Patent Grant
8221972
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 22, 2011, is named 65691537.txt and is 3,465bytes in size.
The invention concerns a method for determining at least one target molecule map in a tissue section, using at least one (A-X)n-B conjugate, wherein A is a tag molecule of known molecular weight, X is a linker that is cleaved during sample desorption/ionization, n is an integer of at least 1, and B is a binding molecule that binds specifically to said target molecule. When using MALDI mass spectrometry, said linker molecule X may be cleaved by photodissociation during sample laser irradiation if photocleavable at the wavelength of said MALDI laser. Alternatively, when using UV-MALDI, IR-MALDI, SIMS or DESI mass spectrometry, said linker molecule X may be cleaved by fragmentation during sample desorption/ionization.
Recently, transcriptome and proteome studies have led to the identification of many proteins implicated in a wide diversity of diseases, such as several kinds of cancers.
However, most of these results have been obtained on purified extracted nucleic acid or protein samples, which do not generate information on the tissue localisation of the incriminated proteins, although this kind of information is crucial for the understanding of physiological processes. Another shortcoming of most of the available data is that few studies have simultaneously analyzed the mRNA and protein expression of molecules of interest, which is nevertheless important to clarify the expression pattern of a particular protein. It would thus be very useful to have combined data of both mRNA and protein expression maps.
Current technologies for the tissue expression mapping of mRNA usually resort to nucleic acid probes coupled to radioactive, fluorescent or chemoluminescent markers after in situ hybridization (ISH). For protein expression mapping in tissue sections, usual technologies include immunohistochemistry and immunofluorescence.
The main drawback of all these technologies of tissue section expression mapping is that the number of target molecules that can be analyzed simultaneously is limited, even with fluorescent probes or antibodies, since no more than 3 or 4 distinct target molecules can be studied in the same experiment.
Mass spectrometry, on the other hand, allows for the simultaneous multiplex analysis of complex mixtures of biomolecules, depending on their molecular weight. In particular, Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry has become a powerful tool in the field of biological researches and is used for the detection, identification and characterization of nucleic acids, peptides and proteins from complex mixtures.
In particular, Olejnik and collaborators describe the synthesis and characterization of photocleavable peptide-DNA conjugates along with their use as photocleavable mass marker (PCMM) hybridization probes for the detection of immobilized synthetic target DNAs by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Olejnik et al. Nucleic Acids Res. 1999 Dec. 1; 27(23):4626-31).
Patent application WO 98/26095 describes the synthesis and use of mass-labeled compounds to specifically interact with biomolecular targets. Patent application WO 00/68434 describes a method of detecting multiple analytes in a sample in a single assay, based on encoding target molecules with signals followed by decoding of the encoded signal.
However, no document in the prior art describes a method for the MALDI mass spectrometry (MALDI-MS) analysis of nucleic acids comprised in tissue sections.
The inventors have set up a new method for the detection of biomolecules, in particular mRNA, in tissue sections by MALDI-MS, using conjugates composed of a moiety that binds specifically to the target molecule(s), a moiety of known molecular weight (the “tag” moiety), and a photocleavable linker that is directly cut by the MALDI laser during the ionization process (see
In addition, when this new method is used to map tissue expression of mRNA, a common picture of mRNA and corresponding protein expression can be obtained in two consecutive tissue sections. Indeed, several publications have shown that MALDI-MS could become an efficient tool for direct analysis of peptides and proteins in tissue sections (Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760; Stoeckli, M.; Farmer, T. B.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496; Chaurand, P.; Schwartz,. S. A.; Caprioli, R. M. Anal. Chem. 2004, 87A-93A).
Finally, the inventors have found that such a method is also transposable to mass spectrometry in general provided that the photocleavable linker is replaced by a linker which is cleaved by fragmentation during sample desorption/ionization. In this case, MALDI (UV-MALDI, or IR-MALDI), SIMS (Second Ion Mass spectrometry) or DESI (Desorption Electrospray Ionization) mass spectrometry may be used instead of MALDI, and preferably UV-MALDI, only. In addition, the inventors have found that conjugates with photocleavable linkers suitable for UV-MALDI analysis may also be used with other mass spectrometry technologies, since the chosen photocleavable linkers are also cleaved by fragmentation during sample desorption/ionization. The particular conjugates set up by the inventors may thus be used with any mass spectrometry technology suitable for tissue section analysis, notably with MALDI (UV-MALDI, or IR-MALDI), SIMS (Second Ion Mass spectrometry) or DESI (Desorption Electrospray Ionization) mass spectrometry.
The invention thus concerns a method for determining at least one target molecule map in a tissue section, comprising:
a) hybridizing said tissue section with at least one (A-X)n-B conjugate, wherein
b) scanning the tissue section surface and analyzing each adjacent spot with a mass spectrometer, wherein said linker X is cleaved during sample ionization, and wherein the resulting data of each spot is saved; and
c) analyzing the obtained data in the molecular mass window(s) of each distinct tag molecule to create as many maps of the tissue section as the number of distinct studied target molecules.
According to the invention, a “tissue section” preferably has the following properties: it may be frozen or paraffin-embedded, its thickness is preferably in the order of a mammalian cell diameter, thus comprised between 5 and 20 μm. In the case of a frozen section that was obtained from a frozen tissue using a cryostat, OCT (optimal cutting temperature polymer) is preferably used only to fix the tissue but the frozen tissue is not embedded in OCT, so that tissue sections were not brought into contact with OCT. The tissue section may then be transferred on a MALDI plate composed of any material suitable for further MALDI analysis, including metals, inorganic or organic materials, such as gold, steel, glass fiber, glass, nylon 6/6, silicon, plastic, polyethylene, polypropylene, polyimide, polyvinylidenedifluoride or a glass slice of any thickness coated with conductive metal keeping transparency properties such as nickel or ITO.
According to the invention, a “target molecule” means a molecule of interest that is capable to specifically bind to another molecule, which is referred to as a “binding molecule”. Such tandem target/binding molecules may display any chemical structure, providing that they are capable to generate a specific hybridization in a tissue section. A large variety of tandem target/binding molecules, as well as tandem binding/target molecules, are comprised in the scope of the present invention, including nucleic acids/nucleic acids, nucleic acids/peptides, nucleic acids/proteins, nucleic acids/antibodies, peptides/peptides, peptides/proteins, peptides/antibodies, proteins/proteins (in particular ligands/receptors), proteins/sugars, antigens/antibodies, haptens/antibodies, organic compounds/receptor (see some example in
In particular, target nucleic acid sequences may be specifically detected using single stranded nucleic acid probes with a nucleic sequence that is complementary to the single-stranded target nucleic acid or to one of the strands of a double-stranded target nucleic acid (see
Particular nucleic acid sequences may be detected using proteins or protein fragments (peptides) that specifically bind to the target sequence, such as for instance transcription factors or antibodies or antibody fragments that are specific for particular DNA sequences. For instance, auto-immune anti-DNA antibodies may be used.
