WO2011128648A1 - Procédé d'identification de peptides quantifiables - Google Patents

Procédé d'identification de peptides quantifiables Download PDF

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Publication number
WO2011128648A1
WO2011128648A1 PCT/GB2011/000586 GB2011000586W WO2011128648A1 WO 2011128648 A1 WO2011128648 A1 WO 2011128648A1 GB 2011000586 W GB2011000586 W GB 2011000586W WO 2011128648 A1 WO2011128648 A1 WO 2011128648A1
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Prior art keywords
peptide
mixtures
samples
peptides
ionic intensity
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PCT/GB2011/000586
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English (en)
Inventor
Pedro Rodriguez Cutillas
Bart Vanhaesebroeck
Pedro Casado Izquierdo
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Queen Mary & Westfield College
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Priority claimed from GBGB1006429.3A external-priority patent/GB201006429D0/en
Priority claimed from GBGB1012805.6A external-priority patent/GB201012805D0/en
Application filed by Queen Mary & Westfield College filed Critical Queen Mary & Westfield College
Publication of WO2011128648A1 publication Critical patent/WO2011128648A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the present invention relates to a method for identifying target peptides for quantification by mass spectrometry (MS), and in particular to identifying target peptides for quantification by liquid chromatography-mass spectrometry (LC-MS).
  • MS mass spectrometry
  • LC-MS liquid chromatography-mass spectrometry
  • Mass spectrometry 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.
  • MS mass to charge
  • 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).
  • ESI electrospray ionization
  • MALDI matrix assisted laser desorption/ionization
  • 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 a combination of these.
  • LC-MS liquid chromatography-mass spectrometry
  • LC-MS assumes that peptide ion intensities are a measure of peptide abundance. However, this assumption does not always hold; it has been shown that, when quantifying proteins, the ionic intensities of just a subset of peptides reflect protein abundance accurately (Malmstrom, J. et al., Nature 460 (7256), 762 (2009); Silva, J. C. et al., Mol Cell Proteomics 5 (1), 144 (2006)), while other peptides, derived from the same protein, poorly reflect protein amounts.
  • the present inventors have devised a method for identifying target peptides that can be quantified accurately using MS.
  • the approach does not require the production of calibration curves using synthetic or recombinant standards and is therefore suitable for high-content and large-scale experiments.
  • the method of the invention involves mixing samples at different proportions prior to MS analysis.
  • the present invention provides a method of identifying a target peptide for quantification by mass spectrometry (MS), comprising: (i) mixing peptides from a pair of samples at y different ratios to produce y mixtures, wherein , y is 2 or greater than 2; (ii) carrying out MS on each of the y mixtures to determine the ionic intensity of a peptide in each of the y mixtures;
  • the present invention provides a method of identifying a target peptide for quantification by mass spectrometry (MS).
  • the present invention provides a method of identifying a target peptide for quantification by mass spectrometry (MS), comprising:
  • a "target peptide for quantification by MS” is a peptide, as defined herein, that can be quantified accurately using MS. Such a peptide is therefore suitable for accurate quantification by MS.
  • peptide ion intensities are a measure of peptide abundance. However, this does not always hold and there is thus a need in the art for a method for determining which peptides can be quantified accurately using MS.
  • the method of the invention is thus useful for identifying peptides that are amenable to quantification by MS.
  • Such peptides are described herein as "proteotypic peptides".
  • Such peptides are also described herein as analytically well-behaved peptides.
  • peptides that are not amenable to quantification by MS can be described as non-proteotypic peptides or analytically badly-behaved peptides.
  • the method of the invention allows the identification of peptides that can be quantified accurately using MS techniques such as LC-MS, and therefore allows the researcher to make decisions regarding data quality for each of the peptides that can be quantified by such techniques.
  • the method of the invention can therefore also be described as a method for determining the accuracy of quantification of peptides by MS.
  • the method of the invention can therefore be used to assess the reliability of data obtained by MS.
  • the method of the invention can be used to identify target peptides for quantification by any method involving mass spectrometry (MS), such as liquid chromatography- mass spectrometry (LC-MS).
