WO2010119261A1 - Method for quantifying modified peptides - Google Patents

Abstract

The present invention provides a method for quantifying modified peptides in a sample, the method comprising: (a) obtaining peptides from the sample; (b) adding reference modified peptides to the peptides obtained in step (a) to produce a mixture of peptides and reference modified peptides; (c) carrying out mass spectrometry (MS) on said mixture of peptides and reference modified peptides to obtain data relating to the peptides in the sample; and (d) comparing the data relating to the peptides in the sample with data in a database of modified peptides using a computer programme; wherein the database of modified peptides is compiled by a method comprising: (i) obtaining peptides from a sample; (ii) enriching modified peptides from the peptides obtained in step (i); (iii) carrying out liquid chromatography-tandem mass spectrometry (LC- MS/MS) on the enriched modified peptides obtained in step (ii); (iv) comparing the modified peptides detected in step (iii) to a known reference database in order to identify the modified peptides; and (v) compiling data relating to the modified peptides identified in step (iv) into a database.

Description

METHOD FOR QUANTIFYING MODIFIED PEPTIDES

The present invention relates to methods for quantifying modified peptides, and in particular to methods for quantifying phosphorylated peptides. Such methods are used, for example, to identify and quantify phosphorylation sites on proteins and to quantify the activity of protein kinases.

Most proteins are modified in some way by the addition of functional groups and these modifications can be detected by mass spectrometry.

Protein modifications that can be detected by mass spectrometry include phosphorylation, nitration, glycosylation, acetylation, methylation and lipidation. These protein modifications have various biological roles in the cell.

Mass spectrometry (MS) is an analytical technique that measures the mass to charge (m/z) ratio of the ions formed when a molecule or atom is ionized, vaporized and introduced into an instrument capable of separating these ions according to their m/z ratios. Mass spectrometry may also involve breaking molecules into fragments, thus enabling the structure of the molecules to be determined. The combination of MS with the physical separation technique of liquid chromatography is known as liquid chromatography-mass spectrometry (LC-MS).

In a typical MS procedure, a sample is loaded onto the MS instrument and compounds present in this sample are ionized, for example by electrospray ionization (ESI) or matrix assisted laser desorption/ionization (MALDI). The mass to charge ratio of the ions is then calculated by different forms of mass analysers such as time of flight, ion traps or quadrupoles, or combination of these.

The homeostasis of normal cells is controlled by the action of cell signalling pathways, which, when deregulated, also contribute to many diseases including cancer, neurodegeneration, allergy and diabetes. Protein and lipid kinases are prominent members of these pathways, and therefore, these enzymes represent one of the most important classes of drug targets for the treatment of many diseases.

Approaches for the unbiased detection of enzymatic activities have been reported. It is possible to use chemical probes to covalently link reactive amino acids in enzyme active sites (Blethrow, J.D. et al. Proc NatlAcadSci USA 105, 1442-1447 (2008)) or on the substrates (Barglow, K.T. & Cravatt, B.F. Nat Methods 4, 822-827 (2007)).

Proteins linked to the probes are affinity purified and their identities determined by mass spectrometry. This approach can. detect activities and substrates, but it requires a large number of cells and the information provided is only qualitative.

As a more quantitative approach, quantification of phosphorylation sites on proteins known to be substrates of specific kinases serves as a measure of kinase activity. When performed using mass spectrometry as the readout, this approach allows the quantification of hundreds to thousands of phosphorylation sites in a single experiment. For example, using metabolic labelling with stable isotopes (the SILAC approach) it was possible to quantify >2000 phosphorylation sites that showed altered levels of expression upon treatment of HeLa cells with EGF (Olsen, J.V. et al. Cell 127, 635-648 (2006)). However, since cells need to be metabolically active to incorporate labelled amino' acids, this approach cannot be used as a general tool to quantify cell signalling in primary tissues, and its low throughput limits its usefulness. a x ;

The use of isotope labelled internal standard peptides to measure phosphorylated peptides could be an alternative (Gerber, S. A. et al. Proc Natl Acad Sci U S A 100, 6940-6945 (2003)) but, although theoretically possible, the synthesis of thousands of phosphorylated peptides labelled with stable isotopes to use as internal standards is not possible in practice.

iTRAQ reagents can be used to label peptides chemically with stable isotopes (Ross, P. L.; et al. MoI Cell Proteomics 2004, 3, (12), 1154-69.). This technique can be used for relative quantification of peptides, including modified peptides. Its limitation is that the number of samples that can be compared is limited by the number of isotope labels, putting a limit to its usefulness for obtaining statistical validation of the data. Chemical labelling is also not practical in a clinical setting and can introduce variability at the chemical reaction step.

There is thus a need in the art for a method which can be used for unbiased, comprehensive and accurate measurement of signalling pathways in primary tissues.

The present inventors have devised an improved method for quantifying modified peptides and in particular phosphorylated peptides. The method involves the preparation of a database of modified peptides and the comparison of the database with data obtained from biological samples, typically using a computer programme.

In a first aspect, the present invention provides a method for quantifying modified peptides in a sample, the method comprising: (a) obtaining peptides from the sample;

(b) adding reference modified peptides to the peptides obtained in step (a) to produce a mixture of peptides and reference modified peptides;

(c) carrying out mass spectrometry (MS) on said mixture of peptides and reference modified peptides to obtain data relating to the peptides in the sample; and

(d) comparing the data relating to the peptides in the sample with data in a database of modified peptides using a computer programme; ^■ • wherein the database of modified peptides is compiled by a method comprising: i) obtaining peptides from a sample; ii) enriching modified peptides from the peptides obtained in step (i); iii) carrying out liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the enriched modified peptides obtained in step (ii); iv) comparing the modified peptides detected in step (iii) to a known reference database in order to identify the modified peptides; and v) compiling data relating to the modified peptides identified in step (iv) into a database.

The present invention provides a method for quantifying modified peptides in a sample. The method of the invention is suitable for quantifying peptides which contain any modifications which can be detected by mass spectrometry.

The method of the invention is used to quantify modified peptides which are derived from longer proteins. The method of the invention can be used to simultaneously quantify thousands of modified peptides.