In the case of peptides and proteins, all peptidic ligand/peptidic receptor tandem molecules that are known in the art are enclosed in the scope of the present invention. Such peptidic ligand/peptidic receptor tandem molecules includes peptidic antigens/antibodies or antibody fragments, as well as any hormone/hormone receptor, cytokine/cytokine receptor tandem, chemokine/chemokine receptor, aptamer/peptide, aptamer/protein. Membrane sugars that are implicated in cell migration and their proteic receptors are also in the scope of the present invention.
Also, antigens of any kind (such as nucleic acids, haptens, peptides or proteins) and their specific antibodies are included in the tandem target/binding and binding/target molecules according to the invention. In particular, the use of an antibody or antibody fragment as binding molecule in a conjugate allows for a novel kind of immunocytochemistry in tissue sections using MALDI-MS analysis as revelation technology thanks to the antibody photocleavably linked tag molecule.
Organic compounds may also be mapped using methods according to the invention. In particular, the in vivo distribution of administered organic drugs may be monitored using methods according to the invention.
In a particular embodiment of the above described method according to the invention, each target molecule is independently chosen in the group constituted of nucleic acids, in particular mRNA molecules, peptides, proteins, in particular receptors and ligands, antibodies, antigens, haptens, and organic compounds. In a preferred embodiment, at least one target molecule is a mRNA molecule. In another preferred embodiment, at least one target molecule is selected from a peptide, a protein, an antigen or a hapten.
In a particular embodiment of any above described method according to the invention, each binding B molecule that binds specifically to a target molecule is independently chosen in the group constituted of nucleic acids, particularly oligonucleotides, peptides, proteins, in particular receptors and ligands, antibodies, antigens, haptens, aptamers and organic compounds. In a preferred embodiment, when at least a target molecule is a mRNA molecule, each B molecule that binds specifically to a target nucleic acid is a nucleic acid probe with a sequence that is complementary to said target mRNA sequence. In another preferred embodiment, when at least a target molecule is a peptide, protein, antigen or hapten, each B molecule that binds specifically to a target peptide, protein, antigen or hapten is an antibody directed against said peptide, protein, antigen or hapten. In particular, when an antibody or antibody fragment is used as binding molecule B, the target molecule against which said antibody or fragment thereof is specifically directed may be the frame-work region of a particular antibody subclass, such as IgM, IgD, IgG, or IgE. Alternatively, when an antibody or antibody fragment is used as binding molecule B, the target molecule against which said antibody or fragment thereof is specifically directed may be the frame-work region of antibodies generated in a particular species, such as for instance rabbit, mouse, rat, goat, hamster, sheep and human. In these two cases, a 2 steps indirect detection may be performed using a primary non modified antibody of a specific subclass directed against any desirable target molecule, said primary antibody being then recognized by an antibody-tag conjugate according to the invention (see Example 2 and
According to the invention, a “tag molecule” refers to a molecule of known molecular weight that is detectable by mass spectrometry using MALDI (UV/IR), SIMS and DESI. A MALDI mass spectrometer usually allows for the detection of compounds in a wide range of m/z ratios. For instance, MALDI-Time of Flight (TOF) analyzers can detect compounds of m/z ratios up to 1 000 000. A suitable tag molecule also needs not to interfere with the specific binding of the target and the binding molecules. Preferably, a suitable tag molecule thus has limited steric volume to avoid steric hindrance of the binding of the target and binding molecules. For both above described reasons, suitable tag molecules thus preferably have a m/z ratio inferior to 10 000. Particular examples of suitable tag molecules include peptides, nucleic acids, sugars, polymers, lipids, and organic compounds. Among these, labelling molecules that are used to reveal a complex formation between a target molecule and a binding molecule, may also be used as tag molecules, including fluorochromes such as for instance usual fluorescein isothyocanate (FITC), R-phycoerythrin (PE), Cy3, Cy5, Cy7, PerCP, Allophycocyanin (APC), Texas Red, TRITC, PE-Cy5 conjugates, PE-Cy7 conjugates, or APC-Cy7 conjugates; enzymes such as alkaline phosphatase or peroxydase; biotin; gold; or all MALDI matrices or even all compounds that can be analyzed in pure laser desorption mode and for example pre-ionized molecules. When a mass spectrometry technology suitable for the detection of very small molecules is used, atoms may also be used as tag molecule.
All tag molecules described above may be used in the invention, and in any case, depending on the mass spectrometry technology used, a person even moderately skilled in the art of mass spectrometry analysis will know what type of tag molecule to choose to optimize the detection step. For instance, when MALDI mass spectrometry is used, tag molecules preferably have a m/z ratio<5000. Alternatively, when SIMS is used, tag molecules preferably have a m/z ratio<500, and when DESI is used, tag molecules preferably have a m/z ratio<5000.
In a particular embodiment of any above described method according to the invention, each A tag molecule is chosen in the group constituted of peptides, nucleic acids, sugars, and organic compounds. In a preferred embodiment, at least one A tag molecule is a peptide.
In a preferred embodiment of an above described method according to the invention, target molecule(s) are mRNA molecules, binding B molecule(s) are nucleic acid probes with a sequence complementary to mRNA sequences, and A tag molecule(s) are peptides. In a preferred embodiment of an above described method according to the invention, target molecule(s) are peptides, proteins (including antibodies), antigens, or haptens, binding molecules are antibodies or antibody fragments and tag molecules are peptides. In another preferred embodiment of an above described method according to the invention, target molecule(s) are peptides, proteins (including antibodies), antigens, or haptens, binding molecules are antibodies or antibody fragments and tag molecules are usual antibody labelling molecules such as fluorochromes, including for instance usual fluorescein isothyocanate (FITC), R-phycoerythrin (PE), Cy3, Cy5, Cy7, PerCP, allophycocyanin (APC), Texas Red, TRITC, PE-Cy5 conjugates, PE-Cy7 conjugates, or APC-Cy7 conjugates; enzymes such as alkaline phosphatase or peroxydase; or biotin or gold; or all MALDI matrices or even all compounds that can be analyzed in pure laser desorption mode and for example pre-ionized molecules.