  • MS mass spectrometry
  • LC-MS liquid chromatography- mass spectrometry
  • the LC-MS or LC-MS/MS is typically label-free MS.
  • the method of the invention can also be used to identify target peptides for quantification by MS techniques that use isotope labels for quantification, such as metabolic labeling (e.g., stable isotope labeled amino acids in culture, (SILAC); Oisen, J.V. et al Cell 127, 635-648 (2006)), and chemical derivatization (e.g., iTRAQ (Ross, P. L.; et al.
  • metabolic labeling e.g., stable isotope labeled amino acids in culture, (SILAC); Oisen, J.V. et al Cell 127,
  • the method of the invention can be used with LC-MS techniques that measure the intensities of the unfragmented ions or with LC-MS/MS techniques that measure the intensities of fragment ions (such as Selected Reaction Monitoring, SRM).
  • a "peptide” as defined herein is a short amino acid sequence and includes oligopeptides and polypeptides. Typically, such peptides are between about 5 and 30 amino acids long, for example from 6 or 7 to 25, 26 or 27 amino acids, from 8, 9 or 10 to 20 amino acids, from 1 1 or 12 to 18 amino acids or from 1 to 16 amino acids, for example 15 amino acids. However, shorter and longer peptides, such as between about 2 and about 50, for example from about 3 to about 35 or 40 or from about 4 to about 45 amino acids can also be used. Typically, the peptide is suitable for mass spectrometric analysis, that is the length of the peptide is such that the peptide is suitable for mass spectrometric analysis.
  • the length of the peptide that can be analysed is limited by the ability of the mass spectrometer to sequence such long peptides.
  • polypeptides of up to 300 amino acids can be analysed, for example from 50 to 250 amino acids, from 100 to 200 amino acids or from 150 to 175 amino acids.
  • the method of the invention is suitable for identifying both unmodified and modified target peptides for quantification by MS.
  • the method of the invention can be used to simultaneously identify thousands of target peptides, such as modified peptides, for quantification by MS.
  • the method of the invention is useful for identifying modified target peptides for quantification by MS, which peptides contain any modifications that can be detected by mass spectrometry. Such modifications are known in the art.
  • the method of the invention is particularly useful for identifying target phosphorylated peptides, i.e. phosphorylated peptides that can be quantified accurately using MS.
  • the target peptides are phosphorylated peptides.
  • Phosphorylated peptides are those peptides to which a phosphate (P0 4 ) group has been added.
  • 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 identifying target peptides that have been modified by means of other post-translational modifications, for example, acetylation, nitration, glycosylation, methylation and/or lipidation.
  • Step (i) of the method of the invention involves mixing peptides from a pair of samples at y different ratios to produce y mixtures, wherein y is 2 or greater than 2.
  • step (i) of the method of the invention involves mixing peptides obtained from a pair of samples at y different ratios to produce y mixtures, wherein y is 2 or greater than 2.
  • the samples used in the method of the invention can be any samples which contain peptides.
  • the samples are typically biological samples 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 samples used in the method of the present invention can be from any species of interest.
  • the samples used in the method of the invention are from a human or animal.
  • the animal is typically a mammal, for example a rodent such as a mouse, rat or guinea pig, or an ungulate such as a 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 samples used in the method of the invention can also be from other life-forms such as bacteria and yeast.
  • the samples used in the method of the invention are typically 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 samples used in the method of the invention can alternatively be from a plant or fungus or a virus.
  • 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.
  • the biological sample is a cell line or a tissue, typically a primary tissue.
  • the sample can be a tissue from a human or animal.
  • the human or animal can be healthy or diseased.
  • the method of the invention is an in vitro method and therefore does not comprise the step of obtaining a sample from an organism such as an animal.
  • Step (i) of the method of the invention involves mixing peptides from a pair of samples, i.e. 2 samples.
  • step (i) of the method of the invention involves mixing peptides obtained from a pair of samples, i.e. 2 samples.
  • the samples may be the same or different, but are typically different. This allows the comparison of different samples.
  • One embodiment of the invention allows the comparison of samples from more than 2 different sources, or the comparison of more than 2 samples.
