Patent Grant
11561226
The instant application contains a sequence listing which has been submitted electronically in ASCI format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 27, 2018, is named LT01152US SL.txt and is 154,793 bytes in size.
This disclosure relates to the field of detection and quantification of AKT-mTOR pathway proteins, including phosphorylated proteins, by immunoprecipitation and mass spectrometry.
The AKT-mTOR pathway plays a central role in tumor progression and anti-cancer drug resistance. The quantitative measurement of protein expression and post-translational modifications of the AKT-mTOR pathway is necessary for precisely characterizing cancer, monitoring cancer progression, and determining treatment responses. See Logue, J. S.; Morrison, D. K.; Genes Dev. Apr. 1 2012, 26 (7), 641-50.
A major limitation in the detection and quantitation of AKT-mTOR pathway proteins is the lack of rigorously validated methods and reagents. Currently, only semi-quantitative results from Western blotting, ELISA, and Luminex assays are available. Mass spectrometry (MS) is increasingly becoming the detection methodology of choice for assaying protein abundance and post-translational modifications. However, to date, MS has not been successful in quantifying AKT-mTOR pathway proteins, possibly due to their low abundance and significant post-translational modification profiles.
Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. See, Gingras et al., Nat. Rev. Mol. Cell. Biol., Aug. 2007, 8 (8), 645-54; and Carr, S. A. et al., Mol. Cell. Proteomics Mar. 2014, 13 (3), 907-17. The identification of appropriate antibodies for use in IP upstream of MS is important, as not all antibodies that bind to protein will be effective immunoprecipitation tools, and further, not all antibodies that are effective immunoprecipiation tools will lead to successful identification via MS.
The present disclosure provides reagents and methods for detecting and quantifying AKT-mTOR pathway proteins via immunoprecipitation (IP), mass spectrometry (MS), and immunoprecipitation followed by mass spectrometry (IP-MS).
In some embodiments, methods for immunoprecipitating an AKT-mTOR pathway protein (target protein) are provided, comprising contacting a biological sample with any one of the antibodies recited in Table 1. In some embodiments, the antibodies useful in the IP methods comprise the antibodies recited in Table 8. In some embodiments, the antibodies useful in the IP methods comprise the antibodies recited in Table 9. The methods may comprise washing the contacted biological sample to enrich for antibody-protein conjugates. Further methods include detecting the antibody-protein conjugates (the immunoprecipitated target protein) to determine the AKT-mTOR pathway protein in the biological sample. In some embodiments, the antibody is labelled. In some embodiments, a detection reagent is provided to the enriched antibody-protein conjugate. In some embodiments the label is biotin and the detection reagent is streptavidin.
In some embodiments the IP is single-plex. In some embodiments the IP is multi-plex. The antibodies useful in multi-plex IP may comprise the antibodies of Table 8 and Table 9.
In some embodiments a method for detecting AKT-mTOR pathway proteins via MS is provided, comprising isolating proteins from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for AKT-mTOR pathway protein(s), and determining the identity of one or more AKT-mTOR pathway protein(s) in the sample. In some embodiments, the peptide for AKT-mTOR pathway protein(s) comprises a sequence of SEQ ID NO: 1-SEQ ID NO: 424. In some embodiments the peptide is less than 40 amino acids in length. In some embodiments, the peptide for AKT-mTOR pathway protein(s) consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424.
In some embodiments a method for quantifying AKT-mTOR pathway proteins via MS is provided, comprising isolating proteins from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for AKT-mTOR pathway protein(s), and determining the quantity of one or more AKT-mTOR pathway protein(s) in the sample. In some embodiments, the peptide for AKT-mTOR pathway protein(s) comprises a sequence of SEQ ID NO: 1-SEQ ID NO: 424. In some embodiments the peptide is less than 40 amino acids in length. In some embodiments, the peptide for AKT-mTOR pathway protein(s) consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments a method for detecting AKT-mTOR pathway proteins via IP-MS is provided, comprising treating a biological sample with at least one antibody capable of immunoprecipitating AKT-mTOR target pathway protein(s) from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for AKT-mTOR pathway protein(s), and determining the identity of one or more AKT-mTOR pathway protein(s) in the sample. In some embodiments, the peptide for AKT-mTOR pathway protein(s) comprises a sequence of SEQ ID NO: 1-SEQ ID NO: 424. In some embodiments the peptide is less than 40 amino acids in length. In some embodiments, the peptide for AKT-mTOR pathway protein(s) consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments a method for quantifying AKT-mTOR pathway proteins via IP-MS is provided, comprising treating a biological sample with at least one antibody capable of immunoprecipitating AKT-mTOR target pathway protein(s) from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for AKT-mTOR pathway protein(s), and determining the quantity of one or more AKT-mTOR pathway protein(s) in the sample. In some embodiments, the peptide for AKT-mTOR pathway protein(s) comprises a sequence of SEQ ID NO: 1-SEQ ID NO: 424. In some embodiments the peptide is less than 40 amino acids in length. In some embodiments, the peptide for AKT-mTOR pathway protein(s) consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments the AKT-mTOR pathway target protein is phosphorylated.