The method of the invention is particularly useful for quantifying phosphorylated peptides in a sample and thus in^1' 'one embodiment the modified peptides are phosphorylated peptides. In relation to this embodiment of the invention, the term "phosphoprotein" is used herein to refer to a phosphorylated protein and the term "phosphopeptide" is used herein to refer to a phosphorylated peptide.

The method of the invention is also useful for quantifying peptides that have been modified by, for example, acetylation, nitration, glycosylation, methylation and/or lipidation.

The method of the invention can be used to quantify modified peptides in any sample which contains peptides. The sample is typically a biological sample and can thus be any type of sample obtained from a biological source, for example a sample obtained from a human, animal, plant or bacterium. The invention thus encompasses the use of samples obtained from human and non-human sources.

The method of the present invention finds use in the detection and quantification of modified peptides in a sample from any species of interest. Typically, the method of the invention is used to analyse a sample from a human or animal. The animal is typically a mammal, such as a mouse, rat, guinea pig, cow, sheep or goat. The animal is alternatively a bird, such as a chicken, a fish, such as a zebra fish, a nematode, such as the worm Caenorhabditis elegans, or an insect, such as the fruit fly Drosophila melanogaster. The method of the invention can also be used to analyse samples from other life-forms such as bacteria and yeast. The method of the invention can be used to analyse samples from an experimentally important species of bacterium such as Escherichia coli, Salmonella enterica, Streptococcus pneumoniae or Staphylococcus aureus, or of yeast such as the baker's yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. The method of the invention can also be used to analyse a sample from a plant or fungus or a virus.

Typically, the biological sample is derived from a human, and can be, for example, a sample of a bodily fluid such as urine or blood, or another tissue. Typically, the biological sample is a cell line or a tissue, typically a primary tissue. For example, the sample can be a tissue from a human or animal. The human or animal can be healthy or diseased.

In some embodiments of the invention, the sample itself or the organism from which the sample is obtained is treated with a test substance prior to carrying out the method of the invention. Thus, in this embodiment, a cell line or an organism from which a tissue is obtained is treated with a test substance prior to carrying out the method of the invention on the cell line or tissue. The test substance is typically an exogenous chemical or drug, such as small molecule inhibitors, RNAi, therapeutic peptides, and antibodies. This embodiment of the invention allows the investigation of the effects of the test substance on peptide modification. For example, in one embodiment, where the method of the invention is used to quantify phosphorylated peptides, a cell line can be treated with agonists of pathways and/or kinase inhibitors prior to carrying out the method of the invention. Typical kinase inhibitors include inhibitors of PB K such as wortmannin and PI-103, as used in the Examples. At least 80 kinase inhibitors are in different stages of clinical development (Zhang, J.; et al Nat Rev Cancer 2009, 9,

(1), 28-39.) The technique is also useful to investigate other types of inhibitors suspected to have an effect on kinase pathways, such as HSP90 inhibitors, phosphatase inhibitors and antibody drugs.

Step (a) of the method of the invention involves obtaining peptides from the sample. Peptides can be obtained from the sample using any suitable method known in the art.

In one embodiment, step (a) of the method of the invention comprises:

(1) Iy sing cells in the sample;

(2) extracting the proteins from the lysed cells obtained in step (1); and

(3) cleaving said proteins into peptides,

In step (1) of this embodiment of the invention, the cells in the sample are lysed, or split open. The cells can be lysed using any suitable means known in the art, for example using physical methods such as mechanical lysis (for example using a Waring blender), liquid homogenization, sonication or manual lysis (for example using a pestle and mortar) or detergent-based methods such as CHAPS or Triton-X. Typically, the cells are lysed using a denaturing buffer such as a urea-based buffer.

In step (2) of this embodiment of the invention, proteins are extracted from the lysed cells obtained in step (1). In other words, the proteins are separated from the other components of the lysed cells.

In step (3) of this embodiment of the invention, the proteins from the lysed cells are cleaved into peptides. In other words, the proteins are broken down into shorter peptides. Protein breakdown is also commonly referred to as digestion. Protein cleavage can be carried out in the present invention using any suitable agent known in the art.

Protein cleavage or digestion is typically carried out using a protease. Any suitable protease can be used in the present invention. In the present invention, the protease is typically trypsin, chyrnotrypsin, Arg-C, pepsin, V8, Lys-C, Asp-C and/or AspN. Alternatively, the proteins can be cleaved chemically, for example using hydroxylamine, formic acid, cyanogen bromide, BNPS-skatole, 2-nitro-5- thiocyanobenzoic acid (NTCB) or any other suitable agent.

The peptides used in the present invention and which are typically produced by protein cleavage or digestion as in step (3) described above are suitable for mass spectrometric analysis. Typically, such peptides are between about 5 and 30 amino acids long, for example from 7 to 25 amino acids, from 10 to 20 amino acids, from 12 to 18 amino acids or from 14 to 16 amino acids. However, shorter and longer peptides, such as between about 2 and about 50, for example from about 3 to about 40 or from about 4 to about 45 amino acids can also be used. The length of the peptide that can be analysed is limited by .the ability of the mass spectrometer to sequence such long peptides. In certain cases polypeptides of up to 300 amino acids can be analysed.

In step (b) of the method of the invention, reference modified peptides are added to the peptides obtained from the sample to produce a mixture of peptides and reference modified peptides. Step (b) thus results in one mixture of peptides (including modified ones) per sample. The reference modified peptides are also referred to herein as "internal standards" (ISs). Typically, 5 to 10, for example 6 to 9 or 7 to 8, reference modified peptides are added.