According to the invention, “hybridizing said tissue section with at least one (A-X)n-B conjugate” refers to a reaction in which the tissue section and the conjugate(s) are brought into contact in such conditions that the binding B molecule of the conjugate is able to bind specifically to its target molecule in the tissue section. Depending on the nature of the binding and the target molecule, well-known hybridization protocols are available to the ones skilled in the art. Indeed, in the case when the binding molecule is a nucleic acid probe, in situ hybridization in tissue section has been well documented and is thus easily available to a man skilled in the art. Antibody staining in tissue sections is also a routine technology which is easily available to a man skilled in the art.
Using a method according to the invention, it is thus possible to obtain a map of at least one target molecule in a tissue section. By a target molecule “map” in a tissue section is meant a two dimensional representation of the expression of said target molecule in said tissue section. This two dimensional representation is obtained by scanning the tissue section surface with the MALDI analyzer at a defined spot density, performing MALDI analysis on each successive spot and storing both the obtained data and the coordinates of each spot. The higher the spot density, the more precise is the resulting map. The diameter of a MALDI laser is generally between 50-200 μm depending on the focalisation of the system, so that two adjacent irradiation spots are preferably separated of the laser beam diameter (i.e. 50-200 μm). To allow for the acquisition of precise target molecule map, adjacent spots are preferably separated of at most 300 μm, at most 200 μm, more preferably at most 100 μm, at most 80 μm, at most 60 μm, at most 50 μm, at most 40 μm, most preferably of the diameter of the MALDI laser.
Each spot data is then analyzed in the molecular window of the tag molecule and the signal intensity of the tag molecule is reported at the spot coordinates. Such image reconstruction may be performed automatically using any suitable image reconstruction software known in the art or commercially available. Examples of suitable softwares are the IDL (Interactive Data Language) software, which is commercialized by RSI (RSI Corporate Headquarters. 4990 Pearl East Circle. Boulder, Colo. 80301), flexImaging (Bruker Daltonics, Bremmen, DE), MIT (M. Stoeckli, Novartis, Bâle, Switzerland).
In a method according to the invention for determining at least one target molecule map in a tissue section, several distinct target molecules can be mapped simultaneously. Indeed, it is sufficient to use several conjugates with distinct tag A molecules, thus displaying distinct molecular weights, to allow for the detection of several distinct target molecules. Using tag molecules with widely dispersed molecular weights, it is thus possible using any above described method according to the invention to map simultaneously the expression of many distinct target molecules in the same tissue section. In particular embodiments, at least 2, at least 3, at least 5, at least 8, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 75, or at least 100 distinct target molecules may be mapped simultaneously. In particular, in the case of mRNA target molecules, at least 2, at least 3, at least 5, at least 8, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 75, or at least 100 distinct target mRNA molecules may be mapped simultaneously in the same tissue section.
In a particular embodiment of any above described method in which target molecule(s) are mRNA molecule(s), said method may further comprise a step d) consisting in analyzing the obtained data in the molecular mass windows of each mRNA molecule corresponding protein to create each corresponding protein expression map of the tissue section.
In a first preferred embodiment, MALDI mass spectrometry is used and the X linker molecule is photocleavable at the wavelength of a MALDI laser. The invention then concerns a method for determining at least one target molecule map in a tissue section, comprising:
a) hybridizing said tissue section with at least one (A-X)n-B conjugate, wherein
b) scanning the tissue section surface and analyzing each adjacent spot with a MALDI mass spectrometer, wherein the MALDI laser is used both to release the tag molecule A and to induce the sample ionization, and wherein the resulting data of each spot is saved; and
c) analyzing the obtained data in the molecular mass window(s) of each distinct tag molecule to create as many maps of the tissue section as the number of distinct studied target molecules.
According to the invention, a “linker that is photocleavable at the wavelength of a MALDI laser” refers to a chemical compound that separates two other chemical moieties, and that may be cleaved at least at one site under exposition to a MALDI laser. Most MALDI lasers usually have an ultraviolet (UV: inferior to 500 nm) wavelength, usually between 300 and 500 nm. For instance, many UV-MALDI analyzers have a pulsed nitrogen laser with a wavelength of 337 nm. Thus, a linker according to the invention is, in a particular embodiment, efficiently cleaved at least at one site under exposition to a wavelength of 250 to 500 nm, preferably a wavelength of 320 to 360 nm or 320 to 350 nm, more preferably a wavelength of 337 nm, so that the UV-MALDI laser acts both to cleave the linker and to ionize the sample. Other MALDI analyser display an infrared (IR: superior to 770 nm) laser. For instance, a Nd:YAG laser (wavelength=1060 nm), Er:YAG laser (wavelength=2940 nm), a mid-infrared optical parametric oscillator (OPO) (wavelength=2940 nm) or a TEA-CO2 laser (wavelength=10600 nm) may be used as IR-MALDI lasers. In other particular embodiments, a linker according to the invention is thus efficiently cleaved at least one site under exposition to a wavelength of 1000 to 1100 nm, preferably a wavelength of 1060 nm, or a wavelength of 2900 to 3000 nm, preferably a wavelength of 2940 nm, or a wavelength of 10500 to 10700 nm, preferably a wavelength of 10600 nm, so that respectively a Nd:YAG, a Er:YAG, or a TEA-CO2 IR-MALDI laser acts both to cleave the linker and to ionize the sample.
In the case of UV-MALDI lasers, several linkers have been described (Olejnik et al. Nucleic Acids Res. 1999 Dec. 1; 27(23):4626-31; Bai X, et al. Nucleic Acids Res. 2004 Jan. 26; 32(2):535-41; Wenzel T et al. Nucleosides Nucleotides Nucleic Acids. 2003 May-August; 22(5-8):1579-81), and several reagents useful to introduce photocleavable linkers in organic molecules are commercially available, notably for instance from AmberGen™ (1106 Commonwealth Avenue. Boston, Mass. 02215, USA), Link Technologies (3 Mallard Way, Strathcycle Business Park, Bellshill, Lanarkshire ML4 3BF, Scotland), Integrated DNA Technologies (1710 Commercial Park o Coralville, Iowa 52241, USA), Glen Research (22825 Davis Drive, Sterling, Va., 20164, USA), Eurogentec (EUROGENTEC s.a. Headquarters. LIEGE Science Park. Rue Bois Saint Jean 5. 4102 SERAING. BELGIUM).
In a particular embodiment of any above described method according to the invention in which MALDI mass spectrometry and a linker molecule X that is photocleavable at the wavelength of a MALDI laser are used, the X linker molecule comprises a moiety chosen in the group constituted of:
wherein R is a C1-C6 alkyl group and m is an integer comprised between 1 and 4.