Methods for determining the ratio of phosphorylated to non-phosphorylated AKT-mTOR pathway proteins are provided, comprising any of the above IP, MS, or MS-IP methods, wherein a further step of determining the ratio of phosphorylated to non-phosphorylated protein is provided. In some embodiments, the method is an MS-IP method comprising treating a biological sample with one or more antibodies capable of immunoprecipitating one or more phosphorylated AKT-mTOR pathway proteins, and separately treating the same biological sample with one or more antibodies capable of immunoprecipitating at least one or more of the same or different non-phosphorylated AKT-mTOR pathway proteins; digesting the immunoprecipitated AKT-mTOR pathway proteins; adding a first and a second detectably labelled internal standard peptide of known amount to the digested proteins, wherein the first internal standard peptide has the same amino acid sequence as a phosphorylated AKT-mTOR pathway peptide used to identify the phosphorylated protein, and the second internal standard peptide has the same amino acid sequence as the non-phosphorylated AKT-mTOR pathway peptide used to identify the non-phosphorylated protein; assaying the digested protein and internal standards via mass spectrometry to determine the presence and amount of phosphorylated and non-phosphorylated AKT-mTOR pathway proteins, wherein the AKT-mTOR pathway peptide comprises a peptide of SEQ ID NO: 1-SEQ ID NO: 424, and is less than 40 amino acids in length; determining the quantity of AKT-mTOR phosphorylated and non-phosphorylated pathway proteins in the sample, and determining the ratio of phosphorylated to non-phosphorylated pathway proteins. In some embodiments, the peptide for AKT-mTOR pathway protein(s) consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments, the biological sample is human. In some embodiments, the biological sample is non-human. In some embodiments, the biological sample is mammalian. In some embodiments, the biological sample is from rat, mouse, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat, or non-human primate.
In embodiments utilizing an AKT-mTOR pathway peptide, the peptide may be modified with a detectable label. The detectable label may comprise an isotope, such as a heavy isotope, such as those known to those of skill in the art, including 13C, 15N, 2H and 180. In some embodiments, the modified/labelled peptide comprises a peptide of SEQ ID NO: 213-424. In some embodiments the peptide is less than 40 amino acids in length. In some embodiments the modified/labelled peptide consists of a peptide of SEQ ID NO: 213-424. In some embodiments the modified/labelled peptide consists of a peptide of SEQ ID NO: 213-424, wherein the peptide is further modified.
In some embodiments, the antibody for IP is selected from the group consisting of the antibodies recited in Table 1. In some embodiments, the antibody for IP is an antibody having the six CDRs of any of the antibodies of Table 1. The antibody may be capable of immunoprecipitating more than one AKT-mTOR pathway protein. In some embodiments the antibody is labelled or capable of being labelled. The label may be any label known to those of skill in the art including enzymatic and fluorescent labels, such as biotin. In some embodiments more than one antibody is used in a multi-plex IP. In some embodiments, the multi-plex IP comprises the antibodies of Table 8. In some embodiments, the multi-plex IP comprises the antibodies of Table 9.
In some embodiments, two or more antibodies are utilized to analyze one biological sample. For example, a first antibody is capable of immunoprecipitating a phosphorylated AKT-mTOR pathway protein, and a second antibody is capable of immunoprecipitating a non-phosphorylated version of the AKT-mTOR pathway protein precipitated by the first antibody. In some embodiments, a single antibody is capable of immunoprecipitating a phosphorylated and non-phosphorylated AKT-mTOR pathway protein.
In some embodiments, the immunoprecipitation comprises treating a sample with a labelled antibody capable of binding to an AKT-mTOR pathway protein to provide a labelled antibody-protein conjugate. The method may further comprise contacting the labelled antibody-protein conjugate with a capture agent capable of binding to the labelled antibody to isolate the pathway protein from the sample. The label may be biotin and the capture agent may be streptavidin.
The quantity of an AKT-mTOR pathway protein may be determined by adding an internal standard peptide of known amount to the digested protein prior to mass spectrometry. In some embodiments, the internal standard peptide has the same amino acid sequence as the AKT-mTOR pathway peptide. In some embodiments, the internal standard is detectably labeled. The method may further comprises determining the quantity of an AKT-mTOR pathway peptide by comparison to the internal standard.
In some embodiments, the internal standard peptide comprises a sequence of SEQ ID NO: 1-SEQ ID NO: 424. In some embodiments the peptide is less than 40 amino acids in length. In some embodiments, the peptide consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments, quantifying the AKT-mTOR pathway protein comprises comparing an amount of an AKT-mTOR pathway peptide in the sample to an amount of the same AKT-mTOR pathway peptide in a control sample.
Quantifying an AKT-mTOR pathway protein may comprise comparing an amount of an AKT-mTOR pathway peptide to an internal standard peptide of known amount, wherein both the peptide in the biological sample and the internal standard peptide comprise SEQ ID NO: 1-SEQ ID NO: 424, wherein the standard peptide is detectably labeled, and wherein the peptide is less than 40 amino acids long. In some embodiments, the standard peptide consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments the mass spectrometry is selected from tandem mass spectrometry and discovery mass spectrometry. The targeted mass spectrometry may be selected from multiple reaction monitoring (MRM), selected reaction monitoring (SRM), and parallel reaction monitoring (PRM), or combinations thereof.