In the present invention, the reference modified peptides are typically reference phosphorylated peptides. Such reference phosphorylated peptides are typically derived from a reference protein of defined nature and concentration, often referred to as an internal standard (IS) protein. ISs can be commercially available proteins, for example casein. Alternatively, ISs are synthesised specifically for use in the invention. In this embodiment of the invention, reference phosphorylated peptides are typically synthesised with the same sequence as some of the phosphorylated peptides that it is desired to quantify but which are enriched in stable heavy isotopes of carbon and nitrogen. The peptides are typically synthesised using solid phase chemistry in which one amino acid is added at a time to form an amino acid chain or polypeptide. Typically, such peptides are enriched in ¹³C and ¹⁵N that substitute the common ¹²C and ¹⁴N. This enrichment results t in the reference phosphorylated peptides being approximately 6 to 10 daltons heavier than the endogenous phosphorylated peptides with the same sequence so that they can be distinguished using a mass spectrometer. 5

In another embodiment of the invention, when the method of the invention is used to quantify acetylated peptides, the reference modified peptides are reference acetylated peptides. Such reference acetylated peptides are typically synthetic peptides containing acetylated amino acids. lθ^"

The reference modified peptides are typically added at a known amount in each of the samples to be compared. The signals of the endogenous modified peptides are normalised to the signal of the reference modified peptides in downstream analysis.

15 In one embodiment, step (b) of the method of the invention further comprises enriching modified peptides from the mixture of peptides and reference modified peptides obtained in step (b) to produce a mixture of enriched modified peptides. This additional step thus results in a single mixture of enriched modified peptides per sample. In this embodiment of the invention, step (c) thus comprises carrying out 0 mass spectrometry (MS) on said mixture of enriched modified peptides to obtain data relating to the peptides in the sample. In this embodiment of the invention, step (b) typically results in a mixture of enriched phosphorylated peptides.

The step of enriching modified peptides is typically carried out using 5 chromatography. In one embodiment, the chromatography is immobilized metal ion affinity chromatography (IMAC), titanium dioxide (TiO₂) chromatography, and/or zirconium dioxide (ZrO₂) chromatography. Typically, the chromatography is IMAC and TiO₂ chromatography. 0 Alternatively, the step of enriching modified peptides is carried out using antibody- based methods. In one embodiment of the invention, when the peptides being quantified are phosphorylated peptides, antibodies with affinity to phosphorylated amino acids such as tyrosine, threonine, serine or histidine are linked (immobilised) to a solid matrix. Phosphorylated peptides are enriched by the ability of these antibodies to specifically bind phosphorylated peptides. Non-phosphorylated peptides are then washed away while phosphorylated peptides are retained on the antibody coated matrices. Elution of phosphorylated peptides from the immobilised antibody is typically carried out using low pH solvents or by any other suitable method that denatures the interaction between antibody and phosphorylated peptides.

In another embodiment of the invention, when the peptides being quantified are acetylated peptides, acetylated peptides are enriched by the use of specific antibodies against acetylated amino acid residues. Such antibodies are linked to a solid matirx and then enriched by the ability of the antibodies to specifically bind acetylated amino acid residues. Non-acetylated peptides are then washed away while acetylated peptides are retained on the immobilised antibody.

In step (c) of the method of the invention, mass spectrometry (MS) is carried out on the mixture of peptides and reference modified peptides obtained in step (b) to obtain data relating to the peptides in the sample. Typically, this data is in the form of an MS datafile for the sample. In one embodiment of the invention, when step (b) of the method of the invention further comprises enriching modified peptides from the mixture of peptides and reference modified peptides obtained in step (b) to produce a mixture of enriched modified peptides, step (c) comprises carrying out mass spectrometry (MS) on said mixture of enriched modified peptides to obtain data relating to the peptides in the sample, typically an MS datafile for the sample. Typically, the mass spectrometry is liquid chromatography-mass spectrometry (LC- MS). Step (c) thus typically results in an LC-MS datafile (one from each sample).

The data relating to the peptides in the sample typically comprises the mass to charge (m/z) ratio, charge (z) and/or relative retention time of the peptides. In step (d) of the method of the invention, the data relating to the peptides in the sample (typically in the form of an MS datafile and more typically an LC-MS datafile) is compared with data in a database of modified peptides using a computer programme. For example, the mass to charge (m/z) ratio, charge (z) and relative retention time of the peptides in the sample are compared with the mass to charge (m/z) ratio, charge (z) and relative retention time of the modified peptides in the database. This enables the identification and quantification of each modified peptide in the sample using the database of modified peptides.

Typically, the computer programme is the programme termed PESCAL (Cutillas, P. R.; Vanhaesebroeck, B. MoI Cell Proteomics 6(9), 1560-73, 2007). PESCAL constructs extracted ion chromatograms (XIC, i.e, an elution profile) for each of the modified peptides present in the database across all the samples that are to be compared. This is done by centring the XIC on the m/z and retention time of the peptide previously identified to be phosphorylated (i.e, present in the database constructed in the first step of the procedure). PESCAL also

the peptide to help in the correct assignment of identity. The program also calculates the peak height and area under the curve of each XIC. The data is normalised by dividing the intensity reading (peak areas or heights) of each modified peptide that is being analysed by those of the reference modified peptides.

In the method of the invention, the database of modified peptides is compiled by a method comprising the following steps:

(i) obtaining peptides from a sample; (ii) enriching modified peptides from the peptides obtained in step (i);

(iii) carrying out liquid chromatography-tandem mass spectrometry (LC- MS/MS) on the enriched modified peptides obtained in step (ii);

(iv) comparing the modified peptides detected in step (iii) to a known reference database in order to identify the modified peptides; and (v) compiling data relating to the modified peptides identified in step (iv) into a database. Step (i) of the method of the invention involves obtaining peptides from a sample. Peptides can be obtained from the sample using any suitable method known in the art.

The sample is typically a biological sample and can thus be any type of sample obtained from a biological source, as described above. Typically, the sample is a cell line or a primary tissue.

In some embodiments of the invention, where the sample used in step (i) is a cell line, the sample is treated with an inhibitor prior to carrying out step (i). The inhibitor can be any suitable type of inhibitor. Typically, when the method of the invention is used to quantify phosphorylated peptides, the inhibitor is a phosphatase inhibitor.

Treatment with phosphatase inhibitors increases the stoichiometry of phosphorylation and results in a greater number of phosphorylated peptides that can be included in the database. In addition, methyl transferase or acetyl hydrolase inhibitors can be used when the purpose is to quantify methylated and acetylated peptides, respectively.

In one embodiment, step (i) of the method of the invention comprises:

(1) lysing cells in a sample; (2) extracting the proteins from the lysed cells obtained in step (1); and

(3) cleaving said proteins into peptides using the same method as in step (3) of step (a) described above.

