WO2008148645A1

WO2008148645A1 - Separation of mono- from multi-phoshorylated peptides

Info

Publication number: WO2008148645A1
Application number: PCT/EP2008/056308
Authority: WO
Inventors: Martin R Larsen; Tine E Thingholm
Original assignee: Syddansk Universitet
Priority date: 2007-06-07
Filing date: 2008-05-22
Publication date: 2008-12-11

Abstract

The present invention relates to a new method for large scale phosphoproteome analysis in which the mono-phosphorylated peptides are separated from the multiply phosphorylated peptides. This method involves IMAC alone or IMAC in combination with TiO2 chromatography. The present inventors have found that mono-phosphorylated peptides can be separated from multiply phosphorylated peptides by IMAC alone or in combination with TiO2 chromatography. The mono-phosphoryated peptides are eluted off the IMAC material with an acidic solution having a pH of from about 0.7 to about 1.0, such as 1 % TFA, and the multiply phosphorylated peptides are subsequently eluted using an alkaline reagent, such as ammonia water (pH 11), an acidic solution having a pH of less than 0.7 or a chelating agent, such as EDTA, with high affinity for the metal ions onto which the phosphomolecules are bound.

Description

SEPARATION OF MONO- FROM MULTI-PHOSPHORYLATED PEPTIDES

FIELD OF THE INVENTION

The present invention relates to a new method for large scale phosphoproteome analysis in which the mono-phosphorylated peptides are separated from the multiply phosphorylated peptides. This method involves IMAC alone or IMAC in combination with TiO₂ chromatography.

BACKGROUND OF THE INVENTION

Reversible protein phosphorylation is an important post-translational modification in most biological processes that takes place in the cell. It plays a key role in cellular communication and signalling, in metabolism, homeostasis, transcriptional and translational regulation etc. (Graves, 1999). Many proteins carry several consensus sites for phosphorylation, indicating a high level of multi-phosphorylation of proteins in biological processes, e.g. phosphorylation is used in the cell to fine-tune regulatory mechanisms, influence protein-protein or protein-nucleic acid interactions or for receptor desensitization (REF). However, not many studies have been performed on elucidating the biological significance of multi-phosphorylation in biological processes due to the highly challenging task of characterizing multi-phosphorylated proteins.

Given the important role of phosphorylation in signal transduction pathways, analysis of phosphorylation events that occur within the entire complement of proteins expressed by cells (phosphoproteome analysis), is useful for understanding a range of cellular processes. Phosphoproteome analysis likely will reveal insight into complex biological processes, such as differentiation, growth control and regulated cell death.

Accordingly, phosphoproteome analysis is expected to contribute to development of diagnostic and prognostic tests, improve aspects of clinical trials, and provide indications of drug safety and efficacy during drug development.

One challenge in the field of phosphoproteome analysis is developing accurate methods for global evaluation of protein phosphorylation levels. Global analysis of protein phosphorylation is an analytical challenge because signaling phosphoproteins are typically present in low abundance within cells. Analytical methods that improve global analysis of protein phosphorylation can contribute to development of medical tests, such as tests that can simultaneously test for multiple phosphoprotein biomarkers. This type of test is expected to be helpful for detecting diseases and conditions for which single diagnostic markers are unfeasible or unavailable.

Thus, the ability to detect and/or isolate phosphoproteins is useful for cellular research as well as medical test development, given the central role of phosphorylation in many disease processes. Improved approaches for phosphomolecule isolation and detection would accelerate protein phosphorylation global analysis and related general and biomedical phosphomolecule research.

Numerous methods are available for characterization of phosphorylated proteins including specific antibodies against serine, threonine or tyrosine phosphorylation (Grønborg, 2002 and Pandey, 2000), two-dimensional gel electrophoresis in combination with ^32/33P labeling (Guy, 1994) or phosphorylation specific fluorescent labeling (REF) as well as amino acid sequencing in combination with ³²P labeling or fluorescent beta-elimination labeling (REF). Due to its unique sensitivity and accuracy, mass spectrometry (MS) has recently become the method of choice for the analysis of phosphorylated proteins/peptides. However, due to the large dynamic range of most complex biological samples and due to the lower ionization efficiency of phosphorylated peptides, signals from these peptides often tend to be suppressed by signals from the more abundant non-phosphorylated peptides. This statement has recently been challenged by (Hanno Sten et al), however, their study was performed using non-tryptic peptides whereas most proteomics studies are performed on tryptic peptides where most peptides only carry one basic amino acid residue. To some degree the suppression effect can be decreased by fractionation of the peptide sample using liquid chromatography (LC)-tandem MS or Strong Ion Exchange chromatography (SA/CX) (Beausoleil, 2004). However, when dealing with complex biological samples "shot-gun" proteomic analysis rarely provide knowledge on the phosphorylation status of proteins unless significant amount of material is used.