In some embodiments the biological sample is selected from isolated human cells, plasma, serum, whole blood, CSF, urine, sputum, tissue, and tumorous tissue.
In some embodiments, the method further comprises quantifying the relative amount of AKT-mTOR pathway protein. In some embodiments, the method further comprises quantifying the absolute amount of AKT-mTOR pathway protein.
In some embodiments, the digesting comprises a protease or chemical digest. In some embodiments the digestion may be single or sequential. The protease digestion may comprise trypsin, chymotrypsin, AspN, GluC, LysC, LysN, ArgC, proteinase K, pepsin, clostripain, elastase, GluC biocarb, LysC/P, LysN promisc, protein endopeptidase, staph protease or thermolysin.
The chemical cleavage may comprise CNBr, iodosobenzoate or formic acid.
In some embodiments the digestion is a protease digest with trypsin.
In some embodiments the methods further comprise desalting after digestion and prior to mass spectrometry.
The AKT-mTOR pathway protein may be selected from AKT1 (UniProtKB—P31749), AKT2 (UniProtKB—P31751), IR (also known as INSR) (UniProtKB P06213), IGF1R (UniProtKB—P08069), IRS1 (UniProtKB—P35568), TSC2 (UniProtKB—P49815), mTOR (UniProtKB—P42345), GSK3a (UniProtKB—P49840), GSK3b (UniProtKB—P49841), GSK3a/GSK3b, p70S6K (also known as RPS6KB1) (UniProtKB—P23443), RPS6 (UniProtKB—P62753), PRAS40 (also known as AKT1S1) (UniProtKB—Q96B36), and PTEN (UniProtKB—P60484).
In some embodiments, the AKT-mTOR pathway is a protein that interacts with any of AKT1 (UniProtKB—P31749), AKT2 (UniProtKB—P31751), IR (also known as INSR) (UniProtKB P06213), IGF1R (UniProtKB—P08069), IRS1 (UniProtKB—P35568), TSC2 (UniProtKB—P49815), mTOR (UniProtKB—P42345), GSK3a (UniProtKB—P49840), GSK3b (UniProtKB—P49841), GSK3a/GSK3b, p70S6K (also known as RPS6KB1) (UniProtKB—P23443), RPS6 (UniProtKB—P62753), PRAS40 (also known as AKT1S1) (UniProtKB—Q96B36), and PTEN (UniProtKB—P60484).
In some embodiments, the AKT-mTOR pathway protein is phosphorylated.
In some embodiments, the concentration of AKT-mTOR protein that may be detected ranges from about 0.08 fmol to about 2000 fmol.
In some embodiments, the lower limit of detection is about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 fmol. The lower limit of detection may be within the range of about 0.05-0.25 fmol.
In some embodiments the lower limit of quantification is about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, or 0.75 fmol. The lower limit of quantification may be within the range of about 0.05-0.75 fmol.
Kits comprising one or more antibodies capable of immunoprecipitating an AKT-mTOR pathway protein are encompassed.
Kits comprising one or more antibodies capable of immunoprecipitating an AKT-mTOR pathway protein, and reagents useful for performing mass spectrometry to detect an AKT-mTOR pathway protein are also provided.
Also encompassed are kits comprising one or more antibodies capable of immunoprecipitating an AKT-mTOR pathway target protein, and reagents useful for performing mass spectrometry to quantify an AKT-mTOR pathway protein.
The antibody to be included in the kit may be selected from any one or more of the antibodies recited in Table 1. In some embodiments the antibody is labelled or capable of being labelled. The label may be any label known to those of skill in the art including enzymatic and fluorescent labels, such as biotin. In some embodiments, the kit comprises more than one antibody. In some embodiments, the kit comprises two or more of the antibodies selected from the antibodies recited in Table 8. In some embodiments, the kit comprises two or more of the antibodies selected from the antibodies recited in Table 9. In some embodiments, the kit comprises two or more of the antibodies selected from the antibodies recited in Table 8 and two or more of the antibodies selected from the antibodies recited in Table 9. In some embodiments, the kit comprises each of the antibodies recited in Table 8, Table 9, or Tables 8 and 9.
The kits may further comprise an AKT-mTOR pathway peptide. In some embodiments, the peptide comprises a sequence of SEQ ID NO: 1-SEQ ID NO: 424. In some embodiments, the peptide is less than 40 amino acids in length. In some embodiments, the peptide consists of a sequence of SEQ ID NO: 1-SEQ ID NO: 424. The peptides of SEQ ID NO: 1-SEQ ID NO: 212 may be labelled. In some embodiments, the label on SEQ ID NO: 1-SEQ ID NO: 212 differs from the label shown on the peptides of SEQ ID NO: 213-SEQ ID NO: 424. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204).
In some embodiments, the kit may comprise at least one peptide selected from peptides of SEQ ID NO: 213-SEQ ID NO: 424, wherein the peptide is less than or equal to 40 amino acids. In one embodiment, the kit comprises at least one peptide consisting of the peptides of SEQ ID NO: 213-SEQ ID NO: 424.