In step (1) of this embodiment of the invention, the cells in the sample are lysed, or split open. The cells can be lysed using any suitable means known in the art, for example using physical methods such as mechanical lysis (for example using a Waring blender), liquid homogenization, sonication or manual lysis (for example using a pestle and mortar) or detergent-based methods such as CHAPS or Triton-X. Typically, the cells are lysed using a denaturing buffer such as a urea-based buffer. In step (2) of this embodiment of the invention, proteins are extracted from the lysed cells obtained in step (1). In other words, the proteins are separated from the other components of the lysed cells.

In step (3) of this embodiment of the invention, the proteins from the lysed cells are cleaved into peptides using the same method as in step (3) of step (a) described above. Step (3) of step (i) thus results in a mixture of peptides including modified ones.

Protein cleavage can be carried out in the present invention using any suitable agent known in the art. However, as set out above, the method of cleavage used in step (3) of step (i) must be the same as the method of cleavage used in step (3) of step (a) described above. Protein cleavage is typically carried out using a protease. Any suitable protease can be used in the present invention, hi the present invention, the protease is typically trypsin, chymotrypsin, Arg-C, pepsin, V8, Lys-C, Asp-C or AspN. Alternatively, the proteins can be cleaved chemically, for example using hydroxylamine, formic acid, cyanogen bromide, BNPS-skatole, 2-nitro-5- thiocyanobenzoic acid (NTCB) or any other suitable agent.

The peptides used in the present invention and which are typically produced by protein cleavage as in step (3) described above are suitable for mass spectrometric analysis. Typically, such peptides are between about 5 and 30 amino acids long, for example from 7 to 25 amino acids, from 10 to 20 amino acids, from 12 to 18 amino acids or from 14 to 16 amino acids. However, shorter and longer peptides, such as between about 2 and about 50, for example from about 3 to about 40 or from about 4 to about 45 amino acids can also be used. The length of the peptide that can be analysed is limited by the ability of the mass spectrometer to sequence such long peptides. In certain cases longer polypeptides, up to 300 amino acids, can be analysed.

In step (ii) of the method of the invention, modified peptides are enriched from the peptides obtained in step (i). Step (ii) thus results in several fractions enriched in modified peptides. The enrichment of modified peptides in step (ii) is typically carried out using multidimensional chromatography. In one embodiment, the multidimensional chromatography is carried out using strong cation exchange high performance liquid chromatography (SCX-HPLC), immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography. In another embodiment, the multidimensional chromatography is carried out using anion exchange high performance liquid chromatography (SAX-HPLC), immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography. In these embodiments of the invention, the chromatographical techniques are carried out sequentially.

Alternatively, the enrichment of modified peptides in step (ii) is carried out using antibody-based methods.

In one embodiment of the invention, when the peptides being quantified are phosphorylated peptides, antibodies with affinity to phosphorylated amino acids such as tyrosine, threonine, serine or histidine are linked to a solid matrix. Phosphorylated peptides are enriched by the ability of these antibodies to specifically bind phosphorylated peptides. Non-phosphorylated peptides are then washed away while phosphorylated peptides are retained on the antibody coated matrix. Elution of phosphorylated peptides from the immobilized antibody is typically carried out using low pH solvents or by any other suitable method that denatures the interaction between antibody and phosphorylated peptides.

In another embodiment of the invention, when the peptides being quantified are acetylated peptides, acetylated peptides are enriched by the use of specific antibodies against acetylated amino acid residues. Such antibodies are linked to a solid matrix and then enriched by the ability of the antibodies to specifically bind acetylated amino acid residues. Non-acetylated peptides are then washed away while acetylated peptides are retained on the antibody $όated matrix. In step (iii) of the method of the invention, liquid chromatography-tandeni mass spectrometry (LC-MS/MS) is carried out on the enriched modified peptides obtained in step (ii).

In step (iv) of the method of the invention, the modified peptides detected in step (iii) are compared to a known reference database in order to identify the modified peptides. This step is typically carried out using a commercially available search engine, such as, but not restricted \$, the MASCOT, ProteinProspector Phenyx, or Sequest search engines.

In step (v) of the method of the invention, data relating to the modified peptides identified in step (iv) is compiled into a database. This database lists all the parameters needed for the quantification of phosphorylated peptides in subsequent biological experiments. Typically, the data relating to the modified peptides includes identity of the modified peptide, mass to charge (m/z) ratio, charge and/or relative retention time. This allows data relating to the peptides in the sample, typically the mass to charge (m/z) ratio, charge (z) and relative retention time of the peptides in the sample, to be compared to the values for the modified peptides in the database and thus allows the identification and quantification of the modified peptides in the sample.

There is no limit to the number of samples that can be compared using the method of the present invention.

The method of the present invention is typically used to quantify phosphorylated peptides. In this embodiment, the method of the invention is a technique for targeted and in-depth quantification of signalling (named TIQUAS), which allows for sensitive, rapid and comprehensive quantification of signalling pathway activity. The method can, in one simple assay, simultaneously measure the amounts of thousands of phosphorylation sites on proteins. The method of the invention thus finds use in the analysis of signalling pathways, because the fluxes of signalling pathways are controlled to a great extent by lipid and protein kinases, enzymes which phosphorylate proteins.

The method according to this embodiment of the present invention has advantages over other methods that use mass spectrometry to quantify phosphorylated peptides (see Table 1). These advantages include comprehensiveness (thousands of phosphorylated peptides can be quantified), throughput (the analysis time is about 2 hrs/sample or about 20 samples/day/LC-MS), the ability of the technique to compare an unlimited number of samples, and its superior sensitivity (because quantification does not require identification of peptides by tandem mass spectrometry (MS/MS)). For comparison a SILAC experiment needs about three weeks to compare up to 3 samples; this is because cells need to grow in SILAC media for about two weeks and extensive separation is needed for each experiment.

