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Lou X, Zhang G, Herrera I, Kinach R, Ornatsky O, Baranov V, Nitz M, Winnik MA. Polymer-based elemental tags for sensitive bioassays, Angew Chem Int Ed Engl. 2007 May 29;46(32):6111-6114.
Tanner SD, Ornatsky O, Bandura DR, Baranov VI. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology, Spectrochimica Acta Part B: Atomic Spectroscopy, 2007 Mar;62(3):188-195.
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“If cancer stem cells lie at the heart of some cancers, then being able to predict the behavior of tumors and providing effective therapies against them means understanding the abnormal growth pathways within the stem cells themselves.”
- John Dick, Project Leader (UHN)
“The only reason that I'm willing to take a risk on this project is my confidence in the quality and camaraderie of my colleagues.”
- Scott Tanner, Principal Investigator (University of Toronto)
“We're examining DNA not for its well-recognized role as the genetic material to store and transmit genetic information, but rather for its less-known potential to act as a novel cancer diagnostic tool.”
- Yingfu Li, Principal Investigator (McMaster University)
“We're gaining more understanding of cell defects and how to modify them, so that treatment is no longer a shotgun approach, but more like an arrow aimed at a specific target.”
- Mark Minden, Principal Investigator (OCI)
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The University of Toronto group is responsible for the construction of the Inductively Coupled Plasma Mass Spectrometer (ICP-MS) with a flow cytometric cell injector, and for the development of a suite of affinity products able to detect targets of interest by ICP-MS.
In previous installments (see links at bottom of page), we described the development of the instrumentation and reagents, and presented examples of data obtained using our system. We continue to refine our methods and improve the power of our instrument.
Here, we provide an overview of our workflow and examples of recent proof-of-principle experiments in bulk format showing multiplexed protein and DNA detection. We also demonstrate the ability of our instrument to perform multiplexed analysis of single cells.
Using Metal Tags to Detect Proteins
Click here to view a recent poster describing our work on elemental tags.
The key to our method of simultaneously measuring multiple biomarkers lies in our ability to produce element-tagged affinity products. Because of the high resolution of detection channels in ICP-MS, multiplex assays of the order of 50-100 are possible. This technology allows for rapid protein expression profiling of both cell surface and intracellular biomarkers.
Figure 1: Protocol for tagging antibodies with metals. Antibodies are partially reduced and reacted with polymer-ligand conjugate. Metal atoms are then incorporated into the tagged antibodies.
Figure 2: Protocol for labeling cells with metal-tagged antibodies. Metal-tagged antibodies are incubated with cell samples, mixed with concentrated HCl, and measured by ICP-MS.
Figure 3: Multiplex ICP-MS assay in K562 cells. Expression of various cell surface and intracellular markers in the erythroleukemia cell line K562 was analyzed using ICP-MS and metal-tagged antibodies as indicated. Values were normalized by using a rhodium-containing DNA intercalator, which measures the number of cells in the sample.
Figure 4: Multiplex ICP-MS assay in MBA-4 cells. Expression of various cell surface and intracellular markers in the megakaryocytic cell line MBA-4 was analyzed using ICP-MS and metal-tagged antibodies as indicated. Values were normalized by using a rhodium-containing DNA intercalator, which measures the number of cells in the sample.
Figure 5: Measurement of differentiation markers by ICP-MS. Differentiation of megakaryocytic cells was induced by treatment with PMA, and changes in protein expression were measured by ICP-MS. Increases in expression of the megakaryocytic markers CD54 and CD61 were detected upon PMA treatment, as well as in expression of CD117, Ki-67 and beta-tubulin.
Using Metal Tags to Detect DNA
The ICP-MS instrument and element-tagged reagents developed by the U of T group can also be used to measure DNA. In situ hybridization experiments have been successfully performed, making it possible to use our technology to carry out powerful multiplex assays that examine gene expression.
Figure 6: Protocol for measuring in situ hybridization by ICP-MS. Cell samples are mixed with biotin-labeled oligonucleotide probes, followed by incubation with streptavidin-conjugated metal-containing reagents. Analysis by ICP-MS generates a rapid read-out of the levels of complementary mRNA transcripts in the sample.
Figure 7: In situ hybridization measured by ICP-MS allows rapid quantitation of mRNA expression. In situ hybridization was performed with adherent A431 cells using probes complementary to expressed sequences of EGFR, D-cyclin and ribosomal 28S RNA. B/A, a nonsense oligo probe, was used as a negative control.
Figure 8: In situ hybridization measured by ICP-MS allows comparison of mRNA expression in different cell types. In situ hybridization was performed with K562 cells and KG-1a cells using probes complementary to expressed sequences of oncogenic BCR/abl and ribosomal 28S RNA. B/A, a nonsense oligo probe, was used as a negative control. A two-fold difference in expression of BCR/abl compared to control B/A probe was observed in K562 cells, whereas no difference in binding of the two probes was observed in KG-1a cells.
Multiplex Single Cell Detection by ICP-MS
By combining ICP-MS with a flow cytometric injector, our technology will permit multiplexed assays at the level of a single cell.
Figure 9: First multiplex analysis of a single cell by the Research Prototype instrument. The research prototype instrument is a Time Of Flight (TOF) configuration, for which ions are extracted from the plasma and collected in an acceleration region. A pulse in the acceleration region accelerates the ions into the flight tube, and the arrival time of the ions is recorded. The "time of flight" of the ions through the tube is proportional to the square root of the ions' mass. In this figure, the time of flight for each pulse (i.e., deconvolutes to ion mass) is shown on the horizontal axis, and the pulse number (the sequence number of mass spectra) is shown on the vertical axis. The mass spectra are recorded at 12 microsecond intervals, and the duration of a cell vaporization/ionization event is approximately 100-150 microseconds: therefore, some 10 spectra are recorded for each cell event. In the data shown, the single and double dense bars can be regarded as the start and end of each pulse, and the spectra are grouped in roughly 80 spectra groupings: the shift that is evident in groupings is a result of the data download time (1 MB per download). The circled data shows the appearance of a transient signal that has a duration (vertical scale) comparable to a cell vaporization event and contains element signatures that correspond to the tag elements that marked the antibodies introduced to bind to specific cell proteins (MBA-4 cells).
Figure 10: Expansion of multiplexed single cell signature. The upper figure shows data similar to the circled region in Figure 9, but in this case is colour-coded for intensity. At the left is seen the signature for Rh, which was introduced as a metallointercalator to bind DNA in the MBA-4 cell. The other elements (e.g., two Eu isotopes and Tm are indicated) correspond to element-tags attached to the antibodies against cellular proteins. (The large Pb signal is due to an impurity in the sample introduction materials.) The lower figure provides the mass spectrum integrated over many similarly immuno-tagged MBA-4 cells, and the specific tag elements are indicated. The Rh metallointercalator is the strongest signal, followed by Eu and Tm. The other elements also appear at appropriately less intensity in the upper figure.
Click here for the first installment of this page
Click here for the second installment of this page
Click here for the third installment of this page
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