Phosphopeptide enrichment prior to MS analysis is essential for large scale phosphoproteomics studies. A widely used enrichment technique for phosphorylated peptides is the use of metal ions for the binding of the negatively charged phosphopeptides i.e., Immobilized Metal ion Affinity Chromatography, IMAC. IMAC was introduced to the characterization of phosphorylated proteins by Andersson and Porath (Andersson and Porath, 1986) and later this technique has been used extensively for enrichment of phosphorylated peptides prior to mass spectrometric analysis (Neville, et al., 1997; Figeys, et al., 1998; Posewitz and Tempst, 1999; Li, et al., 1999; Stensballe, et al., 2001 ; Nϋhse, et al., 2003).

The IMAC technique significantly improves identification of phosphopeptides from complex biological mixtures (Figarro, 2002; Nuhse, 2003; Gruhler, 2005). However, non-phosphorylated peptides containing multiple acidic amino acid residues co-purify with the phosphopeptides in IMAC. O-methyation of these residues have been suggested, but this step may introduce unwanted side reactions (Seward, et al., 2004) and losses of peptides due to extesive lyophilizaiton (Speicher, et al., 2000).

A common effect of IMAC is that it seems to have a stronger selectivity towards multiply phosphorylated peptides in biological buffers (Figarro 2002), and a buffer exchange is therefore in many cases introduced prior to IMAC (White FW) with high risk of loosing phosphopeptides (Larsen MR et al., 2003). Titanium dioxide (TiO₂) chromatography has proven to be an efficient alternative to IMAC (Pinkse et al, 2004; Larsen et al, 2005). It has a higher selectivity for phosphorylated peptides than IMAC and unspecific binding from non-phosphorylated peptides can be significantly reduced by including 2,5-dihydroxybenzoic acid (DHB), phthalic acid or glycolic acid and high concentration of TFA in the loading buffer (Larsen et al, 2005; Thingholm et al, 2006; Larsen et al, 2007). Furthermore, TiO₂ chromatography of phosphorylated peptides is extremely tolerant towards most biological buffers (Larsen et al, 2007).

In contrast to IMAC the present inventors have not observed any bias towards mono- phosphorylated or multiply phosphorylated peptides using the TiO₂ chromatography. For this reason mono-phosphorylated peptides are predominantly identified in large scale phosphoproteomics experiments as the multiply phosphorylated peptides are suppressed in the MS analysis. In addition, most mass spectrometers are only able to perform a limited number of tandem MS experiments in a given time period and will therefore in most cases only fragment the abundant mono-phosphorylated peptides in a complex mixture. Furthermore, the fragmentation of phosphorylated peptides is biased to mono-phosphorylated peptides as the loss of the phosphate group(s) is the predominant fragmentation pathway for phosphopeptides. In practise this indicates that the more phosphate groups that are in a peptide the lower the number of backbone cleavages will be. The fragmentation of phosphorylated peptides can be improved significantly by using phosphorylation directed multistage tandem MS (pdMS³) (REF), where the neutral loss signal detected in the MS² is selected for a second round of CID (MS³) to obtain more sequence information. However, his strategy is favors the analysis of mono-phosphorylated peptides as multiply phosphorylated peptides will subsequently lose another phosphoric acid.

Alternatively, electron capture/transfer dissociation (ECD/ETD) can be applied for multiply phosphorylated pepitdes as these methods primarily result in peptide backbone fragmentation without concomitant loss of the phosphate group (Chalmers, 2004 and Schroeder, 2005). However, these methods are still not routine in most research groups.