The peptides provided in the kit may be detectably labeled or capable of being modified to be detectably labeled. In some embodiments, the kit may comprise at least one peptide selected from peptides of SEQ ID NO: 1-SEQ ID NO: 212, wherein the peptide is detectably labeled or capable of being modified to be detectably labeled.
In some embodiments, the kit further comprises a protease or chemical agent capable of digesting an immunoprecipitated protein sample. The protease agent may be trypsin, chymotrypsin, AspN, OluC, Lyse, LysN, ArgC, proteinase K, pepsin, clostripain, elastase, GluC biocarb, LysC/P, LysN promisc, protein endopeptidase, Staph protease or thermolysin. The chemical agent may be CNBr, iodosobenzoate or formic acid.
The kits may be utilized to detect AKT-mTOR pathway proteins, including AKT1 (UniProtKB—P31749); AKT2 (UniProtKB—P31751), IR (also known as INSR) (UniProtKB P06213), IGF1R (UniProtKB—P08069), IRS1 (UniProtKB—P35568), TSC2 (UniProtKB—P49815), mTOR (UniProtKB—P42345), GSK3a (UniProtKB—P49840), GSK3b (UniProtKB—P49841), GSK3a/GSK3b, p70S6K (also known as RPS6KB1) (UniProtKB—P23443 (KS6B1_HUMAN)), RPS6 (UniProtKB—P62753), PRAS40 (also known as AKT1S1)(UniProtKB—Q96B36), and PTEN (UniProtKB—P60484).
The AKT-mTOR protein to be detected and quantified by the kits may be phosphorylated.
Also encompassed are antibodies recited in Table 1 for use in immunoprecipitating an AKT-mTOR pathway protein. The antibody may be used in methods comprising immunoprecipitating an AKT-mTOR pathway protein prior to analyzing the protein via mass spectrometry.
AKT-mTOR pathway peptides selected from the peptides of Table 3 are encompassed. The AKT-mTOR pathway peptides may be used in methods of detecting and quantifying AKT-mTOR pathway proteins in biological samples. The AKT-mTOR pathway peptides may be used in methods comprising immunoprecipitating the AKT-mTOR pathway protein from the biological sample, and analyzing the immunoprecipitated protein via mass spectrometry.
This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
As used herein, an “AKT-mTOR pathway protein” includes, but is not limited to, AKT1 (UniProtKB—P31749), AKT2 (UniProtKB—P31751), IR (also known as INSR) (UniProtKB P06213), IGF1R (UniProtKB—P08069), IRS1 (UniProtKB—P35568), TSC2 (UniProtKB—P49815), mTOR (UniProtKB—P42345), GSK3a (UniProtKB—P49840), GSK3b (UniProtKB—P49841), GSK3a/GSK3b, p70S6K (also known as RPS6KB1) (UniProtKB—P23443), RPS6 (UniProtKB—P62753), PRAS40 (also known as AKT1S1) (UniProtKB—Q96B36), and PTEN (UniProtKB—P60484).
As used herein “protein”, “peptide”, and “polypeptide” are used interchangeably throughout to mean a chain of amino acids wherein each amino acid is connected to the next by a peptide bond. In some embodiments, when a chain of amino acids consists of about two to forty amino acids, the term “peptide” is used. However, the term “peptide” should not be considered limiting unless expressly indicated.
The term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments so long as they exhibit the desired immunoprecipitating activity. As such, the term antibody includes, but is not limited to, fragments that are capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, di-scFv, sdAb (single domain antibody) and (Fab′)2 (including a chemically linked F(ab′)2). Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment. Pepsin treatment yields a F(ab′)2 fragment that has two antigen-binding sites. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, goat, horse, sheep, chicken, etc. Furthermore, for all antibody constructs provided herein, variants having the sequences from other organisms are also contemplated, such as CDR-grafted antibodies or chimeric antibodies. Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments also include nanobodies (sdAb, an antibody having a single, monomeric domain, such as a pair of variable domains of heavy chains, without a light chain). An antibody fragment can be referred to as being a specific species in some embodiments (for example, human scFv or a mouse scFv). This denotes the sequences of at least part of the non-CDR regions, rather than the source of the construct. The antibodies provided herein are referred to by reference to name and catalog reference. The skilled artisan, holding this name and catalog information, is capable of determining the sequence of the antibody, and therefore the disclosure encompasses any antibody having at least partial sequence of a reference antibody so long as the antibody maintains its ability to immunoprecipitate its antigen protein. In some embodiments, the antibodies comprise antibodies having the same CDRs as the antibodies provided in Table 1.
Mass spectrometry (MS) is a primary technique for analysis of proteins on the basis of their mass-to-charge ratio (m/z). MS techniques generally include ionization of compounds and optional fragmentation of the resulting ions, as well as detection and analysis of the m/z of the ions and/or fragment ions followed by calculation of corresponding ionic masses. A “mass spectrometer” generally includes an ionizer and an ion detector. “Mass spectrometry,” “mass spec,” “mass spectroscopy,” and “MS” are used interchangeably throughout.
