HIGHLY MULTIPLEXED ABSOLUTE QUANTIFICATION OF MOLECULES ON THE SINGLE CELL LEVEL

Abstract

The invention relates to a method for determining a biomarker on single cell level by counting a first portion of a cell sample, subjecting said first portion to conditions whereby the biomarker is fragmented, adding to a known number n(k) of labelled biomarker fragments, measuring a first and a second parameter of said first portion, wherein said first parameter corresponds to the amount of said biomarker fragment and said second parameter corresponds to the amount of said labelled biomarker fragment yielding a biomarker fragment value, v(u), and a labelled biomarker fragment value v(k), respectively, and relating v(u) and v(k) with n(k) and c(1), thereby determining an average number of biomarker molecules per cell, m(u), of said first portion. Subsequently, a second portion of the cell sample is contacted with a label specific for the biomarker, a value of the label is determined for the second portion, yielding a single cell measurement value s(u) for the cells of the second portion, a mean measurement value m(s) is determined, and a number of biomarker molecules, n(u) is computed from s(u), m(s) and m(u) for each cell.

Claims

1. A method for determining the number of molecules of a biomarker, said method comprising the steps of: a) obtaining a first portion of a cell population, wherein said cell population comprises a plurality of single cells characterized by the presence of said biomarker, b) obtaining a cell number, c(1), of said first portion, c) i. subjecting the cells of said first portion to conditions whereby said biomarker is fragmented, yielding a biomarker fragment, in a fragmentation step; ii. adding to said biomarker fragments a known number n(k) of labelled biomarker fragments differing from said biomarker fragment only in a detectable label, iii. measuring a first and a second parameter of said first portion, wherein said first parameter corresponds to the amount of said biomarker fragment and said second parameter corresponds to the amount of said labelled biomarker fragment yielding a biomarker fragment value, v(u), and a labelled biomarker fragment value v(k), respectively, and iv. in a first computational step, relating v(u) and v(k) with n(k) and c(1), thereby determining an average number of biomarker molecules per cell, m(u), of said first portion, d) repeating the steps under b) and c) for sets of cells that express the biomarker at different levels, generating a set of values m(u)1 . . . n then e) v. subjecting the cells of a second portion of said cell population to conditions whereby a labelled affinity binder specifically binds to said biomarker, vi. measuring a value of said bound labelled affinity binder for a plurality of said cells of said second portion, wherein said value corresponds to the amount of said biomarker, yielding a single cell measurement value s(u) for a plurality of cells of said second portion, and determining from said single cell measurement value a mean measurement value m(s) for said plurality of cells, and vii. performing step v and vi) for said sets of cells that express the biomarker at different levels, viii. relating, in a second computational step, the mean measurement values m(s)1 . . . n and the average number of biomarker molecules per cell, m(u)1 . . . n and computing a calibration curve, ix. relating s(u) with the calibration curve yielding a number of biomarker molecules, n(u), for each single cell of said plurality of single cells.

2. A method for determining the number of molecules of a biomarker, said method comprising the steps of: a) obtaining a first portion of a cell population, wherein said cell population comprises a plurality of single cells characterized by the presence of said biomarker, b) obtaining a cell number, c(1), of said first portion, c) i. subjecting the cells of said first portion to conditions whereby said biomarker is fragmented, yielding a biomarker fragment, in a fragmentation step; ii. adding to said biomarker fragments a known number n(k) of labelled biomarker fragments differing from said biomarker fragment only in a detectable label, iii. measuring a first and a second parameter of said first portion, wherein said first parameter corresponds to the amount of said biomarker fragment and said second parameter corresponds to the amount of said labelled biomarker fragment yielding a biomarker fragment value, v(u), and a labelled biomarker fragment value v(k), respectively, and iv. in a first computation step, relating v(u) and v(k) with n(k) and c(1), thereby determining an average number of biomarker molecules per cell, m(u), of said first portion, d) v. subjecting the cells of a second portion said cell population to conditions whereby a labelled affinity binder specifically binds to said biomarker, vi. measuring a value of said bound labelled affinity binder for a plurality of said cells of said second portion, wherein said value corresponds to the amount of said biomarker, yielding a single cell measurement value s(u) for a plurality of cells of said second portion, and determining from said single cell measurement value a mean measurement value m(s) for said plurality of cells, and vii. in a second computation step, relating, s(u), m(s) and m(u), yielding a number of biomarker molecules, n(u), in each of said plurality of single cells.