In Larsen et al, 2005 (see above) a selective enrichment of phosphorylated peptides from peptide mixtures is disclosed, wherein the titanium dioxide affinity chromatography is used. As briefly mentioned above this method relies on the use of DHB to obtain a reduction in the binding of nonphosphorylated peptides to TiO2 and the resulting purified peptides are analyzed/characterized by MS. The enrichment method disclosed is described as an alternative to IMAC and was found to be superior in terms of selectivity and sensitivity of phosphorylated peptide binding. It may be noted that in LC-ESI-MSMS a bias for monophosphorylated peptides has been observed and it is recommended to use also MALDI-MSMS which does not appear to have the same bias.

In order to better understand the impact and perspectives of the present invention notice should be taken of the following prior art literature.

In a recent review article authored by Rossignol et al (Current Opinion in Plant Biology, 2006, 9:538-543) several technologies for phosphopeptide selection/fractionation such as IMAC, TiO2 and ion chromatography are discussed. The review is, however, silent with respect to a selection strategy for phosphopeptides employing both IMAC to separate mono- and poly- phosphorylated peptides.

Klemm et al (J Mass Spectrometry 2006, 41 , 1623-1632) discloses methods employing TiO2 chromatography and MS for analysis of polypeptide. The disclosure does not relate to a method for enrichment and analysis of phosphorylated peptides/proteins employing IMAC. Furthermore, the method does not rely on separation of mono- and poly-phosphorylated peptides. Ndassa et al (J Proteome Res 2006, 5, 2789-2799) describe an improved IMAC for large-scale proteomics applications. One aspect under investigation was a previously reported bias against singly phosphorylated peptides in IMAC-based phosphopeptide enrichment. It is suggested that an overall phosphopeptide enrichment by IMAC may be followed by strong cation exchange (SCX) to fractionate single and multiply phosphorylated peptides. Although the disclosure considers shortcomings of IMAC in relation to a bias for multiple-phosphorylated peptides over monophosphorylated peptides and suggests that a combination with SCX may be useful, there appear to be no mentioning of a method according to the present invention.

Moser et al (J Proteome Res. 2006, 5, 98-104) discloses phosphoproteomic analysis by high capacity IMAC and LC-MS/MS. The method disclosed therein does not rely on separation of mono- and poly-phosphorylated peptides. It may be noted, however, that an increase in the IMAC column capacity appeared to decrease the bias for polyphosphorylated peptides as compared to mono-phosphorylated.

Raggiaschi et al (Bioscience Reports 2005, 25) review recent trends of phosphoproteome analysis and technologies therefor. The methods disclosed therein are not aimed at separation of mono- and poly-phosphorylated peptides.

EP1477800A1 describes a method for analysing an amino acid, peptide, protein, sugar, or lipid comprising the use of reverse phase HPLC with pre-treatment column of TiO2. The analysis can subsequently be carried out by MS. The disclosure does not relate to a method involving IMAC and furthermore appears not to be aimed at separating mono- from poly-phosphorylated proteins.

WO 2004/01 1902 A2 discloses methods for identifying modified amino acids within a protein by combining affinity purification and mass spectrometry. In one aspect of the invention a method for analyzing a prosphoproteome is provided. This method comprises chemically modifying the side chains of glutamic acid and aspartic acid residues to neutral derivatives, and subsequently isolating the phosphorylated proteins by IMAC and analysing the phosphorylated proteins by MS. The disclosure is not aimed at separating mono- from poly-phosphorylated proteins in accordance with the present invention.

WO 2005/1 1 1062 A1 discloses methods for characterization of phosphorylated polypeptides in a sample. The methods comprise separation and enrichment of phosphorylated polypeptides. In one aspect of this prior art invention the separation comprises an affinity purification such as IMAC. However, the disclosed methods do not make use of the steps of the method according to the present invention.

Finally, WO 2006/014424 A2 discloses methods for detecting and isolating phosphomolecules using phosphoaffinity materials that comprise a hydrated metal oxide. Preferred hydrated metal oxides include titanium dioxide. The disclosed technology is described as differing from isolation methods using IMAC. It is contemplated that a sample can be fractionated prior to use in a methods of the invention if desired. However, IMAC is not specifically disclosed as a fractionation in accordance with the present invention. Thus, the separation of monophosphorylated from polyphosphorylated peptides/proteins is not the aim of this disclosure.