  • one of the pair of samples used in the method of the invention is typically a pool of all of the (multiple) samples to be compared.
  • the samples to be compared will be from several different sources.
  • the method of the invention is then carried out in turn with each of the samples from different sources being mixed with the pool of samples at different proportions.
  • the pair of samples used in the method of the invention therefore consists of one sample from a particular (single) source and one sample from the pool of all of the samples (multiple samples).
  • an aliquot of peptides from each of the samples is mixed together to produce a sample pool and an aliquot from each of the individual samples is then mixed at y different ratios with peptides from the sample pool to produce y mixtures.
  • the method of the invention is then repeated for each of the samples, which are typically from different sources.
  • one of the samples can be selected as a "master sample” so that each sample to be analysed is mixed with this "master sample” at different proportions.
  • the pair of samples used in the method of the invention therefore consists of one sample from a particular source and one sample which is the "master sample”.
  • each sample from a particular source is mixed in turn with each other sample from a different source; so if there are samples from 3 different sources, sample 1 is mixed with sample 2 at different proportions and, in a different experiment, with sample 3 at different proportions. In a further different experiment, sample 2 is mixed with sample 3 at different proportions.
  • the pair of samples used in the method of the invention therefore consists of sample 1 and sample 2 in the first experiment, sample 1 and sample 3 in the second experiment and sample 2 and sample 3 in the third experiment. In each case, only 2 samples are used in the method of the invention but the method of the invention is repeated a number of times. The results from all of the experiments can then be compared.
  • Step (i) of the method of the invention involves mixing peptides from a pair of samples at different ratios to produce y mixtures, wherein y is 2 or greater than 2.
  • step (i) of the method of the invention involves mixing peptides obtained from a pair of samples at y different ratios to produce y mixtures, wherein y is 2 or greater than 2.
  • the 2 samples are therefore mixed together in various different proportions to produce various different mixtures.
  • y is at least 3 and the peptides from the pair of samples are therefore mixed in at least 3, typically in 4 or 5 different ratios, or even in 6, 7, 8, 9, or 10 different ratios. It is preferable to use a greater number of different ratios. Any different ratios can be used.
  • y is 3 and the peptides from the samples are mixed at ratios of 100:0, 50:50 and 0: 100. In another embodiment, y is 4 and the peptides from the samples are mixed at ratios of 100:0, 60:40, 40:60 and 0: 100. In another embodiment, y is 5 and the peptides from the samples are mixed at ratios of 100:0, 70:30, 50:50, 30:70 and 0: 100. Put another way, with samples A and B, the samples are mixed in the following proportions: 100% A and 0% B, 70% A and 30% B, 50% A and 50% B, 30% A and 70%) B, 0% A and 100% B.
  • y is 5 and the peptides from the samples are mixed at ratios of 100:0, 80:20, 50:50, 30:70 and 0:100.
  • samples A and B the samples are mixed in the following proportions: 100% A and 0% B, 80% A and 20% B, 50% A and 50% B, 30% A and 70% B, 0% A and 100% B.
  • any different ratios can be used.
  • the peptides from the samples can be mixed by any suitable method known in the art. Mixing typically involves simply adding the samples or peptides (pair of samples or pair of peptides) to the same vessel and, optionally, agitating or stirring the resulting mixture.
  • the pair of samples are mixed at y different ratios and then peptides are obtained from the resulting y mixtures.
  • the y mixtures are mixtures of samples.
  • the peptides are obtained from the pair of samples and then the peptides are mixed at y different ratios to produce y mixtures.
  • the y mixtures are mixtures of peptides.
  • the steps of obtaining the peptides from the samples and mixing the peptides or samples can be carried out in either order.
  • the samples are mixed prior to the peptides being obtained from the samples.
  • Peptides can be obtained from the samples using any suitable method known in the art.
  • the cells in the samples 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.
  • the cells are lysed using a denaturing buffer such as a urea-based buffer.
  • proteins are extracted from the lysed cells.
  • the proteins are separated from the other components of the lysed cells. Protein solubilisation can be achieved by sonication.