3. The method according to claim 1, wherein the first computation step is calculated using formula I: $\frac{\frac{v\left(u\right)}{v\left(k\right)}*n\left(k\right)}{c\left(1\right)}=m\left(u\right).$

4. The method according to claim Error! Reference source not found, wherein the second computation step is calculated using the formula: $\frac{s\left(u\right)}{m\left(s\right)}*m\left(u\right)+d\left(s\right)=n\left(u\right)$ wherein the term d(s) is the lowest detectable copy number of a molecule.

5. The method according to claim 4, wherein d(s) is determined by analysing separate cell samples known to express different levels of the biomarker, particularly wherein a calibration curve or -line is determined yielding d(s), or wherein d(s) is determined based on the measurement of a single cell sample, the detection limit of the instrument and the average signal expected per affinity binder.

6. The method according to claim 1, wherein for the second computation step the ratio between s(u) and m(u) is defined by a response function defined by the instrument response (and/or antibody binding behaviour) over the measured dynamic range (non-linear calibration curve).

7. A method for quantifying a biomarker on a single cell level, comprising the steps of: a) providing a first cell population comprising said single cells, wherein said single cells comprise said biomarker, b) taking a first and a second portion of said cell population and obtaining the cell number of the first portion, c) i. adding a known number of molecules of a labelled biomarker or fragments thereof to said first portion, ii. obtaining a relative quantity value for said biomarker or fragment thereof and said labelled biomarker or fragment thereof in said first portion, and iii. relating the relative quantity of said biomarker or fragment thereof and said labelled biomarker or fragment thereof with said known number of molecules of the labelled biomarker or fragment thereof and said number of cells of said first portion yielding an average number of biomarker molecules per cell in said first portion; d) iv. subjecting the cells of said second portion to conditions whereby a labelled affinity binder specifically binds to said biomarker, v. measuring a value of said bound labelled affinity binder for a plurality of cells of said second portion, wherein said value corresponds to the amount of said biomarker, yielding a single measurement value for a plurality of cells of said second portion, and determining from said single measurement value a mean measurement value for said plurality of cells, and e) repeating the steps under b), c) and d) for sets of cells that express the biomarker at different levels, f) creating a calibration curve relating the average number of biomarker molecules to the mean measurement value for a number of different cell populations, g) relating said single measurement value, said mean measurement value and said average number of biomarker molecules per cell, yielding a number of biomarker molecules in said single cell.

8. (canceled)

9. The method according to claim 1, wherein the biomarker is a peptide derived from a protein, a peptide, an organic small molecule compound, a DNA molecule, an RNA molecule, a oligoribonucleotide, a mono-sachharide, a poly-saccharide or a metalo-organic compound.

10. The method according to claim 1, wherein the biomarker is a posttranslationally modified peptide.

11. The method according to claim 1, wherein the amino acid sequence of said biomarker fragment is selected from the second column in the table shown in FIG. 5.

12. The method according to claim 1, wherein the method of fragmentation in the fragmentation step is enzymatic digestion.

13. The method according to claim 1, wherein the biomarker is fragmented to yield a plurality of biomarker fragments (fragment 1, fragment 2, fragment n) in the fragmentation step, and a known number n(k1), n(k2), n(k3) of labelled biomarker fragments is added for each biomarker fragment in step ii.

14. The method according to claim 1, wherein the biomarker fragments resulting from the fragmentation step are subjected to mass spectrometry, based methods particularly collision induced dissociation, infrared multiphoton dissociation, blackbody infrared radiative dissociation, electron-capture dissociation, negative electron-transfer dissociation, electron-detachment dissociation or surface induced dissociation.

15. The method according to claim 1, wherein the labelled biomarker fragment is labelled with a stable isotope.

16. The method according to claim 1, wherein said first and second parameter are MS1 mass spectrometry signal intensity values at a given m/z value or given m/z values, or MS2 peptide fragment intensity values at a single or multiple m/z value, or MS3 peptide fragment intensity values at a single or multiple m/z value.

17. The method according to claim 1, wherein said first and said second parameter can be the intensity and m/z values, and/or the retention time constraint of a fragment in a chromatographic system or a combination thereof.

18. The method according to claim 1, wherein said value is selected from fluorescence intensity value, mass spectrometry signal intensity value, spectrophotometric values, western blot intensity values, RNA sequencing values and quantitative PCR values.