Alternatively, said linker molecule X that is photocleavable at the wavelength of a MALDI laser are used, the X linker molecule comprises a moiety chosen in the group constituted of:
Linker molecules X linking a tag molecule A and a binding molecule B may be obtained between A and B using commercially available linkers comprising such moieties. For instance, linkers suitable for preparing conjugates with photocleavable linkers for use in a method according to the invention may be obtained from Glen Research Corporation (22825 Davis Drive, Sterling, Va. 20164, USA), including linkers named:
PC Linker (ref. 10-4920-02),
PC Spacer Phosphoramidite (ref. 10-4913-02), and
PC Amino Modifier (ref. 10-4906-02),
or from Pierce (Pierce Biotechnology, Inc. Customer Service Department P.O. Box 117 Rockford, Ill. 61105 U.S.A), for instance linker Sulfo-KMUS (ref. #21111);
or from Merck Biosciences Ltd (Boulevard Industrial Park, Padge Road. Beeston, Nottingham NG9 2JR, United Kingdom) trade mark Novabiochem®, for instance linker
(ref. 01-60-0042);
or from Solulink under reference B1001, named Chromalink Biotin 354S, which is cleavable at a wavelength of 354 nm:
In a preferred embodiment where target molecule(s) are mRNA molecules, binding B molecule(s) are nucleic acid probes with a sequence complementary to mRNA sequences, and A tag molecule(s) are peptides, n is 1 is the (A-X)n-B conjugate(s), and the resulting (A-X)-B conjugate(s) have the following structure:
Such conjugates may for instance be obtained by amplification of a desired nucleic sequence using a forward (5′) primer of the following structure:
In another preferred embodiment where target molecule(s) are mRNA molecules, binding B molecule(s) are nucleic acid probes with a sequence complementary to mRNA sequences, and A tag molecule(s) are peptides, n is 1 is the (A-X)n-B conjugate(s), and the resulting (A-X)-B conjugate(s) have the following structure:
Such conjugates may for instance be obtained by amplification of a desired nucleic sequence using a forward (5′) primer of the following structure:
Alternatively, in another preferred embodiment where target molecule(s) are mRNA molecules, binding B molecule(s) are nucleic acid probes with a sequence complementary to mRNA sequences, and A tag molecule(s) are peptides, n is superior to 1 and the nucleic acid probe(s) comprise at least one modified base of the following structure:
wherein “dNTP” means any triphosphate deoxynucleotide, including dATP, dCTP, dGTP, dTTP, and dUTP. In a preferred embodiment, a modified dUTP base is used.
In another preferred embodiment where target molecule(s) are mRNA molecules, binding B molecule(s) are nucleic acid probes with a sequence complementary to mRNA sequences, and A tag molecule(s) are peptides, n is superior to 1 and the nucleic acid probe(s) comprise at least one modified base of the following structure:
wherein “dNTP” means any triphosphate deoxynucleotide, including dATP, dCTP, dGTP, dTTP, and dUTP. In a preferred embodiment, a modified dUTP base is used.
With the use of such a modified peptide-tagged dUTP, peptide-tagged nucleic acid hybridization probes can be very simply generated using a simple PCR amplification in the presence of dATP, dCTP, dGTP and the modified peptide-tagged dUTP. This way, a specific hybridization probe can be easily synthesized for any target mRNA sequence.
Moreover, it allows for a signal amplification since a given hybridization probe synthesized with the modified peptide-tagged dUTP will carry as many tag peptides as the number of U bases in its sequence.
Finally, the use of hybridization probes synthesized with the modified peptide-tagged dUTP allows for a quantitative analysis of mRNA expression in tissue sections. Indeed, the signal generated by the corresponding tag(s) of one or several studied mRNA(s) can be compared with that obtained for a reference mRNA sequence (for instance a house-keeping gene such as actin of HPRT). As the number of U bases in each hybridization probe is known, the expression ratio between each studied mRNA sequence and the reference mRNA sequence can be calculated.
In a method where target molecule(s) are mRNA molecules, binding B molecule(s) are nucleic acid probes with a sequence complementary to mRNA sequences, and A tag molecule(s) are peptides, using conjugates described above, several distinct target mRNA molecules can be mapped simultaneously. Indeed, it is sufficient to use several conjugates with distinct nucleic acid probes and distinct tag peptides displaying distinct molecular weights, to allow for the detection of several distinct target molecules. Using tag peptides with widely dispersed molecular weights, it is thus possible using any above described conjugate to map simultaneously the expression of many distinct target mRNA molecules in the same tissue section. In particular embodiments, at least 2, at least 3, at least 5, at least 8, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 75, or at least 100 distinct target mRNA molecules may be mapped simultaneously.
In addition, stored spectrum data also display direct peptide/protein analysis of the tissue section, so that the tissue section analysis method of target mRNA molecules using the conjugates described above may further comprise a last step d) consisting in analyzing the obtained data in the molecular mass windows of each mRNA molecule corresponding protein to create each corresponding protein expression map of the tissue section.
In a preferred embodiment where target molecule(s) are peptide(s), protein(s) (including antibodies) or hapten(s), binding B molecule(s) are antibodies directed against said target molecules, A tag molecule(s) are peptides, n is 1 and the (A-X)-B conjugate(s) have the following structure:
In any method according to the invention in which MALDI mass spectrometry is used, the matrix that is used for MALDI-MS analysis may be any classical MALDI matrix (e.g. CHCA, 2,5-DHB, SA, HABA, 3-HPA . . . ), or 2,4-DNPH.
By “matrix” is meant any material that, when mixed with the analyte, generates crystalline matrix-embedded analyte molecules that are successfully desorbed by laser irradiation and ionized from the solid phase crystals into the gaseous or vapour phase and accelerated as molecular ions. Commonly used MALDI-MS matrices are generally small, acidic chemicals absorbing at the laser wavelength, including nicotinic acid, cinnamic acid, 2,5-dihydroxybenzoic acid (2,5-DHB), α-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA), 3-methoxy-4-hydroxycinnamic acid (ferulic acid), 3,4-dihydroxycinnamic acid (caffeic acid), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 3-hydroxy picolinic acid (HPA), 2,4,6-trihydroxy acetophenone (THAP) and 2-amino-4-methyl-5-nitropyridine. Protocols for the preparation of these matrices are well-known in the art, and most of these matrices are commercially available. Current commonly used matrices for peptide/protein analysis include α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB) and sinapinic acid (SA). DNPH is 2,4-Dinitrophenylhydrazine and is used for aldehydes and ketones detection.
In a second preferred embodiment of the invention, a linker molecule X that is cleaved by fragmentation during sample desorption/ionization is used, and the analysis is then not restricted to MALDI mass spectrometry, but generally transposable to any mass spectrometry technology suitable for tissue section analysis. In particular, UV-MALDI, IR-MALDI, SIMS or DESI mass spectrometry may be used.