  • the method of the invention is typically used to identify target peptides which are derived from longer proteins and thus peptides are typically obtained from the samples by breaking down longer proteins in the samples into shorter peptides. Protein breakdown is also commonly referred to as digestion. Thus, in one embodiment, peptides are obtained from the samples by digestion of proteins in the samples. Protein digestion can be carried out in the present invention using any suitable agent known in the art. ⁇
  • Protein digestion is typically carried out using a protease.
  • Any suitable protease can be used in the present invention.
  • the protease is typically trypsin, chymotrypsin, Arg-C, pepsin, V8, Lys-C, Asp-C and/or AspN.
  • 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 described above are typically suitable for mass spectrometric analysis.
  • such peptides are between about 5 and 30 amino acids long, for example from 6 or 7 to 25, 26 or 27 amino acids, from 8, 9 or 10 to 20 amino acids, from 11 or 12 to 18 amino acids or from 14 to 16 amino acids, for example 15 amino acids.
  • shorter and longer peptides such as between about 2 and about 50, for example from about 3 to about 35 or 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, for example from 50 to 250 amino acids, from 100 to 200 amino acids or from 150 to 175 amino acids.
  • step (i) of the method of the invention comprises the following steps:
  • step (l) extracting the proteins from the lysed cells obtained in step (l);
  • the cells in the samples 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.
  • the cells are lysed using a denaturing buffer such as a urea-based buffer.
  • step (2) of this embodiment of the invention proteins are extracted from the lysed cells obtained in step (1).
  • the proteins are separated from the other components of the lysed cells. Protein solubilisation can be achieved by sonication.
  • the proteins from the lysed cells are mixed at y ratios to produce y mixtures.
  • the proteins are typically mixed simply by combining the chosen ratios of proteins from each sample to arrive at the y mixtures.
  • the cell lysates from the lysed cells are mixed at a range of different proportions.
  • step (4) of this embodiment of the invention the proteins in the y mixtures are cleaved into peptides.
  • the proteins are digested using a protease, for example trypsin.
  • Step (i) of the method of the invention optionally further comprises a step of enriching peptides from each of the y mixtures. This step is typically used when the target peptide is a phosphorylated peptide.
  • the step of enriching peptides is typically carried out using chromatography.
  • the chromatography is immobilized metal ion affinity chromatography ( AC), for example the adapted AC enrichment protocol described in Alcolea, M. P. et al., J Proteome Res 8 (8), 3808 (2009).
  • AC immobilized metal ion affinity chromatography
  • Other types of chromatography can alternatively be used, such as titanium dioxide (Ti0 2 ) chromatography, and/or zirconium dioxide (Zr0 2 ) chromatography.
  • the step of enriching peptides is carried out using antibody-based methods.
  • the target peptides 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.
  • 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 immobilised antibody.
  • Step (ii) of the method of the invention involves carrying out MS on each of the y mixtures to determine the ionic intensity of a peptide in each of the y mixtures. MS is therefore carried out y times when using the method of the invention to identify target peptides. MS is carried out to determine the ionic intensity of at least one peptide in each of the y mixtures.
  • step (ii) of the method of the invention involves carrying out MS on each of the y mixtures to determine the ionic intensity of multiple peptides in each of the y mixtures.
  • the MS is LC-MS and/or LC-MS/MS.
  • the peptides are detected by LC-MS/MS, for example nanoflow LC-MS/MS, and then quantified by LC-MS to give the ionic intensity of the peptide.
  • ionic intensity or "ion intensity” means the intensity value of recorded ions.
  • the intensity value represents the signal intensity of the ions recorded by MS, in other words the intensity of ion current recorded by the mass spectrometer. Typically, this value is given by MS software. This value is recorded by the mass spectrometer for each ionized molecule. This typically happens after mass analysis, detection and amplification of each ionized molecule. Mass analysis takes place in the analyzer part of the mass spectrometer, detection is carried out by the ion detector of the mass spectrometer, and amplification is carried out in devices such as electron multipliers.
  • the present invention finds use in identifying target peptides for quantification by the TIQUAS technique, as described in UK patent application no. 0906698.6 (International patent application no. PCT/GB2010/000770, published as WO 2010/1 19261) and in Alcolea et al (in press).