19. The method according to claim 1, wherein the labelled affinity binder is selected from an antibody, RNA/DNA binder particularly aptamers and a DARPINs (designed ankyrin repeat proteins).

20. The method according to claim 1, wherein the labelled affinity binder is labelled with a fluorophore, a stable marker isotope, a DNA or RNA marker, a protein marker, an enzyme marker or any other marker, which particularly may be detectable by a second marker.

21. The method according to claim 1, wherein the number of biomarker molecules is determined for a plurality of different biomarkers.

22. (canceled)

23. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0080] FIG. 1 shows the approach for absolute quantification of the number of biomarker molecules on the single cell level for single population (A) and multiple population (calibration curve, B) relation building of copy number and mean cytometry intensity values.

[0081] FIG. 2 shows 2 shows a generalized workflow for an assay for absolute quantification of biomarkers of interest (BOI) on the single cell level.

[0082] FIG. 3: shows example calibration curves for CD44 (left), vimentin (middle) and c-Met (right) X-axis: mean cytometry intensity values; Y-axis: copy number determined by mass spec; different colour/data point shapes correspond to different cell populations; multiple data points of similar colour/data point shapes correspond to data replicates.

[0083] FIG. 4 shows an example for an assay to quantify the number of Her2 marker molecules in cells.

[0084] FIG. 5 is a list of representative peptide fragments characteristic of biomarkers.

EXAMPLES

[0085] The invention discloses a general approach to develop assays that allow to absolutely quantify biomarkers on the single cell level. This approach uses cell population based techniques such as protein mass spectrometry to generate cell standards, which can be the cells studied or “standard cells” that express the biomarker to determine the average biomarker copy number per cell over a cell population. These standard cells are then analysed by the single cell analysis technology alone or concomitantly with other samples of interest to determine the single cell copy numbers of all analysed single cells. Typically sets of standard cells that express the biomarker of interest to generate calibration curves for the single cell copy number determination are used. As a result, the current lack of absolute quantification in affinity binder based single cell analysis technologies is overcome, and we thus realize an important measurement parameter which is highly relevant in many clinical, pharmaceutical and research applications.

[0086] As a result of this approach, the inventors disclose a list of validated assays for biomarker quantification on the single cell level.

DETAILED DESCRIPTION OF THE FIGURES

[0087] The general concept to determine the absolute number of biomarker molecules on the single cell level is described in FIG. 1. First a mean copy number (CN) per cell is determined with a population based quantification method using a labelled synthetic peptide (SP). In a second step in a single cell analysis method a relative quantity value for the biomarker of interest is determined and calibrated with the mean copy number per cell value to yield a single cell copy number.

[0088] To determine the absolute numbers for a biomarker of interest (BOI) first the average copy number per cell is determined using cell population measurement techniques, such as mass spectrometry. These average copy numbers are determined for several cell samples that differ in the abundance of the molecule of interest. Then these cells are analysed using the single cell analysis technique and the mean number of biomarker molecules per cell. Relating the average single copy number with the mean single cell copy number allows to generate a calibration line/curve. Based on this calibration curve/line the single cell number of biomarker molecules can be computed.

[0089] FIG. 2 describes the approach to determine the average single cell copy number for a BOI. Biomarkers are determined for the clinical, biomedical or research question of interest and the corresponding affinity binders are selected. Then, for each BOI peptides are selected that univocally identify and describe the protein or the protein modification. For these peptides then mass spectrometry assays are developed to find and measure those peptides in complex peptide mixtures. In an exemplary set-up, those mass spectrometry assays are for a single reaction monitoring measurement, in which the mass-over-charge ratio (m/z) for the peptide and the m/z ratios of a set of peptide fragments is defined to identify and measure the peptide by LC-MS/SRM (SRM: selected reaction monitoring). To then quantify those peptides in a cell lysate, first proteins are isolated, digested using a protease and a known amount of a synthetic peptide is spiked into the peptide mixture. Then both the intensity of the endogenous and spike-in standard are measured by LC-MS/SRM and the ratio between those two is used to determine the number of the endogenous biomarker peptide molecules. The analysed cells can either be part of the sample that then is analysed by the single cell analysis technology, or can be other cells (standard cells) that then are co-measured with a sample of interest to calibrate the signal of these. In the next step then the cells are prepared for single cell analysis (optionally the standard cells are spiked in), stained with the affinity binders of interest, and are analysed by the single cell analysis technology, e.g. mass cytometry. Based on the signal of the standard cells (that can also be cells which are part of the sample of interest) the mean signal intensity (MSI) is computed. This MSI corresponds to the copy number per cell determined by the cell population assay (SRM analysis). Given that the signal response line or curve is known, the copy number can be computed for each single cell.