Indeed, indirect detection of compounds necessitates a release of the tag molecule at the very beginning of the desorption and ion production in the source of the instrument. When using MALDI mass spectrometry, which uses a laser for ion desorption/ionization via energy absorption by the matrix molecules, several ways to induce the tag molecule release can be thought of. The first, most elegant, approach is to use photodissociation of the tag molecule by the MALDI laser irradiation (see
However, fast fragmentation can also be considered as a way of releasing the tag molecule. This phenomenon is generally present in mass spectrometry technologies, and its use is thus compatible with MALDI and with other mass spectrometry sources. Notably, it can be taken advantage of this fragmentation phenomenon with other mass spectrometry technologies such as SIMS (Secondary Ion Mass Spectrometry), where the source is a primary ion beam, or with a recently described technology named DESI (Desorption Electrospray Ionisation). Moreover, as for MALDI/MS, these two technologies can be used for the direct analysis of tissue sections (Touboul, D.; Kolmer, F.; Niehis, E.; Brunelle, A.; Laprevote, O. Journal of the American Society for Mass Spectrometry, (JASMS) 2005, 15, 1608-1618) (Cooks, R. G. M.; Ouyang, Z. Takats, Z.; Wiseman, J. M.; Science, 17 Mar. 2006, Vol 311, 1566-1570).
Thus, the invention concept of using conjugates of a specific binding molecules linked to a tag molecule via a linker molecule can be transposed to other mass spectrometry technologies than MALDI, such as SIMS or DESI, provided that a linker that is cleaved by fast fragmentation is used, resulting in a fast fragmentation release of the tag molecule. Considering this phenomenon, it is possible to design other conjugates without more simple linkers, since they do not need to be photocleavable but only to be cleaved by fast fragmentation, which can be easily obtained. In fact, all sorts of linkers may be cleaved by fragmentation, and the capacity of any linker may be easily tested
In addition, linkers that were described above as photocleavable at the wavelength of a MALDI laser were found by the inventor to be cleaved also by fast fragmentation. Alternatively, such linkers may thus be used in conjugates for any method according to the invention described above, including when other mass spectrometry technologies than MALDI are used, notably for SIMS or DESI mass spectrometry.
However, it must be clearly understood that the (A-X)n-B conjugates usable by taking advantage of the fast fragmentation phenomenon are not limited to the particular suitable linkers described above that may be cleaved by photodissociation and fragmentation. Indeed, the capacity to be cleaved by fast fragmentation of any linker that would permit an easy linkage between a A tag molecule and a B binding molecule is easily tested by simply analyzing the synthesized conjugate in solution and monitoring the presence of a cleaved A tag molecule. This way, any potential linker can be easily tested and approved or refused for use in the present invention. In addition, any person skilled in the art of mass spectrometry will know, for the great majority of potential linkers, if their chemical structure will or not permit there cleavage by fragmentation, so that the easy to perform test described above will even not be necessary in most cases.
In particular, conventional, and even commercial, labelled antibodies may be used as conjugates when fast fragmentation is used to cleave the linker. For instance, fluorescent conjugated antibodies may be used, the fluorescent molecule being used as tag molecule. Notably, FITC-conjugated, PE-conjugated, PerCP-conjugated, APC-conjugated, Cy3-conjugated, Cy5-conjugated, Cy7-conjugated, Texas red-conjugated, TRITC-conjugated, PE-Cy5-conjugated, PE-Cy7-conjugated, or APC-Cy7-conjugated antibodies may be used. Alternatively, antibodies conjugated to other molecules such as enzymes alkaline phosphatase or peroxydase may also be used, the enzyme being used as tag molecule. This possibility has the crucial advantage that all sorts of antibodies, with a very wide range of antigenic specificities, are commercially available as labelled antibodies. In addition, technologies to attach a labelling molecule via conventional linkers to any antibody are well-known routine technologies. This way, indirect mass spectrometry analysis of biomolecules present in tissue sections can be performed for a very large number of distinct biomolecules using the methods according to the invention.
Thus, in a preferred embodiment, when fast fragmentation is used, target molecule(s) are peptides, proteins (including antibodies), antigens, or haptens, binding molecules are antibodies or antibody fragments, n is 1 and tag molecules are fluorochromes, enzymes, biotin or gold. Atoms such as Br may also be conjugated to antibodies, in particular when SIMS mass spectrometry is used (see Example 4). In this case, a linker named EDAC commercially available from Sigma under reference E1769 may be used to conjugate the antibody to the Br atom (see Example 4).
In addition, when such a linker molecule X that is cleaved by fast fragmentation is used, potentially any mass spectrometry technology, provided that it is suitable for tissue sections analysis (which is immediately apparent for any skilled person), may be used to implement the method according to the invention. In particular, several additional mass spectrometry technologies than UV-MALDI or IR-MALDI may be used, such as SIMS or DESI mass spectrometry. Thus, in a preferred embodiment of a method in which a linker molecule X that is cleaved by fast fragmentation is used, UV-MALDI, IR-MALDI, SIMS or DESI mass spectrometry is used
The invention further concerns a conjugate suitable for use in any method according to the invention, wherein said conjugate is selected from:
Each of the above conjugates is individually preferred as a conjugate according to the invention
Having generally described this invention, a further understanding of characteristics and advantages of the invention can be obtained by reference to certain specific examples and figures which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Oligo-peptide conjugates with a photocleavable linker were tested for their capacity to allow indirect detection of specific mRNA in tissue sections by MALDI-MS.
1.1 Materials and Methods
1.1.1 Oligo-Peptide Conjugates
Structure
Oligo-peptide conjugates that were studied displayed the following structure:
Five oligo-peptide conjugates with distinct peptide moieties were synthesized. The peptide amino acid sequence, monoisotopic (Mmono) and average (Marg) molecular weights, and the nucleic sequence of the oligonucleotide of each conjugate are displayed in the following Table 2.
Synthesis
Oligo-peptide conjugates were synthesized using the following protocol:
The peptide is synthesized on Symphony (Protein Technologies Inc) and purified on a Delta-Pak C18 15 μm 100A column (Waters).
The oligonucleotide is synthesized from 3′ to 5′ on Expedite (ABI). The amine function with photocleavable linker is added in 5′ before cleavage and deprotection. These steps are performed using a NH4OH 28% solution during 24 hours in the dark. The amino oligonucleotide is then purified on a Delta-Pak C18 15 μm 300A column (Waters). The amino function of the oligonucleotide is coupled to a heterobifunctional reagent comprising a maleimide function. The maleimido oligonucleotide is solubilized in water and added to an 1.2 equivalent of peptide in solution. The mixture is let under stirring for 16 hours.