References for Table 1

1. Olsen, J.V. et al. Cell 127, 635-648 (2006)

2. Cutillas, P.R et al. MoI Cell Proteomics 4, 1038-1051 (2005)

3. Gygi, S.P. et al. Nat Biotechnol 11,, 994-999 (1999) 4. Ross, P.L. et al. MoI Cell Proteomics 3, 1154-1169 (2004)

5. Gerber, S.A. et al. Proc Natl Acad Sci USA 100, 6940-6945 (2003)

6. Barglow, K.T. & Cravatt, B.F. Nat Methods 4, 822-827 (2007)

7. Cutillas, P.R. et al. Proc Natl Acad .Sci USA 103, 8959-8964 (2006)

Table 1

In one embodiment, the present invention provides a method for quantifying phosphorylated peptides in a sample, the method comprising:

(a) obtaining peptides from the sample;

(b) optionally, adding reference phosphorylated peptides to the peptides obtained in step (a) to produce a mixture of peptides and reference phosphorylated peptides and⁵ enriching phosphorylated peptides from the mixture of peptides and reference phosphorylated peptides obtained in step (b) to produce a mixture of enriched phosphorylated peptides;

(c) carrying out mass spectrometry (MS) on said mixture of peptides and reference phosphorylated peptides or said mixture of enriched phosphorylated peptides to obtain data relating to the peptides in the sample; and

(d) comparing the data relating to the peptides in the sample with data in a database of phosphorylated peptides using a computer programme; wherein the database of phosphorylated peptides is compiled by a method comprising: (i) obtaining peptides from a sample;

(ii) enriching phosphorylated peptides from the peptides obtained in step

(0;

(iii) carrying out liquid chrόmatography-tandem mass spectrometry (LC- MS/MS) on the enriched phosphorylated peptides obtained in step (ii); (iv) comparing the phosphorylated peptides detected in step (iii) to a known reference database in order to identify the phosphorylated peptides; and (v) compiling data relating to the phosphorylated peptides identified in step (iv) into a database.

In one embodiment, the present invention provides a method for quantifying phosphorylated peptides in a sample, as set out in Figure 1.

In one embodiment, the present invention provides a method of quantifying phosphorylated peptides in a sample, as follows. The method is referred to herein as TIQUAS. The basis of the TIQUAS technique is the construction of a database of phosphorylated peptides that can be detected and quantified by LC-MS. This database lists all the parameters needed for the quantification of phosphorylated peptides in subsequent biological experiments including the identity of the phosphorylated peptide, mass to charge ratio (m/z), charge, and relative retention time. The database is constructed by enriching phosphorylated peptides using multidimensional chromatography (such as strong cation exchange, IMAC and TiO₂). Fractions of enriched phosphorylated peptides are then analysed by LC-MS/MS for identification of phosphorylated peptides. Constructing the database takes about 2 to 3 weeks of mass spectrometry time but once it is constructed it can be used to perform biological experiments with good throughput and sensitivity.

The inventors have written a computer program named PESCAL (Cutillas and Vanhaesebroeck, Molecular & Cellular Proteomics 6, 1560-1573 (2007)) that automates the quantification of each of the phosphorylated peptides listed in the database in LC-MS runs of phosphorylated peptides taken from biological experiments. For these biological experiments, proteins in cell lysates are digested using trypsin or other suitable proteases. Phosphopeptide internal standards, which are reference phosphorylated peptides, are spiked at known amounts in all the samples to be compared. Phosphorylated peptides in the resultant peptide mixture are enriched using a simple-to-perform IMAC or TiO₂ extraction step. Enriched phosphorylated peptides are analysed in a single LC-MS run of about 120 minutes (total cycle). PESCAL then constructs extracted ion chromatograms (XIC, i.e, an elution profile) for each of the phosphorylated peptides present in the database across all the samples that are to be compared. The program also calculates the peak height and area under the curve of each XIC. The data is normalised by dividing the intensity reading (peak areas or heights) of each phosphopeptide analyte by those of the phosphopeptide ISs.

This phosphoproteomic approach allows the comparison of an unlimited number of samples and replicates.

In another embodiment, the method of the invention is a method for quantifying acetylated peptides in a sample. Quantitative analysis of acetylation involves digesting proteins in a cell lysate with a suitable protease, for example trypsin, to generate thousands of peptides. Acetylated peptides are then enriched by the use of specific antibodies against acetylated amino acid residues. Synthetic peptides containing acetylated amino acids are used as internal standards that are spiked into the samples prior to the enriching step. LC-MS/MS is then used to identify these acetylated peptides, which are then incorporated into a database. Acetylated peptides are identified in individual samples by LC-MS₃ taking the entries in the database as a reference.

The present invention will now be further described by way of reference to the following Examples which are present for the purposes of illustration only. In the Examples, reference is made to a number of Figures in which:

FIGURE 1 is a flowchart illustrating an embodiment of the present invention. Notes to the flowchart are as 'follows: ^l Treatment with phosphatase inhibitors increases the stoichiometry of phosphorylation and results in a greater number of phosphorylated peptides that can be included in the database. ² Proteases are typically trypsin, chymotrypsin, Arg-C, pepsin, V8, Lys-C, Asp-C or AspN. 3 Multidimensional chomatography is typically carried out using strong cation exchange, high performance liquid chromatography (HPLC), immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography or alternatively using anion exchange, high performance liquid chromatography (HPLC), immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography. In another alternative, antibody-based methods can be used. ⁴ Biological samples can be any suitable biological sample and are typically cell lines (optionally treated with investigative agents) or primary tissues from animals or patients.

There is no limit to the number of samples that can be compared. ⁵ The internal standards are reference phosphorylated peptides and are added at a known amount to each of the samples to be compared. The signals of the endogenous phosphorylated peptides are normalised to the signal of the internal standard peptides in downstream analysis. FIGURE 2 shows a strategy for quantitative analysis of cell signalling using TIQUAS in which: (A) shows experimental design; (B) gives a summary of data obtained (4 independent biological replicates), and a snapshot of the data sorted by probability; (C) is an example of extracted ion chromatograms (XIC) of a phosphorylated peptide from protein phosphatase 1 (PPl) regulatory subunit 11; (D) shows examples of phosphorylation sites on proteins previously demonstrated to be downstream of PBK.

FIGURE 3 shows examples of phosphorylated sites identified in the analyses described in Example 1.

