Reagents and methods for elemental mass spectrometry of biological samples
Embodiments of the present invention relate to reagents and their use for elemental imaging mass spectrometry of biological samples. Embodiments include a method for quantifying one or more analytes within a sample. The method may include providing the sample. One or more analytes may be immobilised to a mass cytometry sample carrier. The sample may have been labelled with one or more mass-tagged SBPs including one or more labelling atoms. The method may include performing mass cytometry on the sample to determine a level of the one or more labelling atoms, where the level of the one or more labelling atoms corresponds to the copy number of the one or more analytes to quantify the analytes.
1. A method for quantifying one or more analytes within a sample, the method comprising:
a. providing the sample, wherein the one or more analytes are immobilised to a mass cytometry sample carrier, wherein the sample has been labelled with one or more mass-tagged specific binding pairs comprising one or more labelling atoms, wherein the mass cytometer sample carrier is planar and comprises a polydopamine layer, and
b. performing mass cytometry on the sample to determine a level of the one or more labelling atoms, wherein the level of the one or more labelling atoms corresponds to the copy number of the one or more analytes to quantify the analytes.
2. The method of claim 1 , wherein the quantitation of the analytes resulting from performing mass cytometry is:
a. relative, or
b. absolute, comprising comparison of the level of the one or more labelling atoms to a calibration curve.
3. The method of claim 1 , wherein the mass cytometer sample carrier comprises a substrate and a surface modification, wherein one or more capture elements are immobilised to the surface modification.
4. The method of claim 3 , wherein the one or more analytes are immobilised to the sample carrier via one or more capture elements.
5. The method of claim 1 , wherein the method further comprises sampling and ionizing sample material to form sample ions, and analysing said ions in a mass detector.
6. The method of claim 5 , in which the sampling and ionisation comprises:
a. laser ablation followed by separate ionisation of the sample material in an ICP to form sample ions; or
b. laser desorption ionisation (LDI) to form sample ions.
7. The method of claim 5 , in which the sampling and ionisation comprises particulate mass cytometry sample carriers from a solution and introducing a particulate material individually into an ionisation system to form sample ions.
8. The method of claim 2 , wherein:
the quantitation is absolute, and
the calibration curve comprises at least two points.
9. The method of claim 2 , wherein:
the quantitation is absolute, and
the calibration curve is determined prior to analysis of the sample.
10. The method of claim 2 , wherein:
the quantitation is absolute, and
the calibration curve is determined based on a set of doped polystyrene beads or set of nanoparticles, wherein each member of the set contains a known quantity of one or more labelling atoms.
11. The method of claim 1 , wherein the mass tag of the mass tagged specific binding pair is:
a. a metal chelating moiety which has been loaded with at least one metal labelling atom;
b. a nanoparticle comprising at least one labelling atom; or
c. a polymer comprising at least one labelling atom in its backbone.
12. The method of claim 11 , wherein the metal chelating moiety is a polymer having a degree of polymerization of between 1 and 10,000.
13. The method of claim 11 , wherein the metal chelating moiety is a polymer comprising between 1 and 10,000 metal-chelating groups, wherein each metal chelating group comprise 1,4,7,10-tetraazacycloidodecane-1,4,7,10-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTPA), or combinations thereof, and wherein each metal chelating group is attached to a polymer subunit derived from either a substituted polyacrylate, polyacrylamide, polymethacrylate, or polymethacrylamide.
14. The method of claim 11 , wherein the one or more labelling atoms have an atomic mass in the range 80-250.
15. The method of claim 11 , wherein all labelling atoms in a mass tag are of the same atomic mass.
16. The method of claim 11 , at least one mass tag comprises labelling atoms of differing atomic mass.
17. The method of claim 1 , wherein the one or more mass-tagged specific binding pairs comprise one or more of the following:
a. a protein comprising a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a multispecific antibody, an antibody fusion protein, scFv, antibody mimetic, avidin, streptavidin, neutravidin, biotin, a nanobody, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, kunitz domain peptide, monobody, a receptor, a receptor-Fc fusion, a ligand, a ligand-Fc fusion, a lectin, an agglutinin, wheat germ agglutinin, or a combination thereof;
b. a peptide comprising a linear peptide, a cyclical peptide, a bicyclic peptide, or phalloidin; or
c. a nucleic acid comprising a polynucleotide, oligonucleotide, DNA, RNA, cDNA, a polynucleotide analog, xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), peptide nucleic acids (PNAs), yPNAs, morpholino polynucleotides, locked nucleic acids (LNAs), threose nucleic acid (TNA), 2′-0-Methyl polynucleotides, 2′-0-alkyl ribosyl substituted polynucleotides, phosphorothioate polynucleotides, or boronophosphate polynucleotides.