The oligo-peptide conjugate is then purified on a Delta-Pak C18 15 μm 300A column (Waters) and characterized by mass spectrometry.
1.1.2 Preparation of Samples Before MALDI-MS
Several commonly used matrices were indifferently used for MALDI-MS analysis: α-cyano-4-hydroxycinnamic acid (CHCA), 3-hydroxy picolinic acid (HPA), and sinapinic acid (SA).
For CHCA, 10 mg of the matrix was dissolved in 1 mL of acetonitrile/water (2:1, v/v, 0.1% TFA/H2O). For SA, 20 mg of matrix was dissolved in the same solvent.
For SA, 20 mg of matrix was dissolved in the same solvent.
For HPA, 50 mg/ml of the matrix was dissolved in water.
In some cases, an additive (ammonium citrate or acetate) was added to the matrix.
Before MALDI-MS analysis, 14 of sample solution and 14 of matrix solution were mixed on the MALDI plate according to the procedure of the dried-droplet preparation (Karas, M.; Hillenkamp, F.; Anal. Chem. 1998, 60, 2299-2301).
Three distinct MALDI plate materials have been tested: stainless steel, gold and Teflon.
1.1.3 MALDI-MS Analysis
MALDI-TOF mass spectra were performed on a Voyager-DE STR mass spectrometer (Applied Biosystems, Framingham, Mass., USA) with delayed extraction (DE) and operating with a pulsed nitrogen laser emitting at 337 nm.
Analysis in Linear Mode
For inox MALDI plates, acquisition parameters were set to: acceleration voltage: 20 kV; 1st grid voltage: 94%; guide-wire voltage: 0.05%; extraction delay time: 100-250 ns.
For gold MALDI plates, acquisition parameters were set to: acceleration voltage: 25 kV; 1st grid voltage: 96%; guide-wire voltage: 0.05%; extraction delay time: 600 ns.
Analysis in Reflector Mode:
Acceleration voltage: 20 kV, 1st grid voltage: 70%, guide-wire voltage: 0.05%, extraction delay time: 200 ns.
1.1.4 Software for Image Reconstruction
For image reconstruction, software flexImaging (Bruker daltonics, Bremmen, DE) was used.
1.1.5 PCR Amplifications Using Oligo-Peptide Conjugates
PCR amplifications of proenkephalin were obtained using either tagged (peptide-photocleavable linker-oligonucleotide) or conventional (without the photocleavable linker and the peptide moiety) reverse (5′-CAG-GAC-TCC-CCA-AAG-GAG-AAC-AGG-A-3′, SEQ ID NO:10, see Table 2) and forward (5′-GA-CGT-ACC-AGG-CGG -TAG-CTG-CAT-TT-3′, SEQ ID NO:9, see Table 2) primers. Amplification products were then analyzed by electrophoresis in 2% agarose gel.
1.1.6 Synthesis of a dUTP-Peptide Conjugate with a Photocleavable Linker
A modified peptide-tagged dUTP with the following formula was synthesized:
For the synthesis, the following synthesis scheme was followed:
In order to prepare this triphosphate a Fmoc protected CPG resin was required. Since none of the precursor was available, then the succinylate was prepared from GT115A (100 mg). The sample was relatively pure but contained a small amount (by TLC) of a higher running non-tritylated compound (originates from the Sonogashira reaction and does not interfere with subsequent reactions and was not visible in the nmr spectra of the sample). Since it was not possible to purify the succinate, the reaction was modified slightly. It is normal to add 2 equivalents of succinic anhydride to the reaction to get quantitative yield but if this is not removed completely the amino residues of the cpg resin can become blocked during functionalisation. Therefore, 1.5 equivalents were used since the exact purity of the product is undetermined. The reaction did not go to completion (from TLC this was more than 50% by comparing the intensity of the components on the TLC by UV (254 nm) and the intensity of the DMT cation on treatment with HCl fumes. Since the non-succinylated product will not react, the resin was functionalised using this mixture. The resin was prepared but the loading is very low, 5.4 μmolg−1 (180 mg).
The resin was detritylated using 2% TCA/DCM washed with DCM and the process repeated until no orange colour due to the DMT cation was observed.
This was then dried (suction under argon) and the resin soaked in pyr/DMF 1:3 (0.4 ml) for 5 minutes before a solution of 0.1M Eckstein's reagent in dioxane was added (0.1 ml). The reaction was allowed to stand for 15 minutes after which time the resin was washed (dioxane, MeCN) and dried (suction under argon).
The resin was then soaked in a solution on 0.5M bis-(tributylammonium) pyrophosphate in anhydrous DMF and tri-n-butylamine for 20 minutes and the resin washed (DMF, MeCN) and dried (suction under argon).
The product was oxidised (iodine/water/pyridine/THF for 30 minutes), washed (MeCN) dried (suction under argon).
The Fmoc protecting group was removed (20% piperidine/DMF, 0.5 ml, 20 mins) and the resin washed thoroughly, (DMF, MeCN) and dried (suction under argon). This was then washed with DCI and a solution of DCI/photolabile amino linker CEP (1:1, 0.5 ml) was added and the reaction allowed to stand for 20 minutes. The solution was removed and the resin washed (MeCN) and dried (suction under argon). A mixture of cap A/cap B (1:1, 0.5 ml) was added and the resin soaked for 5 minutes before removing the capping reagents and washing and drying the resin as before. The product was oxidised (I2/THF/pyr/H2O, 5 mins) and the resin washed and dried as before. This was cleaved from the resin with cNH4OH at room temperature for 30 mins, then purified by anion exchange HPLC on a Dionex NucleoPac100 HPLC column using the following solvent system Buffer A:0.1M NH4Cl with 10% acetonitrile; Buffer B: 1M NH4Cl with 10% acetonitrile; flow rate 2.5 mL/min. using 6-Triphos.mth. This gave 3 fractions (A:—7 mins, B:—7.9 mins and C:—10.3 mins). All 3 fractions were lyophilized over night before being desalted by reverse phase HPLC Buffer A: Water; Buffer B: acetonitrile; flow rate 4 mL/min. The 3 fractions were again lyophilized overnight before being suspended in 200 ul of water. M.S. showed that CMM661A pk 1 was definitely not the triphosphate but it could be either CMM661pk 2 or 3 (very similar M.S. profiles). (CMM662A was formed from CMM661A pk 2 and CMM663A was formed from CMM661A pk 3).