FIGURE 4 (A) shows IC50 inhibition of proliferation by PI-103 in cell lines with differing sensitivities to PI-103; and (B) shows the results of quantification of phosphopeptides in the same cell lines using the method of the present invention. The_, table shows phosphopeptides with a positive correlation (high phosphorylation in more resistant cell lines).

FIGURE 5 shows a strategy for accurate phosphopeptide quantification by label-free LC-MS. (A) MS Spectra showing mitogen-activated protein kinase 3 (pERKl) peptide (IADPEHDHTGFLTEEYVATR) at m/z = 751.33939, tR

= 54.54 min, z = 3). (B) The extracted ion chromatogram (XIC) of this m/z ± 25 ppm yields four possible candidates. (C) After applying additional charge and isotope distribution restrictions, the chromatographic elution peak of this peptide can be unequivocally identified (arrowed in the figure) since the XICs for the first three isotopes have relative intensities that correspond to those shown in the spectrum (the relative intensities of the isotopes can also be calculated from sequence data). (D) Further specificity is achieved by narrowing the mass window (± 7 ppm) at which the XIC is constructed. FIGURE 6 shows that AML cell lines with different sensitivity to signaling inhibitors showed markedly different patterns of protein phosphorylation. (A), (B). and (C) AML cell lines were cultured in the presence of the indicated inhibitors for 72 h and survival measured by MTS assay. Data points are shown as the mean ± SEM (n=3) (D), (E) and (F) Examples of phosphopeptides identified b^ TIQUAS to be robustly (as assessed by fold change) and significantly (as assessed by t-test statistics) differentially regulated in cell lines with different sensitivities to the signaling inhibitors named in (A), (B) and (C).

Example 1 - Study on PI3K signalling

The novel quantitative techniques of the invention were used to quantify phosphorylation sites in NIH-3T3 fibroblasts, which were starved and then stimulated with serum with or without pre-incubation with the pan-PI3K inhibitor wortmannin (WM). These experiments were performed 4 times.

Figure 2(A) shows experimental design. Cells were treated as indicated, lysed with a urea-based buffer to obtain nuclear as well as cytosolic proteins and proteins digested. Resultant phosphorylated peptides were enriched by IMAC and TiO₂ chromatography and quantified by LC-MS using a computer program developed by the inventors (PESCAL) (Cutillas and Vanhaesebroeck, Molecular & Cellular Proteomics 6, 1560- 1573 (2007)).

Figure 2(B) gives a summary of the data obtained and a snapshot of the data sorted by probability. 3,100 phosphopeptides were detected in the database, 2,350 phosphorylated peptides were quantified in these analyses, 265 of which were significantly (p<0.05) affected by WM treatment of cells prior to serum stimulation. Further information on the phosphopeptides shown in Figure 2(B) is given in Table 2. Table 2 ts) to

S

Figure 2(C) is an example of extracted ion chromatograms (XIC) of a phosphorylated peptide from PPl regulatory subunit 11 (CCCIpYEKPR; XIC = m/z 683.26), demonstrating that this site is sensitive to WM.

Figure 2(D) shows examples of phosphorylation sites on proteins previously demonstrated to be downstream of PpK. The data of the 4 independent experiments are shown separately to demonstrate the reproducibility of the experiments. These sites are known to be downstream of PBK and thus these data validate the method of the present invention. This phosphoproteomic approach allows the comparison an unlimited number of samples and replicates.

Several sites of phosphorylation were identified on protein kinases and phosphatases, and transcription and translation factors that were affected by WM treatment of cells (Figure 3). Values are the normalized mean phosphorylated peptide levels of 4 independent experiments. The p values were calculated using the student's t test on the means of serum versus WM sample groups. Further information on the phosphopeptides shown in Figure 3 is given in Table 3.

Table 3

Example 2 - Investigation of signalling pathways in cells with a range of sensitivities to a dual inhibitor of PI3K and mTOR

The claimed techniques were also used to profile kinase pathways in a panel of cell lines with a range of sensitivities to PI-103, an inhibitor of the lipid kinase PBK and the protein kinase mTOR.

The cell lines in the panel shown in Figure 4A were incubated with a titration of PI- 103 and their rate of proliferation measured as a function of inhibitor concentration. The inhibitor concentration was expressed as IC50 (concentration of inhibitor giving 50% inhibition) in micromolar units of concentration (Figure 4A).

In a separate experiment (Figure 4B), phosphopeptides in these cell lines were quantified using the disclosed techniques. Experiments were performed three times. As Figure 4B illustrates, several phosphopeptides correlated with the IC50 values, thus suggesting a causal relationship of inhibitor efficacy with kinase pathway activation. These values correlated with statistical significance (R² values above 0.75 were statistically significant). In addition, statistical tests (student's t-test) of the mean phosphopeptide signals of resistant and sensitive cell lines were also significant. These phosphopeptides may represent novel biomarkers of inhibitor efficacy and may also shed light into the mode of action of these inhibitors.

Example 3 - Investigation of kinase pathway activation associated with sensitivity of cancer cells to kinase inhibitors

Analytical strategy

The TIQUAS approach used in this Example consists of using LC-MS for targeting the quantification of phosphopeptides previously identified by LC-MS/MS. This analysis is automated using the computer programme PESCAL described herein (Cutillas, P. R.; Vanhaesebroeck, B. MoI Cell Proteomics 6(9), 1560-73, 2007). PESCAL performs extracted ion chromatograms (XIC) of the first three isotopes of a given molecular ion. This allows both the identification of the charge of the peptide ions and the calculation of the relative intensities of the isotopes that can then be correlated with theoretical isotope distributions.

Figure 5 illustrates the principle of the analysis, which combines high resolution and high mass accuracy mass spectrometry with a new version of Pescal for specific and reliable identification of chromatographic peaks for label-free quantification. Since this is a targeted approach in which samples are analyzed by a single LC-MS/MS run, problems with undersampling in data-dependent experiments are avoided. A feature of the approach is its ability to provide in-depth quantitative information across an unlimited number of samples and replicates with sufficient throughput for its use as a routine phosphoproteomics tool,

TIQUAS analysis of drug response in leukaemia cells

The responses of eight AML cell lines to a panel of inhibitors for kinases in pathways critical for AML development, namely the JAK, MEK and PDK pathways, were investigated. Cells were treated with increasing concentrations of LY294002, PIl 03 and IC87114 (inhibitors that have PI3K as main targets), a MEK inhibitor (MEK I, Calbiochem), a JAK inhibitor (JAK I, Calbiochem) and a FLT3 inhibitor (Calbiochem). These kinases are all potential drug targets for the treatment of diverse forms of cancer; however the mechanisms responsible for making cells susceptible to their inhibition are poorly understood. Proliferation of the cell lines in our panel as a function of these kinase inhibitors showed a wide range of sensitivities, reflecting the clinical situation where patients may respond to cancer drugs to different extents.

