies the MAXPAR™ reagent tags. The instrument data system recognizes single whole cell events and reports multi-parameter signatures for each cell. The data is formatted to be compatible with third party flow cytometry analysis software.