  • MS/MS data can be processed, for example smoothed and centroided, using a programme such as Mascot Distiller.
  • the processed files can then be searched against a database of known peptides, for example using the Mascot search engine.
  • Phosphorylated peptides identified by Mascot with a statistical significant threshold can then be placed in a database of peptides quantifiable by LC- MS.
  • a computer programme can be used to quantify the ionic intensity of a peptide.
  • the computer programme is PESCAL (Cutillas, P. R.; Vanhaesebroeck, B. Mol Cell Proteomics 6(9), 1560-73, 2007).
  • PESCAL quantifies the intensities of the peptides present in the database across all the samples.
  • 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.
  • Step (iii) of the method of the invention involves calculating the relative ionic intensity of said peptide (or peptides if multiple peptides are being identified) in each of the y mixtures by comparing the ionic intensity of said peptide in each of the y mixtures to the maximum ionic intensity of said peptide across all of the y mixtures. A value is therefore obtained for each peptide in each of the y mixtures by dividing the ionic intensity value of the peptide in each of the y mixtures by the maximum ionic intensity of the peptide across all of the y mixtures. This has the effect of normalising the ionic intensity value of the peptide in each of the y mixtures, for example to a value of 100.
  • the relative intensity of each peptide in each of the y mixtures is a value from 0 to 100. This is then correlated with the ⁇ 31 3 ⁇ 4 ⁇ in composition of the y mixtures in step (iv), as described herein.
  • Step (iv) of the method of the invention involves correlating the relative ionic intensity of said peptide (or peptides if multiple peptides are being identified) in each of the y mixtures with the change in composition of the y mixtures.
  • the relative ionic intensity value for the peptide is plotted on a graph against the change in composition of the y mixtures.
  • the relative intensity of each peptide in each of the y mixtures is a value from 0 to 100, y is 5 and the peptides from the samples are mixed at ratios of 100:0, 70:30, 50:50, 30:70 and 0:100.
  • the relative intensity values are plotted on the y axis of a graph, and the ratios are plotted on the x axis of a graph.
  • the relative ionic intensity of said peptide in each of thejy mixtures is then correlated with the change in composition of the y mixtures.
  • said peptide is a target peptide for quantification by MS.
  • the relative ionic intensity of the peptide does not necessarily need to be exactly directly proportional, and the skilled person can determine using his or her judgement based on the results of the data analysis as to whether the proportionality is sufficient to identify a target peptide as suitable for quantification by MS.
  • the relative ionic intensity of said peptide is essentially directly proportional to the change in composition of the y mixtures, then said peptide is a target peptide for quantification by MS.
  • Directly proportional is defined herein as the relative ionic intensity of a peptide showing a correlation coefficient close to 1.
  • An appropriate correlation coefficient is Pearson's correlation coefficient, but other mathematical ways of calculating correlation exist and can be used in the present invention. Such methods would be known to a person skilled in the art. researchers may choose an appropriate correlation coefficient cut off as being significant; this is in analogy to choosing a p value in other statistical tests. In some cases, a correlation coefficient greater than 0.99 can be considered as a cutoff value to define direct proportionality. In some other cases, a cutoff of, for example, 0.60, 0.75, or 0.80, suitably 0.81 may be chosen.
  • %CV percentage confidence of variations
  • the linear regression function is typically used to derive objective measures of accuracy for each peptide.
  • Pearson's correlation coefficients PCC
  • percentage of accuracy Figure I B
  • a Fold Coefficient Fc
  • RI in Figure 1A the change in relative ionic intensity of each peptide
  • PE in Figure 1A the change in relative ionic intensity of each peptide
  • Fc is taken as the slope of the linear regression function and ranges from 0 (no differences in abundance of the phosphorylated peptide between the samples being compared) to 1 (infinite fold change). Fc is a useful measure of differences between samples because it avoids divisions by zero, which happens when a peptide is not detected in one of the samples, or by a negative number, which would in some instances occur if the intercept of the linear regression is taken as the denominator.
  • R2 Pearson correlation coefficient
  • the present invention can be used to simultaneously identify multiple target peptides for quantification by MS. This is particularly the case where high-thoughput methods are used, such as the PESCAL based method described herein and used in the Examples. This finds particular use where the target peptide is a phosphorylated peptide .