Both samples were then used in the subsequent reaction. Bicarbonate buffer (10 ul) and the maleimide NHS ester (50 ul) were added to each sample and the reactions agitated overnight. The samples were diluted with milliQ water (500 ul) and filtered. The samples were purified by RP-HPLC, buffer A: 0.1M TEAA, buffer B: MeCN, flow rate 4 mL/min. using MeCN50.mth and the coupling of the peptide was carried out on these fractions.
1.1.7 Synthesis of a Proenkephalin Nucleic Probe (400 bp) Incorporating the Modified, Peptide-Tagged dUTP
A proenkephalin nucleic probe (400 bp) incorporating the previously synthesized modified, peptide-tagged dUTP (see paragraph 1.1.6) was synthesized by RT-PCR using the conventional forward and reward primers described in paragraph 1.1.5.
1.2 Results
1.2.1 Detection of One or Multiple Oligo-Peptide Conjugates in Various Modes of Analysis
The three oligo-peptides conjugates were analyzed in various analysis modes, either separately or simultaneously (
The analysis of separate oligo-peptide conjugates in linear positive mode (
Sensitivity tests were also performed to assess the detection threshold in the various MALDI analysis modes. The observed thresholds were of 100 fmol with a signal/noise ratio of 8 in linear positive mode (
Oligo-peptides 1, 2 and 3 were further analyzed simultaneously in the linear positive mode, linear negative mode and reflector positive mode (
In any case, these results show that three distinct oligo-peptides can be easily detected simultaneously.
The influence of the MALDI plate material on resolution, sensitivity and signal/noise ratio was investigated using stainless steel, gold and Teflon MALDI plates, with or without an additive composed of ammonium citrate or acetate.
Results show that cleavage of the photocleavable linker is possible no matter which material is used, with or without coating with ammonium citrate or acetate.
Moreover, it appears that the signal/noise ratio and signals intensity are higher on gold MALDI plates (
1.2.2 Mapping of an Oligo-Peptide Conjugate on a Solid Support
To evaluate the possibility to map an oligo-peptide conjugate on a surface, the image of an X letter made of oligo-peptide 3 was drawn using a multipipette on a MALDI plate.
The corresponding area was then analyzed, spot by spot, by MALDI-MS and the repartition of the ion m/z 1626.37 corresponding to the photocleaved fragment of oligo-peptide 3 under its sodium cationized form MNa+ was reconstructed using image reconstruction software.
1.2.3 Synthesis of a Peptide-Tagged Proenkephalin In Situ Hybridization Probe Using Oligo-Peptide Conjugates as Primers
To confirm the possibility to synthesize larger hybridization probes using an oligo-peptide conjugate as forward and/or reward primer, PCR amplifications were carried out using either tagged (peptide-photocleavable linker-oligonucleotide) or conventional (without the photocleavable linker and the peptide moiety) reverse (5′-CAG-GAC-TCC-CCA -AAG-GAG-AAC-AGG-A-3′ (SEQ ID NO: 10)) and forward (5′-GA-CGT-ACC -AGG-CGG-TAG-CTG-CAT-TT-3′ (SEQ ID NO: 9)) primers.
The resulting amplification products are displayed in
After silica column purification, non amplified primer dimers were removed and pure PCR amplification products were obtained (
MALDI analysis of these purified PCR amplification products was carried out. Results obtained with both tagged forward and reverse primers (
1.2.4 Proenkephalin mRNA Analysis in a Deparaffined Rat Brain Section Using a Peptide-Tagged Hybridization Probe
In situ hybridization was performed on a deparaffined rat brain section using a peptide-tagged hybridization probe, as described before.
MALDI analysis was then performed on different tissue localizations and the resulting spectra analyzed.
The control spectrum shows that no signal is detected at the m/z ratios of the cleaved peptide (m/z=1704) or the characteristic ion (m/z=1720) when using a non peptide-tagged hybridization probe.
In contrast, proenkephalin mRNA expression can be clearly identified in the
The mRNA expression of proenkephalin can thus be clearly monitored at distinct spots of the tissue section, thus allowing to further reconstruct an image of the tissue section proenkephalin mRNA expression.
1.2.5 Synthesis of a dUTP-Peptide Conjugate with a Photocleavable Linker and Use of Such a Modified, Peptide-Tagged dUTP to Generate a Multi-Peptide-Tagged Proenkephalin Probe (400 bp).
With the synthesis protocol described in the Material and Methods section, a modified peptide-tagged dUTP nucleotide has thus been synthesized, with the formula displayed on
The modified peptide-tagged dUTP was analyzed by MALDI analysis to confirm the detection of the peptide tag. As shown on
With the use of such a modified peptide-tagged dUTP, a multi-peptide-tagged nucleic acid hybridization probe for proenkephalin has been very simply generated using a simple RT-PCR amplification in the presence of dATP, dCTP, dGTP and the modified peptide-tagged dUTP, as displayed on
To confirm that the modified peptide-tagged dUTP was readily incorporated into the proenkephalin probe, the purified PCR product of proenkephalin amplified with either the modified peptide-tagged dUTP or a non modified dUTP with no tag was analyzed using MALDI analysis.
As shown on
Moreover, the use of a modified, peptide-tagged dUTP allows for a signal amplification since a given hybridization probe synthesized with the modified peptide-tagged dUTP will carry as many tag peptides as the number of U bases in its sequence.
Finally, the use of hybridization probes synthesized with the modified peptide-tagged dUTP allows for a quantitative analysis of mRNA expression in tissue sections. Indeed, the signal generated by the corresponding tag(s) of one or several studied mRNA(s) can be compared with that obtained for a reference mRNA sequence (for instance a house-keeping gene such as actin of HPRT). As the number of U bases in each hybridization probe is known, the expression ratio between each studied mRNA sequence and the reference mRNA sequence can be calculated.
1.3 Conclusion
These results clearly demonstrate the possibility to use nucleic acid-photocleavable linker-peptide conjugates for the indirect simultaneous detection of multiple mRNA target molecules in tissue sections, using hybridization probes synthesized with peptide-tagged primers.
Moreover, a modified peptide-tagged dUTP has been synthesized that allows for an easier synthesis of peptide-tagged hybridization probes with any target mRNA specificity, an amplified tag signal, thus lowering the detection threshold, and the possibility to perform a quantitative mRNA expression analysis in tissue sections.
Thus, the indirect simultaneous mapping of several distinct mRNA target molecules in tissue sections using MALDI-MS analysis is now possible, with low detection threshold, possible multiplex analysis, and even possible quantitative analysis.
An antibody-peptide conjugate with a photocleavable linker was synthesized and tested for its capacity to allow indirect detection of a specific protein in tissue sections using MALDI-MS.