Hence, current methods for phosphopeptide enrichment, e.g. Immobilized Metal ion Affinity Chromatography (IMAC) and titanium dioxide (TiO2) chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. Meanwhile the currently available methods do not focus on the separation of mono- and multiply-phosphorylated peptides. Furthermore, the number of multiply-phosphorylated peptides that are identified in most published studies is rather low.

The methods used in current phosphoproteomic studies for enrichment of phosphorylated peptides, e.g., Immobilized affinity chromatography and titanium dioxide, are based on the high affinity of the iron/gallium and TiO₂ towards the phosphate group on the phosphopeptides. The subsequent mass spectrometric identification of the phosphopeptides is compromised by the fact that mono-phosphorylated peptides ionize better than multiply phosphorylated peptides, resulting in a decrease in the signal intensities of the multiply phosphorylated peptides when analyzed together with mono-phosphorylated peptides. Therefore, when analyzing complex mixtures enriched for phosphorylated peptides mono-phosphorylated peptides are preferentially identified.

It is therefore an object of the present invention to provide a method to effectively separate mono-phosphorylated peptides from multiply-phosphorylated peptides. SUMMARY OF THE INVENTION

The present inventors have found that mono-phosphorylated peptides can be separated from multiply phosphorylated peptides by IMAC alone or in combination with TiO₂ chromatography. The mono-phosphoryated peptides are eluted off the IMAC material with an acidic solution having a pH of from about 0.7 to about 1 .0, such as 1% TFA, and the multiply phosphorylated peptides are subsequently eluted using an alkaline reagent, such as ammonia water (pH 1 1 ), an acidic solution having a pH of less than 0.7 or a chelating agent, such as EDTA, with high affinity for the metal ions onto which the phosphomolecules are bound. It follows therefrom that the elution of the multiply phosphorylated peptides may be based on at least three different mechanisms.

The present inventors have also combined this efficient separation method with TiO₂ chromatography to significantly reduce the level of non-phosphorylated peptides in both the IMAC flow-through and the mono-phosphorylated fraction. The separation of the two species allows for optimizing the subsequent analysis (e.g. mass spectrometric analysis) to favor analysis of either mono-phosphorylated or multiply phosphorylated peptides and thereby avoid suppression effects between them.

Thus, according to the present invention there is provided a method for specific separation of mono-phosphorylated peptides from multiply-phosphorylated peptides for subsequent analysis.

The method conceptually involves the steps:

• establishing a phosphoaffinity environment by washing a phosphoaffinity material based on metal ions with a loading buffer, said buffer comprising an ion pairing agent, such as TFA, dissolved in a solvent, such as acetonitrile, acetic acid, acetone, or combinations thereof,

• contacting the sample with the phosphoaffinity environment to establish a phosphomolecule-phosphoaffinity material complex;

• releasing the mono-phosphorylated peptides from the phosphoaffinity environment by treating with a solution having a pH of from about 0.7 to about 1 .0; and • releasing or eluting the multiply-phosphorylated peptides from the phosphoaffinity environment by treating with an IMAC elution buffer, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent with high affinity for the metal ions onto which the phosphomolecules are bound.

The mono- and multiply-phosphorylated peptides may be further purified by removing unbound sample components from the phosphomolecule-phosphoaffinity material complex prior to eluting. Also the eluted fractions comprising mono-phosphorylated peptides may be purified on TiO2 as tought herein as well as in other publications of the present inventors.

Since the present invention enables the separation of mono- from multiply-phosphorylated peptides it may also be extended to a method for isolating mono-phosphorylated peptides from a sample, comprising:

• contacting a sample with a phosphoaffinity material comprising a metal ion and a support, under conditions wherein phosphomolecules are capable of binding to the phosphoaffinity material to form a phosphomolecule phosphoaffinity material complex in a liquid medium,

• eluting the mono-phosphorylated peptides from the phosphomolecule-phosphoaffinity material complex with a solution having a pH of from about 0.7 to about 1 .0, and

• purifying the eluted mono-phosphorylated peptides on a TiO₂- or ZiO₂-covered substrate thereby isolating the mono-phosphorylated peptides from the sample.