“Targeted mass spectrometry,” also referred to herein as “targeted mass spec,” “targeted MS,” and “tMS” increases the speed, sensitivity, and quantitative precision of mass spec analysis. Non-targeted mass spectrometry, sometimes referred to as “data-dependent scanning,” “discovery MS,” and “dMS” and targeted mass spec are alike in that in each, analytes (proteins, small molecules, or peptides) are infused or eluted from a reversed phase column attached to a liquid chromatography instrument and converted to gas phase ions by electrospray ionization. Analytes are fragmented in the mass spec (a process known as tandem MS or MS/MS), and fragment and parent masses are used to establish the identity of the analyte. Discovery MS analyzes the entire content of the MS/MS fragmentation spectrum. In contrast, in targeted mass spectrometry, a reference spectrum is used to guide analysis to only a few selected fragment ions rather than the entire content.
“Multiple reaction monitoring,” “MRM,” “selected reaction monitoring,” and “SRM” are used interchangeably throughout to refer to a type of targeted mass spectrometry that relies on a unique scanning mode accessible on triple-quadrupole (QQQ) instruments. See, e.g., Chambers et al., Expert Rev. Proteomics, 1-12 (2014).
“Parallel Reaction Monitoring,” and “PRM” are used interchangeably herein to describe another type of targeted mass spec wherein the second mass analyzer used in SRM (quadrupole) is substituted by a high resolution orbitrap mass analyzer in PRM. Unlike SRM, which allows the measuring of one single transition at a given point in time, PRM allows parallel monitoring in one MS/MS spectrum. PRM also allows for the separation of ions with close m/z values (i.e., within a 10 ppm range), and may therefore allow for lower limits of detection and quantification (LOD or LLOD and LOQ or LLOQ).
The methods disclosed herein may be applied to any type of MS analysis. The disclosure is not limited by the specific equipment or analysis used. The use of any equipment with the intent of analyzing the m/z of a sample would be included in the definition of mass spectrometry. Non-limiting examples of MS analysis and/or equipment that may be used include electrospray ionization, ion mobility, time-of-flight, tandem, ion trap, MRM, SRM, MRM/SRM, PRM, and Orbitrap. The disclosure is neither limited by the type of ionizer or detector used in the MS analysis nor by the specific configuration of the MS. The disclosure is not limited to use with specific equipment or software. The disclosure is not limited to the equipment and software described in the Examples.
In some embodiments, methods of immunoprecipitating an AKT-mTOR pathway protein are provided, comprising contacting a biological sample with at least one antibody recited in Table 1. The immunoprecipitating method may be single-plex or multi-plex. A “single-plex” IP utilizes one antibody per assay, whereas a “multi-plex” IP utilizes more than one antibody per assay.
In some embodiments, an IP-MS method for detecting and quantifying phosphorylated and non-phosphorylated AKT-mTOR pathway proteins is provided. The methods may comprise contacting a biological sample with at least one antibody recited in Table 1, digesting the immunoprecipitated protein(s), and assaying the digested proteins via mass spectrometry. The IP and MS may be single-plex or multi-plex. A “single-plex” MS refers to monitoring a single peptide in a single MS run, whereas a “mulit-plex” MS refers to monitoring more than one target peptides in a single MS run.
Table 1 provides a listing of antibodies useful in the IP and IP-MS methods described herein. Table 2 provides a listing of antibodies that are known to bind to their antigen AKT-mTOR protein, but were found to be less useful in the IP and IP-MS methods described herein.
The immunoprecipitated AKT-mTOR pathway proteins may be reduced and alkylated prior to fragmentation (e.g., digestion). Samples that have been reduced and alkylated may comprises modifications, such as to cysteine residues (e.g., CAM). Where an AKT-mTOR peptide of SEQ ID NO: 1-424 shows modification resulting from, for example, reduction/alkylation, the non-modified peptide is also encompassed. For example, in each instance where an AKT-mTOR pathway peptide of SEQ ID NO: 1-424 is referred to, also encompassed are unmodified peptides of SEQ ID NO: 1-424.
The samples may optionally be desalted prior to analysis by mass spectrometry. Both enzymatic and chemical digestion is encompassed. Enzymatic digestion includes, but is not limited to, digestion with a protease such as, for example, trypsin, chymotrypsin, AspN, GluC, LysC, LysN, ArgC, proteinase K, pepsin, Clostripain, Elastase, GluC biocarb, LysC/P, LysN Promise, Protein Endopeptidase, Staph Protease or thermolysin. Chemical digestion includes use of, for example, CNBr, iodosobenzoate and formic acid.
In some embodiments, after fragmentation (e.g., digestion), peptide samples are analyzed by mass spectrometry (MS), and the resulting spectra are compared with theoretical spectra from known proteins to determine the peptides and proteins in a sample. For AKT-mTOR pathway proteins, discovery MS is cumbersome and time consuming and is not a viable clinical method. Therefore, the inventors have identified novel peptides that associate with AKT-mTOR pathway proteins for use in the IP-MS methods of the disclosure. Use of these peptides in targeted MS, and IP-targeted MS methods allows quantitation of even low abundant AKT-mTOR proteins. Moreover, use of these peptides in targeted MS, and in IP-targeted MS methods, allows quantitation of phosphorylated AKT-mTOR proteins.