2.1 Synthesis of the Antibody Peptide Conjugate
A goat antibody specific for the framework region of rabbit antibodies (goat anti-rabbit antibody) has been used to synthesize an antibody-peptide conjugate with a photocleavable linker at UV-MALDI laser wavelength. This way, the conjugate is suitable as secondary antibody for the indirect analysis of any peptide, protein, antigen or hapten for which a primary rabbit antibody is available. In addition, it has to be noted that the protocol of synthesis described below is applicable to any antibody of any specificity, including any peptide, protein, antigen or hapten, so that other conjugates in which the antibody is specific for another type of antibody or for any desirable peptide, protein, antigen or hapten may be easily synthesized using the protocol described below.
As tag molecule, a peptide was used displaying the following formula: DSPEGLNRKQKPA (SEQ ID NO: 11). However, in this case also, the protocol described below is applicable to any other peptide that might be desirable a tag molecule.
The peptide was synthesized in solid phase and directly coupled to the photocleavable linker, resulting in a peptide-phocleavable compound of formula (SEO ID NO: 11):
The antibody-peptide conjugate was synthesized using the following reaction steps:
The main reactions for the synthesis of the anti-body-peptide conjugate with a photocleavable linker are shown on
which is shown in abbreviated form in
2.2 MALDI Analysis of the Obtained Antibody-Peptide Conjugate in Solution
The obtained antibody-peptide conjugate with a photocleavable linker has then been tested in solution first, to verify the ability of the tag peptide to be efficiently cleaved by photodissociation induced by the UV-MALDI laser.
The results are shown on
In addition, a fast fragmentation fragment comprising the tag peptide, the photocleavable linker, and a cystein initially belonging to the antibody (see
2.3 MALDI Direct Analysis of Rat Brain Tissue Sections Using the Obtained Antibody-Peptide Conjugate
The obtained antibody-peptide conjugate with a photocleavable linker has then been tested for indirect UV-MALDI analysis of membrane protein carboxypeptidase D (180 kDa) in rat brain tissue sections.
Rat brain tissue sections were first hybridized with a primary rabbit antibody specific for carboxypeptidase D. In a second step, the antibody-peptide conjugate with a photocleavable linker was added to hybridize with primary antibodies present on the tissue section.
The resulting stained tissue sections were then analyzed using UV-MALDI.
A typical spectrum is displayed on
Using the method according to the invention, the rat brain tissue section was scanned, each spectrum data being stored, and the image of the expression map of carboxypeptidase D was constructed. The result is displayed in
In addition, to further demonstrate the reliability of the method according to the invention to construct expression maps, the rat brain tissue section stained with rabbit primary antibodies specific for carboxypeptidase D where developed using T-chloronaphtol as chromogen after fixation on a classical commercial secondary antibody. The obtained staining colocalized with the MALDI detection of carboxypeptidase D (data not shown), thus demonstrating the reliability of the method according to the invention and MALDI imaging shows to be even more sensitive than classical revelation.
These results demonstrate that the synthesized antibody-peptide conjugate with a photocleavable linker permits the indirect detection of a protein in a tissue section, as well as the construction of an expression map.
Fast fragmentation is usually considered as a detrimental but inevitable phenomenon.
In the present case, the inventors have found that it is possible to take advantage of this necessary phenomenon to implement a method for determining at least one target molecule map in a tissue section using (A-X)n-B conjugates in which the linker X is cleaved by fast fragmentation.
3.1 Fast Fragmentation for Indirect Mass Spectrometry Analysis of Biomolecules Present in Tissue Sections Using Conjugates in which the Linker is Photocleavable at the Wavelength of a UV-MALDI Laser
Although this concept is transposable to other mass spectrometry technologies, such as SIMS or DESI mass spectrometry, the inventors have first proven the possible use of this concept using MALDI mass spectrometry and conjugates in which the linker is photocleavable at the wavelength of a UV-MALDI laser.
Indeed, when using either oligo-peptide conjugates or antibody-peptide conjugates with photocleavable linkers (PC), in addition to expected (peptide+PC) fragments, other fragments comprising the peptide tag are observed, these fragments corresponding to fast fragmentation fragments of the conjugates.
More precisely, when using 3 distinct oligo-peptide conjugates with photocleavable linkers (see Example 1 for a more precise definition of these conjugates), fragments corresponding to the tag peptide 1, peptide 2 or peptide 3 are observed in addition to expected fragments (peptide 1, 2 or 3+PC), as is clearly shown on
The same phenomenon is observable for conjugate 3 on
When using the antibody-peptide conjugate described in Example 2 also, both an expected photodissociation fragment and a fast fragmentation fragment are observed as shown in
3.2 Fast Fragmentation for Indirect Mass Spectrometry Analysis of Biomolecules Present in Tissue Sections Using a FITC Labelled Antibody
The inventors have also analyzed the possibility to use fast fragmentation for indirect mass spectrometry analysis of biomolecules present in tissue sections using conventional, and notably commercial, labelled antibodies.
Indeed, conventional labelling molecules have definite molecular weights and may thus be used as tag molecules. In addition, all sorts of antibody, with a very wide range of antigenic specificities, are commercially available as labelled antibodies. Finally, technologies to attach a labelling molecule via conventional linkers to any antibody are well-known routine technologies, thus ensuring that the possibility to use conventionally labelled antibodies for indirect mass spectrometry analysis of biomolecules present in tissue sections would permit to significantly enlarge the number of distinct biomolecules that may be mapped using the method according to the invention.
A FITC labelled antibody has been used for in solution MALDI analysis of a FITC labelled antibody using CHCA matrix.
Results are displayed in
Although these results have yet to be confirmed on tissue section, all preceding results obtained in solution have been further confirmed on tissue sections. These results thus highly support the possibility to use fast fragmentation and conventional labelled antibodies for indirect detection of target molecules in tissue section using MALDI mass spectrometry, but also other mass spectrometry technologies involving fast fragmentation, and notably SIMS or DESI mass spectrometry.
3.3 Conclusion
The obtained results clearly demonstrate the possibility to take advantage of the usually detrimental phenomenon of fast fragmentation for the indirect detection of target molecules in tissue section using various mass spectrometry technologies, in particular MALDI, SIMS or DESI mass spectrometry.
An antibody-Br conjugate may be synthesized using EDAC linker:
available from SIGMA under reference E1769, using the following protocol:
Such a protocol may be applied using any antibody or antibody fragment, to conjugate it with a Br atom. Such conjugates may be used in a method according to the invention in which fragmentation is used to cleave the linker between the antibody and the Br atom.
This application is a National Stage application of PCT/IB2006/002309, filed Jun. 7, 2006, which claims priority from US Provisional Application No. 60/687,848, filed Jun. 7, 2005.
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