The TIQUAS approach was then used to compare basal phosphorylation levels in HEL versus AML-193 (resistant and sensitive to JAK I), P31/FUJ versus MV4-11 (resistant and sensitive to PI- 103), and HEL versus MV4-11 (resistant and sensitive to MEK I); these experiments were performed in triplicate (Figure 6A, B and C). More than 3000 phosphopeptides were identified in these analyses. A total of 1095 phosphopeptides were quantified across these cell lines, from which hundreds showed significant (p < 0.05) and robust (>2 fold) differences between the sensitive and resistant cell lines (Examples of these are shown in Figure 6D, E and F).

MATERIALS AND METHODS Cell Culture. Acute myeloid leukemia cell lines (AML- 193, CMK, CTS, HEL₅ Kasumi-1, KG-1.MV4-11 and P3.UFUJ) and murine NIH/3T3 fibroblasts were routinely cultured at 37°C in a humidified atmosphere of 5% CO₂ in medium supplemented with 10% bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin. AML cell lines were maintained at about 5 to 10 xl O⁵ cell/ml in RPMI with an additional supplement of 50 μM beta-mercaptoethanol. NIH-3T3 fibroblasts were grown in DMEM medium.

For phosphoproteomic studies, AML cells were seeded at 5x10⁵ cells/ml in fresh medium the day before the experiment. Each culture contained 5x10⁶ cells in 10 ml and was performed in three independent cell cultures. When testing the effect of drug inhibitors, the cells were treated with 1 μM PI-103, 50OnM MEK I inhibitor and 50OnM JAK I inhibitor (528100, 444937 and 420099 from Calbiochem, respectively) for 1 hour prior to cell harvesting.

Cells were harvested by centrifugation at 300xg for 10 min, and washed twice with ice cold PBS containing 1 raM sodium vanadate and 1 mM sodium fluoride. Lysis was performed using a denaturing buffer (20 mM HEPES pH 8.0, 8 M urea, 1 mM sodium vanadate, 1 mM sodium fluoride, 2.5 rnM sodium pyrophosphate, ImM β- glycerol-phosphate) at a concentration of 10x10⁶ cells/ml. Further protein solubilization was achieved by sonication. Lysate debris was cleared by centrifugation at 20000xg for 10 min and protein concentration of the supernatants was determined by Bradford assay. Samples were then kept frozen at -80°C until further analysis.

Sensitivity of AML cell lines to drug treatment. Eight cell lines (AML- 193, CMK, CTS, HEL, Kasumi-1, KG-1,MV4-11 and P31/FUJ) were seeded in 96 well plates at 1x10⁵ cell/ml in triplicate for each condition. After a recovery period of 2 h, cells were treated with increasing concentrations (1 nM, 10 nM, 100 nM, 1 μM and 10 μM) of FLT3 inhibitor, MEK I inhibitor, JAK I inhibitor, LY294002, PI- 103 and IC87114. As controls, cells were both treated with the vehicle (DMSO) and left untreated. After 72 hours treatment, cell viability was assessed by MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation assay, Promega Corporation, Madison, WI, USA). Digestion and solid phase extraction.

Sample reduction and alkylation were performed with 4.1 mM DTT and 8.3 mM iodoacetamide in the dark and at room temperature for 15 min each. After diluting the samples to 2 M urea with HEPES pH 8.0, trypsinization was performed using 10 TAME units of immobilised TLCK-trypsin per 5x10⁶ cells for 16 h at 37°C. Digestion was stopped by adding TFA at a final concentration of 1%. The resultant peptide solutions were desalted using Sep-Pak C₁₈ columns (Waters UK Ltd, Manchester, UK) according to manufacturer guidelines. Peptide elution was carried out with 5 ml 50% ACN / 0.1% TFA.

Immobilized Metal Ion Affinity Chromatography (IMAC). Phosphopeptide separation was achieved using an adapted IMAC enrichment protocol (Alcolea, M. P. et al, JProteome Res 8 (8), 3808 (2009)). In short, each sample was incubated for 30 min at room temperature with 300 μl of Fe(III)-coated sepharose high performance beads used as a 50% slurry in 50% ACN / 0.1% TFA. Unbound peptides were discarded and beads washed with 300 μl of 50% ACN / 0.1% TFA twice. The enriched phosphopeptide fraction was eluted with 300 μl ammonia water 1.5%, pH

11. A second elution using 50 μl ammonia water 1.5%, pH 11 containing 50% ACN allowed further enrichment. Eluted peptides were finally dried in a SpeedVac and stored at -80 °C.

N ano flow-liquid chromatography tandem mass spectrometry (LC-MS/MS). For phosphoproteomic experiments, dried phosphopeptide enriched samples were dissolved in 10 μl of 0.1% TFA and analysed in a LC-MS/MS system. The latter consisted of an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Hemel Hempstead, UK) connected online to a nanoflow ultra-high pressure liquid chromatography (nanoAcquity, Waters/Micromass) that delivered a flow rate of 5 μL/min (loading) and 400 nL/min (elution) with an operating back pressure of about 3,000 psi. Separations were performed in a BEH 100 μm x 100 mm column (Waters/Micromass). The mobile phases were solution A, 0.1% FA in LC-MS grade water, and solution B, 0.1% FA in LC-MS grade ACN. Gradient runs were from 1% B to 35% B in 100 min followed by a 5 min wash at 85% B and a 7 min equilibration step at 1% B. Full scan survey spectra (m/z 350-1600) were acquired in the LTQ- Orbitrap XL with a resolution of 60000 at m/z 400. A data dependent analysis (DDA) was employed in which the 5 most abundant multiply charged ions present in the survey spectrum were automatically mass-selected and fragmented by collision- induced dissociation (normalized collision energy 35%). MS scans were followed by 5 MS/MS scans (m/z 50-2000). Dynamic exclusion was enabled with the exclusion list restricted to 500 entries, exclusion duration of 40 seconds and mass window of 10 ppm.