The present invention also provides a method for detecting a monophosphorylated peptide in a sample, comprising:

• contacting a sample with a phosphoaffinity material comprising a metal ion and a support under conditions wherein the mono-phosphorylated peptide is capable of binding to the phosphoaffinity material to form a phosphomolecule-phosphoaffinity material complex,

• washing with an organic solvent or water, and • eluting the mono-phosphorylated peptides from the phosphomolecule-phosphoaffinity material complex with a solution having a pH of from about 0.7 to about 1 .0, and

• analyzing the eluent for bound peptides, thereby detecting the mono-phosphorylated peptide in the sample.

The present invention furthermore provides a method for detecting a multiply-phosphorylated peptide in a sample, comprising:

• establishing a phosphoaffinity environment by washing a phosphoaffinity material based on metal ions with a loading buffer, said buffer comprising an ion pairing agent dissolved in an organic solvent and/or water; said metal ions are selected from Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Fe(III), Sc(III), AI(III), Lu(III), Th(III), and Ga(III);

• releasing or eluting the mono-phosphorylated peptides from the phosphoaffinity environment by treating with a solution having a pH of from about 0.7 to about 1.0;

• releasing or eluting the multiply-phosphorylated peptides from the phosphoaffinity environment by treating with an IMAC elution buffer, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent with high affinity for the metal ions onto which the phosphomolecules are bound; and

• analyzing the eluent comprising the multiply-phosphorylated peptides, thereby detecting the multiply-phosphorylated peptides in the sample

Finally the present invention provides a kits for performing the above methods. Thus, there is provided a kit for separating mono-phosphorylated peptides from multiply-phosphorylated peptides, said kit comprising: • a phosphoaffinity environment obtainable by washing a phosphoaffinity material based on metal ions with a loading buffer, said buffer comprising an ion pairing agent dissolved in an organic solvent and/or water; said metal ions are selected from Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Fe(III), Sc(III), AI(III), Lu(III), Th(III), and Ga(III);

• first means for releasing or eluting the mono-phosphorylated peptides from the phosphoaffinity environment, said means comprising a solution having a pH of from about 0.7 to about 1.0; and

• second means for releasing or eluting the multiply-phosphorylated peptides from the phosphoaffinity environment by treating with an IMAC elution buffer, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent with high affinity for the metal ions onto which the phosphomolecules are bound.

Similarly the present invention provides kits for isolating and detecting mono- and multiply- phosphorylated peptides, respectively.

In any of the methods and kits described herein, the metal ion can be in, for example, particle form. As such, the phospho-affinity material can comprise a support. The support can be selected from the group of particle, bead, gel, matrix, membrane, filter, fiber, sheet, mesh, frit, resin, sample vessel, column, pipette tip, slide channel and MALDI-TOF plate. The support can include a detectable tag, if desired.

The metal ion of the present invention may be selected from Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Fe(III), Sc(III), AI(III), Lu(III), Th(III), Ga(III), and the like. Particularly preferred metal ions are Fe(III) and Ga(III).

In any of the methods and kits described herein, it is contemplated that the sample to be separated and/or analyzed may be admixed with a reagent having higher affinity for the metal ions of the present invention than is the case for the monophosphorylated peptides but less affinity for the metal ions than is the case for the multiply-phosphorylated peptides. Thereby the step of eluting the mono-phosphorylated peptides may be dispensed with. This is a particularly preferred embodiment of the method of detecting or isolating multiphosphorylated peptides.

As described herein the method of the present invention can be advantageously combined with other methods for purifying phosphomolecules, such as the TiO₂ chromatography method developed by the present inventors (Thingholm et al 2006; Larsen et al, 2007).

DETAILED DESCRIPTION OF THE INVENTION

The technology described herein relates to methods, compositions and commercial packages for separating, isolating and/or detecting mono- and multi-phosphorylated molecules, in particular peptides, using metal ion containing phosphoaffinity materials, such as IMAC. Moreover, the present invention features a subsequent purification on TiO2 or another phosphoaffinity material of the eluted fraction that contains the mono-phosphorylated peptides.