Theoretically, peptides useful in MS to detect and quantify AKT-mTOR pathway proteins can be designed by use of computer software and the like. However, many of these potential peptide sequences are unsuitable or ineffective for use in MS-based assays, including SRM/MRM and PRM. Because it was not possible to predict the most suitable peptides for MS analysis, it was necessary to experimentally identify modified and unmodified peptides to develop as clinical reagents. To complicate the analysis, it was discovered that certain peptides useful when assaying typical samples were not predictive when assaying samples that had undergone immunoprecipitation.
Typically, targeted MS is performed by quantifying specific unique peptides of the protein. In some embodiments, known amounts of isotope-labeled (e.g., heavy isotope-labeled) versions of these targeted peptides can be used as internal standards for absolute quantitation. In some instances, proteins of interest are not detectable even after identifying unique peptide standards. The combination of specific antibodies with specific target peptides has allowed the inventors to improve the sensitivity of detection of AKT-mTOR pathway proteins by MS, and has allowed for lower levels of detection and lower levels of quantification than ever previously seen. See, e.g.,
In some embodiments, the AKT-mTOR pathway peptides provided in the kits, and useful in the described methods, are listed in Table 3. SEQ ID Nos: 1-212 are native peptide sequences useful in identifying the AKT-mTOR pathway proteins recited in the “Target ID” column. Certain peptide sequences are phosphorylated at certain residues as shown in parentheses “(P03H2)” following the modified residue.
Certain peptides are modified at cysteine residues as shown by “(CAM)” following the modified residue. The “CAM” post-translational modification is well known to those of skill in the art to mean carbamidomethylation, resulting from alkylation of the protein/peptide. The peptides may be as shown in Table 3, or may be non-modified version of these peptides lacking carbamidomethylation.
In some embodiments, the peptides reagents are recited in Table 5 (SEQ ID Nos: 98, 96, 157, 163, 40, 42, 37, 25, 73, 80, 52, 57, 59, 208, 209, 16, 23, 124, 120, 195, 200, 129, 133, 1, 6, 27, 91, and 204). In some embodiments, the peptides of Table 5 are useful in multi-plex MS methods.
In some embodiments, protein samples are denatured or solubilized before fragmentation.
In some embodiments, the fragmentation protocol uses chemical cleavage. In some embodiments, the chemical cleavage uses CNBr. In some embodiments, the fragmentation protocol is done using an enzyme. In some embodiments, the fragmentation protocol uses MS-grade commercially available proteases. Examples of proteases that may be used to digest samples include trypsin, endoproteinase GluC, endoproteinase ArgC, pepsin, chymotrypsin, LysN protease, LysC protease, GluC protease, AspN protease, proteinase K, and thermolysin. In some embodiments, a mixture of different proteases are used and the individual results are combined together after the digestion and analysis. In some embodiments, the digestion is incomplete in order to see larger, overlapping peptides. In some embodiments, the antibody digestion is performed with IdeS, IdeZ, pepsin, or papain to generate large antibody domains for “middle-down” protein characterization. In some embodiments, the fragmentation protocol uses trypsin that is modified. In some embodiments, a protein:protease ratio (w/w) of 10:1, 20:1, 25:1, 50:1, 66:1, or 100:1 may be used. In some embodiments, the trypsin used is at a concentration of about 100 ng/ml-1 mg/ml, or about 100 ng/ml-500 μg/ml, or about 100 ng/ml-100 μg/ml, or about 1 μg/ml-1 mg/ml, or about 1 μg/ml-500 μg/ml, or about 1 μg/ml-100 μg/ml, or about 10 μg/mg-1 mg/ml, or about 10 μg/mg-500 μg/ml, or about 10 μg/mg-100 μg/ml. In some embodiments, the digestion step is for 10 minutes to 48 hours, or 30 minutes to 48 hours, or 30 minutes to 24 hours, or 30 minutes to 16 hours, or 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 16 hours, or 1 to 8 hours, or 1 to 6 hours, or 1 to 4 hours. In some embodiments, the digestion step is incubated at a temperature between 20° C. and 45° C., or between 20° C. and 40° C., or between 22° C. and 40° C., or between 25° C. and 37° C. In some embodiments, the digestion step is incubated at 37° C. or 30° C. In some embodiments, a step is included to end the digestion step. The step to end the digestion protocol may be addition of a stop solution or a step of spinning or pelleting of a sample. In some embodiments, the digestion is followed by guanidation.
In some embodiments, the fragmentation protocol includes use of protein gels. In some embodiments, the fragmentation protocol comprises in-gel digestion. An exemplary commercially available kit for performing in-gel digestion is the In-Gel Tryptic Digestion Kit (Thermo Fisher Cat#89871).
In some embodiments, the fragmentation protocol is carried out in solution. An exemplary commercially available kit for performing in-solution digestion is the In-Solution Tryptic Digestion and Guanidiation Kit (Thermo Fisher Cat#89895).
In some embodiments, the fragmentation protocol uses beads. In some embodiments, the fragmentation protocol comprises on-bead digestion. In some embodiments, agarose beads or Protein G beads are used. In some embodiments, magnetic beads are used.
In some embodiments, protein samples are separated using liquid chromatography before MS analysis. In some embodiments, fragmented samples are separated using liquid chromatography before MS analysis.