Data Analysis. LTQ-Orbitrap MS/MS data were smoothed and centroided using Mascot Distiller. The processed files were searched against the human or mouse sequence library in the international protein index (IPI Mouse v3.49, 165169 sequences and IPI Human. v3.56, 76539 sequences) using the Mascot search engine. Searches were automated with Mascot Daemon (v2.2.2; Matrix Science, London, UK). The parameters included, choosing trypsin as digestion enzyme with one missed cleavage allowed, carbamidomethyl (C) was set as fixed modification, and Pyro-glu (N-term), Oxidation (M) and Phospho (STY) were variable modifications. Datasets were searched with a mass tolerance of ± 7 ppm and a fragment mass tolerance of ± 800 mmu. Hits were considered significant when they were above the statistical significant threshold (Expectation value < 0.05) (as returned by Mascot). False positive rate as estimated by searches against a decoy database was about 2%. Sites of modification are reported when these were returned by Mascot and were also present in the phosphoELM database. Otherwise the site of modification was deemed to be ambiguous; in such cases phosphopeptides are reported as the start-end residues within the protein sequence.

Phosphopeptides identified by Mascot with a statistical significant threshold were placed in a database of peptides quantifiable by LC-MS. The computer programme PESCAL, as described herein (Cutillas, P.R. & Vanhaesebroeck, B. Quantitative profile of five murine core proteomes using label-free functional proteomics. MoI Cell Proteomics 6, 1560-1573 (2007)) was then used. PESCAL quantifies the intensities of the peptides present in the database across all the samples one wishes to compare. PESCAL uses the m/z and retention time of the selected peptides to construct extracted ion chromatograms (XICs) for the first three isotopes of each ion. This applies restrictions on the molecular mass, retention time and charge, which permits the unequivocal identification of the LC-MS elution profiles corresponding to the studied phosphopeptides. Windows for XIC construction were 7 ppm and 5 min for m/z and retention time, respectively. The intensity values could then be calculated by determining the peak height of each individual XIC.

Claims

1. A method for .quantifying modified peptides in a sample, the method comprising: (a) obtaining peptides from the sample;

(b) adding reference modified peptides to the peptides obtained in step (a) to produce a mixture of peptides and reference modified peptides;

(c) carrying out mass spectrometry (MS) on said mixture of peptides and reference modified peptides to obtain data relating to the peptides in the sample; and

(d) comparing the data relating to the peptides in the sample with data in a database of modified peptides using a computer programme; wherein the database of modified peptides is compiled by a method comprising: i. obtaining peptides from a sample; ii. enriching modified peptides from the peptides obtained in step

(i); iii. carrying out liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the enriched modified peptides obtained in step (ii); iv. comparing the modified peptides detected in step (iii) to a known reference database in order to identify the modified peptides; and v. compiling data relating to the modified peptides identified in step (iv) into a database.

2. A method as claimed in claim 1, wherein the modified peptides are phosphorylated peptides.

3. A method as claimed in claim 1 or claim 2, wherein the sample is a cell line or a primary tissue.

4. A method as claimed in any one of the preceding claims, wherein step (a) comprises:

(1) Iy sing cells in the sample;

(2) extracting the proteins from the lysed cells obtained in step (1); and

(3) cleaving said proteins into peptides.

5. A method as claimed in claim 4, wherein step (3) is carried out using a protease.

6. A method as claimed in claim 5, wherein the protease is selected from the group consisting of trypsin, chymotrypsin, Arg-C, pepsin, V8, Lys-C, Asp-C and AspN.

7. A method as claimed in any one of claims 4 to 7, wherein step (3) comprises cleaving said proteins into peptides of 5 to 30 amino acids.

8. A method as claimed in any one of the preceding claims, wherein step (b) further comprises enriching modified peptides from said mixture of peptides and reference modified peptides to produce a mixture of enriched modified peptides and step (c) comprises carrying out mass spectrometry (MS) on said mixture of enriched modified peptides to obtain data relating to the peptides in the sample.

9. A method as claimed in claim 8, wherein the step of enriching modified peptides is carried out using chromatography.

10. A method as claimed in claim 9, wherein the chromatography is selected from the group consisting of immobilized metal ion affinity chromatography (IMAC), titanium dioxide (TiO₂) chromatography and zirconium dioxide (ZrO₂) chromatography.

11. A method as claimed in claim 8, wherein the step of enriching modified peptides is carried out using antibody-based methods.

12. A method as claimed in any one of the preceding claims, wherein the data relating to the peptides in the sample comprises the mass to charge (m/z) ratio, charge

(z) and relative retention time of the peptides.

13. A method as claimed in any one of the preceding claims, wherein said mass spectrometry (MS) in step (c) is liquid chromatography-mass spectrometry (LC-MS).

14. A method as claimed in any one of claims 4 to 7, wherein step (i) comprises:

(1) lysing cells in a sample;

(2) extracting the proteins from the lysed cells obtained in step (1); and (3) cleaving said proteins into peptides using the same method as in step (3) of claim 4;

15. A method as claimed in any one of the preceding claims, wherein step (ϋ) is carried out using multidimensional chromatography.

16. A method as claimed in claim 15, wherein the multidimensional chromatography is carried out using strong cation exchange high performance liquid chromatography (SCX - HPLC), immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography.

17. A method as claimed in claim 15, wherein the multidimensional chromatography is carried out using anion exchange high performance liquid chromatography (SAX-HPLC), immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography.

18. A method as claimed in any one of claims 1 to 14, wherein step (ii) is carried out using antibody-based methods.

19. A method as claimed in any one of the preceding claims, wherein step (iv) is carried out using the MASCOT search engine.

20. A method as claimed in any one of the preceding claims, wherein the data relating to the modified peptides is selected from the group consisting of identity of the modified peptide, mass to charge (m/z) ratio, charge (z) and relative retention time of the modified peptide.