  • the present invention finds use in the comparison of different samples. The method of the invention can be repeated a number of times to compare the peptides in different samples. In this embodiment, the present invention finds use for example in the comparison of different samples that are treated with different test substances. In some embodiments of the invention therefore, 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.
  • 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.
  • the test substance is typically an exogenous chemical or drug, such as small molecule inhibitors, RNAi, therapeutic peptides, and antibodies.
  • 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 src and phosphoinositide 3 -kinase (PI3K), such as PP2 and PI- 103, as used in the Examples.
  • Other inhibitors of PI3K include wortmannin.
  • 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.
  • the present invention can also be used to quantify both unmodified and modified peptides. This aspect of the invention finds use particularly for the quantification of peptides present in complex mixtures.
  • Quantification in proteomics typically refers to relative quantification. In other words, the abundance of a particular peptide in sample A is compared to the abundance of the same peptide in sample B. These values (e.g., fold difference of a particular peptide between the two samples) can be taken directly from the regression function or from the actual data.
  • the method of the first aspect of the invention further comprises:
  • the method of the invention can be described as a method of concurrently quantifying a peptide and identifying a target peptide for quantification by mass spectrometry (MS).
  • MS mass spectrometry
  • the present invention therefore provides a method of concurrently quantifying a peptide and identifying a target peptide for quantification by mass spectrometry (MS), comprising:
  • a method of concurrently quantifying a peptide and identifying a target peptide for quantification by mass spectrometry (MS), comprising:
  • Relative quantification is therefore carried out by dividing the intensities of the peptides in one sample by the intensities of peptides in the other sample so that a fold difference between the intensities of the peptides in samples is reported.
  • Another measure of quantification is the Fc.
  • relative quantification and accuracy are determined concurrently, and so the method allows the researcher to obtain relative quantification of peptides, typically modified peptides such as phosphorylated peptides, and simultaneously assess the accuracy of such quantifications.
  • the peptide is typically a modified peptide, typically a phosphorylated peptide.
  • the mean value of intensities of only the proteotypic peptides is selected. This is particularly useful for proteins that generate a low number of peptides upon digestion because in this case it is difficult to exclude outliers based on the standard deviation.
  • Preferred features for the second aspect of the invention are as for the first aspect mutatis mutandis.
  • the present inventors have devised an LC-MS approach for high-content comparison of phosphoproteomes in which fold change and measures of accuracy are determined concurrently. Samples to be compared are mixed at different proportions prior to analysis; fitting of the experimental data into the resultant linear regression function allows calculating objective values of accuracy for each identified phosphorylated peptide.
  • the present invention thus provides a method for measuring linearity and accuracy of quantification for each of the thousands of phosphorylated peptides that can be detected by LC-MS/MS.
  • calibration curves can only be constructed for a limited number of analytes because this entails synthesising standards for each of the compounds one wishes to quantify. Therefore this approach is not suitable for high-content/large-scale experiments, such as in phosphoproteomics, in which the aim is to provide a comprehensive read-out of signal pathway activation and wiring of kinase networks.
  • the present invention therefore provides a solution to this problem, as it does not require labelled standards or the production of calibration curves.
  • the method of the present invention for determining linearity and accuracy of quantitative phosphoproteomic data involves mixing the cell lysates of samples to be quantified at different proportions prior to digestion, enrichment of phosphorylated peptides and LC-MS analysis (Figure 1A).
  • the present invention therefore provides a method of identifying a target peptide for quantification by MS, as set out in Figure 1 A.
  • the present invention provides a method of identifying a target modified peptide for quantification by mass spectrometry (MS), comprising:
  • a method of identifying a target modified peptide for quantification by mass spectrometry comprising:
  • the present invention provides a method of identifying a target phosphorylated peptide for quantification by mass spectrometry (MS), comprising: (i) mixing phosphorylated peptides from a pair of samples at y different ratios to produce y mixtures, wherein .y is 2 or greater than 2;
  • a method of identifying a target phosphorylated peptide for quantification by mass spectrometry comprising:
  • FIGURE 1 shows a scheme for simultaneous calculation of fold changes and accuracy in high-content phosphoproteomics experiments
  • A P31 ⁇ Fuj and kasumi-1 AML cell lines were lysed and the cell lysates were mixed at different proportions and digested. After IMAC enrichment, phosphorylated peptides were quantified by LC-MS/MS and the relative intensity of each phosphorylated peptide (RI) correlated with the proportion of cell extracts (PE).