[0090] In other words, for each BOI peptides that uniquely represent the biomarker (biomarker representing peptide, BRP) in a cell or cell mixture are determined and their abundance is quantified in mass spectrometry. These BRPs can also include and represent protein modifications. For these BRPs, synthetic peptides are synthesized which are chemically identical, but differ in the mass. This is achieved by incorporation of heavy, stable isotopes during peptide synthesis. To determine the abundance of the biomarker via the BRP, a cell sample is split, and part is lysed and peptides are generated via enzymatic digestion. Then the synthetic, isotopically labelled version of the BRP, of which the exact number of molecules is known, is spiked into the peptide sample and concomitantly measured with the BRP. The ratio of labelled and endogenous peptide together with the known starting cell number is used to compute the average number of BOI molecules per cell in the analyzed sample.

[0091] The cell sample left after splitting then is used as a standard in the single cell measurements for the BOI. The BOI is labelled using a reporter carrying affinity binder and the signal is analysed by a single cell analysis technology. After such a measurement, the mean signal of the biomarker over all analysed single cells can be computed. This computed mean is equal to the measured mean in the population-based measurement (i.e. mass spectrometry) and thus the average number of biomarker molecules per cell can be assigned to it. This then allows to compute a calibration curve for single cell copy number determination as described above.

[0092] FIG. 3 shows example calibration curves for CD44, vimentin and c-Met, respectively. The calibration curve values are y=−172256+31482x for CD44, y=756668+24156x for vimentin and y=32692+412x for c-Met. The x-axis shows mass cytometry ion counts, the y-axis shows average single cell copy numbers.

[0093] Technical Specifications

[0094] Samples

[0095] The disclosed invention can be applied to a variety of samples to determine the absolute number of biomarker molecules per single cell. These samples include cells in suspension, cells on surfaces and cells in a three dimensional context such as in a tissues. The cells can come from cells grown in culture, cells from any tissue and can be from any organism.

[0096] To determine the absolute number of biomarker molecules cells can either be in suspension form, in a single cell layer as analysed in immunocytochemical applications, and in the form of sections (in the case of tissues) in immunocytochemical applications. Also, cells in tissues can be dissociated to generate single cell suspensions in order to determine the absolute copy numbers.

[0097] Single Cell Analysis Technologies

[0098] A variety of single cell analysis technologies exist, that rely on the detection of biomarkers using affinity binders—all can be applied to determine the absolute number of biomarker molecules on the single cell level with the instant invention. These methods include flow cytometry, mass cytometry, immunohistochemistry, immunocytochemistry, variants of microscopy, microfluidic devices and combinations thereof. In a preferred embodiment, mass cytometry is used to determine single cell absolute quantities.

[0099] Affinity Binders

[0100] The affinity binders that can be used with the approach include antibodies and parts thereof (e.g. single chain antibody fragments), RNA/DNA binders such as aptamers and variants thereof and DARPINs (Designed Ankyrin Repeat Proteins). The affinity binders can be coupled to a wide range of reporters, including but not limited to fluorophores, pure isotopes, elements with a natural isotopic distribution, an isotope mixture with a defined ratios of the isotopes, DNA, RNA and molecules with a defined mass over charge ratio (m/z) in mass spectrometric applications.

[0101] Staining/Labeling of Cells with Affinity Binders

[0102] The staining of single cells follows standard staining protocols known in the art for binding reagents, such as antibodies, to cells.

[0103] Biomarker Representing Peptide (BRP)

[0104] The BRP to calibrate the single cell analysis epitope signal has a defined and unique m/z allowing its univocal identification. The BRP fragment m/z values and/or their relative intensity are used with the peptides m/z value for its identification. To determine the ratio between the BRP and the BOI peptide, the peptide fragment ion intensities and/or the peptide ion intensity is used. A wide variety of methods can be used to analyse and quantify the peptides using MS. First, the peptides have to be ionized. To ionize the peptide, any of the following methods can be used: electron and chemical ionization, spray ionization (e.g. electrospray ionization), desorption ionization (e.g. matrix-assisted laser desorption ionization), gas discharge ionization, ambient ionization and any other used methods to ionize analytes for MS.