The IP and IP-MS methods described herein are capable of detecting phosphorylated AKT-mTOR pathway proteins, including those described in Table 4.
In some embodiments, the AKT-mTOR pathway peptides used in the MS methods described herein have limits of detection considered useful in clinical and research methods. See, e.g, Table 5. In some embodiments, the AKT-mTOR pathway peptides used in the MS and IP-MS methods comprise or consist of the peptides described in Table 5. In some embodiments, the peptides of Table 5 are detectably labelled. The peptides of SEQ ID NO: 163 may lack the “CAM” modification shown on the fifth amino acid.
In some embodiments, methods for detecting phosphorylated AKT-mTOR pathway proteins are encompassed. In some embodiments, IP, MS, and IP-MS methods to detect phosphorylated AKT-mTOR pathway proteins are conducted separately from methods to detect total (non-phosphorylated) AKT-mTOR pathway proteins. In some embodiments, the IP and IP-MS methods to detect phosphorylated AKT-mTOR pathway proteins utilize the antibodies of Table 9. In some embodiments, the IP and IP-MS methods to detect non-phosphorylated AKT-mTOR pathway proteins utilize the antibodies of Table 8. In some embodiments, the IP-MS methods to detect phosphorylated AKT-mTOR pathway proteins utilize the antibodies of Table 9 and the peptides of Table 5. In some embodiments, the IP-MS methods to detect non-phosphorylated AKT-mTOR pathway proteins utilize the antibodies of Table 8 and the peptides of Table 5.
The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.
AKT-mTOR pathway proteins play central roles in diseases including cancer. The identification of AKT-mTOR pathway proteins, while desired as a means for monitoring disease progression, and as a tool for scientific research, has been limited in part because of the low abundance of AKT-mTOR pathway proteins, and in part due to a lack of validated methods and reagents. Phosphorylated AKT-mTOR pathway proteins are particularly important to identify and quantify as a measure of protein activation status, and also as markers for disease progression. As shown in
Cell Culture
For all assays, HCT116 (ATCC product#CCL-247), MCF7, (ATCC product#HTB-22) and A549 (ATCC product#CCL-185) cells were grown in Hamm's F-12K media, McCoy's 5A Media, and MEM Media, respectively, with 10% FBS/1×PenStrep to approximately 70-80% confluency. Cells were starved in 0.1% charcoal stripped FBS for 24 hours before stimulation with 100 ng/ml of IGF (CST product#8917SF) for 15 minutes.
Controls
Western Blot (WB), ELISA, and Luminex Assays were used as controls to compare to the IP-MS method described herein. The reagents and methods for Western Blots are summarized in Table 6.
Reagents for ELISA kits are shown in Table 7.
For Luminex Assays, AKT Pathway (total) Magnetic 7-Plex Panel (Thermo Fisher Scientific, PN: LH00002M), AKT Pathway (phospho) Magnetic 7-Plex Panel (Thermo Fisher Scientific, PN: LH00001M), Milliplex Map Akt/mTOR Phosphoprotein Magnetic Bead 11-Plex Kit (Millipore, PN: 48-611MAG) and Milliplex Map Total Akt/mTOR Magnetic Bead 11-Plex Kit (Millipore, PN: 48-612MAG) were used as recommended in instruction manuals. Luminex MagPix instrument was used to acquire and analyze Luminex assay data.
Immunoprecipitation and MS Sample Preparation
The Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Protein A/G) was used to screen and validate antibodies for 11 total and 10 phosphorylated AKT-mTOR pathway proteins from 500 μg cell lysate. Validated antibodies were biotinylated with the Thermo Scientific™ Pierce Antibody Biotinylation Kit for IP. The Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Streptavidin) was used to perform the single or multiplex IPs for target enrichment. IP samples were processed by an in-solution digestion method where IP eluates were reconstituted in 6M Urea, 50 mM TEAB, pH 8.5 followed by reduction, alkylation and trypsin digestion overnight at 37° C. The digested samples were acidified with TFA before MS analysis.
Liquid Chromatography and Mass Spectrometry
Prior to MS analysis, tryptic digest samples were desalted on-line using the Thermo Scientific™ Acclaim™ PepMap 100 C18 Trap Column. For discovery MS, the samples were analyzed by nanoLC-MS/MS using a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System and Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. For targeted MS, the samples were analyzed using the UltiMate 3000 RSLCnano System and the Thermo Scientific™ TSQ™ Vantage™ Mass Spectrometer (SRM mode) or the Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (PRM mode).
MS Data Analysis
Discovery MS data were analyzed with Thermo Scientific™ Proteome Discoverer™ 1.4 to assess percent sequence coverage, unique peptides, MS1 intensities, spectral counts and PTMs. The Proteome Discoverer software searches were executed using the Uniprot human protein database. Tryptic peptides with highest MS1 intensity and relevant phosphorylation sites were selected from the discovery data for targeted assay development. For targeted MS data analysis, Thermo Scientific™ Pinpoint software and Skyline software (University of Washington) were used to measure limit of quantitation (LOQ) from the calibration curve and target analyte concentration from unknown samples.