  • RI relative intensity of each phosphorylated peptide
  • PE proportion of cell extracts
  • B Analysis of correlated data allows calculating linearity (correlation coefficient, R2), fold of change (Fold), fold coefficient (Fc) and accuracy CV (% Acc).
  • P31/Fuj31 and Kasumi-1 cells were treated with vehicle (DMSO), 1 ⁇ PI 103 or 10 ⁇ PP2 for 72 h and the relative number of cells was measured.
  • FIGURE 2 demonstrates the identification and quantification of proteotypic phosphorylated peptides in AML cell lines with different sensitivity to kinase inhibitors.
  • Representative examples of phosphorylation sites differentially regulated in P31/Fuj and Kasumi-1 AML cell lines are shown grouped by the known function of the phosphoprotein. Table shows the gene name of the phosphoprotein followed by the position of the phosphorylation site (in cases when the position was ambiguous the start-end residues within the protein sequence are reported) and a heat map of normalized phosphorylated peptide intensities as a function of the proportion of cells in the mixture. Definitions of R2, Fc, Fold and accuracy are as in Figure 1.
  • FIGURE 3 demonstrates the quantification of unmodified peptides using the method of the present invention.
  • Fuji and Kasumi acute myeloid leukaemia (AML) cell lines were routinely grown in RPMI medium supplemented with 10% FBS, 100 units/mL of Penicillin/Streptomycin and b-Mercaptoethanol at 37°C in a humidified atmosphere at 5% C0 2 . Cells were maintained at about 0.5 to 2 xlO 6 cells/mL.
  • P31/Fuj and Kasumi-1 AML cell lines were seeded in 96 well plates at lxlO 4 cell/ml 5 times for each condition. After a recovery period of 24 h, cells were treated with 1 ⁇ PI- 103, 10 ⁇ PP2 or vehicle (DMSO). After 72 h treatment, cell viability was assessed by MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation assay, Promega Corporation, Madison, WI, USA).
  • Immobilized metal ion affinity chromatography IMAC
  • Enrichment of phosphorylated peptides was achieved using an adapted IMAC enrichment protocol (Alcolea, M. P. et al., J Proteome Res 8 (8), 3808 (2009)).
  • IMAC enrichment protocol Alcolea, M. P. et al., J Proteome Res 8 (8), 3808 (2009).
  • each sample was incubated for lh at room temperature with 300 ⁇ 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 sequentially washed with 300 ⁇ of 50% ACN / 0.1% TFA and 300 ⁇ of 50% ACN / 1% TFA.
  • beads were incubated twice with 300 ⁇ 50% ACN / 1.5% ammonia water pH 1 1 for 5 min. Recovered peptides were acidified by the addition of 10% FA, dried in a SpeedVac and stored at -80 °C.
  • 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 Orbitrap 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 thei surve y S p ec trum were automatically mass-selected, fragmented by collision-induced dissociation (normalized collision energy 35%) and analysed in the LTQ.
  • DDA data dependent analysis
  • Full- 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 mouse sequence library in the international protein index (IPI Mouse v3.49, 165169 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 two 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.
  • Phosphorylated peptides identified by Mascot with a statistical significant threshold were placed in a database of peptides quantifiable by LC-MS.
  • PESCAL Cutillas, P. R.; Vanhaesebroeck, B. Mol Cell Proteomics 6(9), 1560-73, 2007
  • This program written in-house for the automation of label-free LC- MS data analysis, quantifies the intensities of the peptides present in the database across all the samples.
  • 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.
  • Phosphorylation was measured in Kasumi-1 and P31/Fuj, two acute myeloid leukaemia (AML) cell lines exhibiting strikingly different sensitivities to PP2 and PI- 103, two kinase inhibitors having src and phosphoinositide 3 -kinase (PI3 ) as main targets, respectively ( Figure 1C).