[0105] The fragments of the peptide can be generated by collision induced dissociation, infrared multiphoton dissociation, blackbody infrared radiative dissociation, electron-capture dissociation, (negative) electron-transfer dissociation, electron-detachment dissociation, surface induced dissociation and combinations and variants thereof.

[0106] Appropriate MS instruments to determine the m/z and intensity of the peptides are time of flight (TOF), quadrupole, ion trap, fourier transform ion cyclotron resonance, orbitrap, sector field, any other mass analyser and combinations thereof.

[0107] One MS instrument set-up which is particularly suitable to measure and quantify the peptides for absolute quantification due to its precision and sensitivity is a triple quadrupole MS instrument. It achieves low attomole sensitivity, allows detection of 1, 2, 3 or at least 5, or at least 10, or at least 50, or at least 100, or at least 200, or at least 300, or at least 500, or at least 1000 peptides in a single analysis via collision induced dissociation (CID) coupled to liquid chromatography.

[0108] The synthetic peptides to perform the absolute quantification are synthesized with defined isotopes of any existing element with 1 or n Dalton mass differences that allow to uniquely identify and quantify them in a MS measurement. Ultimately, the minimal needed mass difference will be defined by the achievable resolution of the MS instrument. Suitable elements with their isotopes include without being limited to hydrogen, carbon, nitrogen, oxygen, sulphur, chlorine, fluorine and bromide.

[0109] Standard Cells

[0110] To generate the protein lysate from the standard cells and subsequently peptides from protein, the following methods can be used: chemical, mechanical, enzymatic, sonic and electromagnetic methods.

[0111] In a certain embodiment, cells are lysed using mechanical force and are digested using the trypsin protease. Other proteases to digest the protein into peptides include LysC, Asp-N, Glu-C, Lys-C, Arg-C, pepsine, chemotrypsin, any other protease and combinations thereof.

[0112] The peptides are either unmodified or modified. In a certain embodiment the peptides are unmodified or phosphorylated on serine, threonine, tyrosine, histidine, aspartate and glutamate residues. Other modifications on any other amino acid residue of the peptides can include (Z)-2,3-didehydrotyrosine,1-thioglycine, 2,3-didehydroalanine (Ser), 2,3-didehydrobutyrine, 2′,4′,5′-topaquinone, 2-oxobutanoic acid, 3-oxoalanine (Cys, Ser), 3-phenyllactic acid, acetylation, acid aspartate ester, ADP-ribosylation, allysine, amidation, beta-methylthiolation, biotin, bromination, cholesterol, cis-14-hydroxy-10,13-dioxo-7-heptadecenoic, citrullination, C-Mannosylation, cysteine persulfide, cysteine sulfenic acid (—SOH, —SO2H), deamidation, deamidation followed by a methylation, dihydroxylation, dimethylation, dimethylation of proline, diphthamide, FAD, FMN conjugation (Cys, His, Ser/Thr), formylation, gamma-carboxyglutamic acid, geranyl-geranylation, glucosylation (Glycation), glutathionylation, hydroxylation, hypusine, lipoyl, methionine sulfone, methylation, myristoylation, N6,N6,N6-trimethyl-5-hydroxylysine, N6-1-carboxyethyl lysine, N6-poly(methylaminopropyl)lysine, n-Decanoate, n-Octanoate, O-GlcNAc, Omega-hydroxyceramide glutamate ester, palmitoylation, phosphatidylethanolamine amidated glycine, phosphopantetheine, phosphorylation, pyridoxal phosphate, pyrrolidone carboxylic acid, pyrrolidone carboxylic acid (Glu), pyrrolysine, pyruvic acid (Cys), pyruvic acid (Ser), S-12-hydroxyfarnesyl cysteine, S-archaeol, S-diacylglycerol cysteine, S-farnesyl cysteine, S-Nitrosylation, S-palmitoleyl cysteine, sulfation, thyroxine, triiodothyronine and trimethylation.

[0113] Besides peptides, other molecules that can be quantified by mass spectrometry and mass cytometry or other single cell analysis techniques can be absolutely quantified using the presented approach here. These include small molecule compound, nucleotide, DNA, RNA, metabolite, mono-saccharide, poly-saccharide, metalo-organic compound and any combination of the above mentioned.