Results
As shown in
Higher numbers of unique peptides were identified in IP enriched samples as compared to neat (non-IP-enriched) lysate. See
Limits of detection (LOD) and lower limits of quantification (LLOQ) were analyzed for twelve AKT-mTOR pathway proteins, including AKT2, AKT1, mTOR, IGF1R, IR, PRAS40, p70S6K, TSC2, PTEN, GSK3alpha, GSK3beta, and IRS1. Results are presented in
Eleven total and ten phosphorylated AKT-mTOR pathway protein targets were enriched simultaneously from unstimulated and IGF stimulated MCF7 lysates with biotinylated antibodies and Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Streptavidin). MCF7 cells were starved in 0.1% charcoal stripped FBS for 24 hours before stimulation with 100 ng/ml of IGF for 15 minutes. Validated IP-MS antibodies are biotinylated for 11 total and 10 phosphorylated AKT-mTOR pathway targets using the Thermo Scientific™ Pierce Antibody Biotinylation Kit for IP (PN: 90407) as recommended in instruction manual. 1 μg of each biotinylated antibody for 11 total targets were added simultaneously to 1000 μg of control and IGF stimulated MCF7 cell lysate in duplicate. 1 μg of each biotinylated antibody for 10 total targets were added simultaneously to 1000 μg of control and IGF stimulated MCF7 cell lysate in duplicate. IP was performed as recommended in the Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Streptavidin) (PN: 90408) with the following modification. 5 microgram of streptavidin magnetic beads per microgram of biotinylated antibody concentration was used for multiplex IP.
IP samples were processed by an in-solution digestion method where IP eluates were reconstituted in 6M Urea, 50 mM TEAB, pH 8.5 followed by reduction (5 mM TCEP for 30 minutes at 35° C.), alkylation (20 mM Iodoacetamide in dark at room temperature for 30 minutes) and trypsin digestion overnight at 37° C. The digested samples were acidified with 3.5 μL of 10% TFA before discovery MS analysis. For discovery MS, the samples were analyzed by nanoLC-MS/MS using a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System and Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Briefly, the digested samples were cleaned on-line using the C18 trap column (Thermo Fisher Scientific, PN: 164564) followed by reversed-phase separation using the analytical C18 column (75 μm i.d.×15 cm, nanoViper, 3 μm particle size, Thermo Fisher Scientific, PN: ES800) with a 2-30% gradient of Buffer B using Buffer A (0.1% formic acid) and Buffer B (0.1% formic acid/99.9% acetonitrile) at 0.300 μL/min.
Next the ability of IP-MS to quantify sub-fmol concentrations of AKT-mTOR pathway proteins via the disclosed IP-MS methods was tested. As shown in
Next, comparison of mIP-tMS assays with current immunoassay techniques to quantitate AKT-mTOR pathway targets from unstimulated and IGF stimulated A549, HCT116 and MCF7 lysates were performed. Western Blot, ELISA, and Luminex assays were performed as described above and according to manufacturer's instructions. mIP-tMS was performed as in Example 2.
A summary of AKT-mTOR pathway proteins that were identified and quantified using the IP-MS methods described herein is provided in
Immunoprecipitation using particular selected antibodies resulted in a higher yield of AKT-mTOR pathway target proteins and less non-specific binding proteins than MS alone. IP-MS assay was also more successful than other commercially available non-MS assays. Furthermore, IP to MS analysis of total and phosphorylated AKT-mTOR pathway proteins enabled identification of multiple isoforms, relevant protein interactions and phosphorylation sites. Total and phosphorylated mIP-tMS assays allowed simultaneous quantitation of 12 total and 11 phosphorylated AKT-mTOR pathway proteins in the low to sub-fmol range from unstimulated and IGF stimulated A549, HCT116 and MCF7 cell lysates. The benchmarking of mIP-tMS assays showed moderate correlation for quantitation of total and phosphorylated target relative abundance compared to WB, ELISA and Luminex assays. The low concordance for a few targets is possibly due to differences in the specificity of antibodies used for each assay. Major advantages of the MS-based assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets, interacting proteins and their phosphophorylated forms.
Tissue lysis protocol was optimized for IP-MS application. Briefly, 50-100 mg of human and murine tissue samples were washed with 5 mL 1× cold PBS three times. Tissue samples was minced in 5 mL 1× cold PS using scissor followed by homogenization in IP lysis buffer (Thermo Fisher Scientific PN: 87788) and electronic Polytron Handheld Tissue Tearer. Homogenized tissue samples were passed through tissue strainer (Thermo Fisher Scientific PN: 87791) to prepare tissue lysates before IP. To validate the IP-MS method in murine and human tissue lysate, eleven total and ten phosphorylated AKT-mTOR pathway protein targets were enriched simultaneously from normal mouse lung tissue lysate, normal mouse kidney tissue lysate, and normal human lung tissue lysate as per Example 2. A549 cell lysate was used as a non-tissue control. As shown in Table 10, the IP-MS method described herein is capable of validating AKT-mTOR pathway proteins in murine and human tissue lysate in addition to cell lysate. Seven out of eleven AKT-mTOR pathway protein targets were identified in normal human lung tissue, and nine out of eleven AKT-mTOR pathway protein targets were identified for normal mouse kidney tissue using our IP-MS method.
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| WO2017/160698 | 9/21/2017 | WO | A |
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