  • Figure I D shows examples of phosphorylation sites that could be accurately quantified in these analyses
  • Figure 2 shows examples of phosphorylation sites on kinases and transcription factors that were differentially regulated in these cells and which correlate with the sensitivity of these AML cells to signalling inhibitors. Because these phosphorylations were proportional to the amount of cells in the mixture - and it was possible to measure levels of accuracy and correlation - these differences in phosphorylations - as given by Fc and fold changes - between samples appeared to be robust and accurate.
  • Extracts from Fuji (P31/Fuj) and Kasumi- 1 cell lines were mixed at different proportions as shown in Figure 3. Proteins present in these mixtures were then digested with trypsin. The resultant peptide mixture was desalted using solid phase extraction (Oasis cartridges, Waters) and peptides analysed by LC-MS/MS. The identity of peptides was determined from MS/MS spectra by Mascot searches against the IPI Human protein database. Quantification was with Pescal software. Columns display the name of the gene product followed by the start-end of the peptide within the amino acid sequence. Expectancy refers to the expectancy value as returned by Mascot (which is related to the statistical probability that the identification was not correct).
  • the next five columns display the percentages of Kasumi cell extracts in the mixture (0, 20, 50, 70 and 100%, with the balance being from P31/Fuji cells).
  • R2 is the Pearson correlation coefficient between the proportion of Kasumi cell extracts and the quantitative values.
  • Fc is the fold coefficient.
  • Fold log2 is the fold difference in intensity between peptides present in Kasumi and Fuji cells in log2. %Accuracy refers to how much the experimental values of intensity deviate from those calculated from the linear regression function.

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Abstract

La présente invention concerne un procédé d'identification d'un peptide cible pour la quantification par spectrométrie de masse (MS), comprenant : (i) le mélange de peptides provenant d'une paire d'échantillons à y rapports différents pour produire y mélanges, dans lequel y est 2 ou plus que 2; (ii) réalisation de MS sur chacun des y mélanges pour déterminer l'intensité ionique d'un peptide dans chacun des y mélanges; (iii) calcul de l'intensité ionique relative dudit peptide dans chacun des y mélanges en comparant l'intensité ionique dudit peptide dans chacun des y mélanges à l'intensité ionique maximale dudit peptide parmi tous les y mélanges; et (iv) corrélation de l'intensité ionique relative dudit peptide dans chacun des y mélanges avec le changement de composition des y mélanges; dans lequel si l'intensité ionique relative dudit peptide est directement proportionnelle au changement de composition des y mélanges, ledit peptide est un peptide cible pour la quantification par MS.
PCT/GB2011/000586 2010-04-16 2011-04-15 Procédé d'identification de peptides quantifiables WO2011128648A1 (fr)

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Citations (4)

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WO2002097703A2 (fr) * 2001-05-30 2002-12-05 Andrew Emili Base de donnees pour profil d'expression de proteine
US20070048752A1 (en) * 2004-07-12 2007-03-01 Applera Corporation Mass tags for quantitative analyses
EP2017622A1 (fr) * 2007-07-19 2009-01-21 DKFZ Deutsches Krebsforschungszentrum, Stiftung des Öffentlichen Rechts Procédé de détermination de la concentration d'une molécule
WO2010119261A1 (fr) 2009-04-17 2010-10-21 Queen Mary & Westfield College Procédé de quantification de peptides modifiés

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2002097703A2 (fr) * 2001-05-30 2002-12-05 Andrew Emili Base de donnees pour profil d'expression de proteine
US20070048752A1 (en) * 2004-07-12 2007-03-01 Applera Corporation Mass tags for quantitative analyses
EP2017622A1 (fr) * 2007-07-19 2009-01-21 DKFZ Deutsches Krebsforschungszentrum, Stiftung des Öffentlichen Rechts Procédé de détermination de la concentration d'une molécule
WO2010119261A1 (fr) 2009-04-17 2010-10-21 Queen Mary & Westfield College Procédé de quantification de peptides modifiés

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