Accordingly, in one embodiment, the present invention is directed to methods for separating and isolating mono- and multi-phosphorylated peptides. This separation method can be used for preparing samples enriched with mono-phosphorylated or multiply-phosphorylated peptides, for example to improve the detection of these in a complex sample. This isolation can be achieved by binding the mono- and multiply-phosphorylated peptides to a metal ion phosphoaffinity material and separating, due to differential affinity to the phosphoaffinity material, the mono- and multiply-phosphorylated peptides. Specifically the mono-phosphorylated peptides are first eluted with an acidic reagent having a pH higher than 0.7, and subsequently the multi-phosphorylated peptides may be eluted with an IMAC elution buffer known in the art, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent. A sulphuric compound is meant to include sulphates and sulphonates as well as derivatives thereof. Similarly a phosphoric compound is meant to include phophates and phosphonates as well as derivatives thereof.

As used herein, the term "isolating" when used in reference to a phosphorylated peptide means the act of separating the phosphorylated peptide from other molecules, substances or materials in the sample. The term "isolated" when used in reference to a phosphorylated peptide, phosphoaffinity material, metal ion or other component useful in a method or commercial package of the invention means that the component is acted upon by the hand of man to remove other molecules, substances or materials with which the component is associated in a sample or preparation. The term isolated does not require absolute purity, but rather is intended as a relative term. As such, the term isolating includes acting on a sample to increase the amount of phosphorylated peptide in the sample relative to the amount of one or more initial sample components or amount of initial phosphorylated peptide, which is sometimes referred to herein as enriching a sample.

The detection methods described herein can be performed in a variety of physical formats. For example, phosphopeptides can be detected when in solution; when in a matrix; when in an array; as well as other formats. A variety of particle-based methods for detecting a phosphopeptide are described herein. A phosphoaffinity particle, which can be for example, a metal ion or a particle support coated with metal ions, can be detected directly; can be labeled prior to detection; or can be used to enrich or isolate a phosphomolecule-phosphoaffinity material complex which is then detected.

Immobilized metal affinity chromatography (IMAC) uses a stationary phase containing organic chelating groups charged with trivalent transition metal ions, such as Ga and Fe, to enrich phosphopeptides prior to microchemical analysis (Posewitz and Tempst, 1999). For IMAC, peptides are conventionally eluted from the resin using a buffer having higher pH or higher concentration of inorganic phosphate with respect to the sample loading buffer.

An example of a commercial IMAC-based procedure is the IMAP assay (Molecular Devices, Sunnyvale, CA). IMAP is a fluorescence polarization homogenous solution assay in which beads derivatized with trivalent transition metal ions are used for binding to phosphate residues. The beads are added to a kinase reaction along with a fluorescently-labeled peptide substrate. If the kinase phosphorylates the substrate, the bead binds to the phosphate residue. Rotation of the fluorescent phosphorylated substrate is slowed by the bead binding, resulting in greater polarization of the emitted light. IMAP appears to be applicable to measurement of phosphopeptides but not phosphoproteins. In IMAP, fluorescence polarization readings are performed at a pH value of less than about 6.0 to preserve interaction of the phosphate group with the trivalent cation. Consequently, continuous monitoring of kinase assays cannot be achieved by IMAP because kinase reactions are typically inhibited at the low pH at which fluorescence polarization is read. A sample can be processed to preserve or stabilize phosphorylated molecules. Methods for preserving the integrity of molecules in a sample are well known to those skilled in the art. Such methods include the use of appropriate buffers and/or inhibitors, including nuclease, protease and phosphatase inhibitors that preserve or minimize changes in the molecules in the sample. Such inhibitors include, for example, chelators such as ethylenediamne tetraacetic acid (EDTA), ethylene glycol bis(P aminoethyl ether)N,N,N1 ,N1 -tetreacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain and the like, and phosphatase inhibitors such as phosphate, sodium fluoride, vanadate and the like.

Appropriate buffers and conditions for allowing selective interactions between molecules are well known to those skilled in the art and can be varied depending, for example, on the type of molecule in the sample to be characterized (see, for example, Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999), Tietz Textbook of Clinical Chemistry, 3rd ea., Burtis and Ashwood eds W. B. Saunders, Philadelphia, (1999)).

A sample also can be processed to reduce the presence of interfering substances and/or reduce non-selective binding of sample components to a phosphoaffinity material. Exemplary agents useful for improving solubility of phosphorylated molecules include detergents such as TRITON X-100, sodium deoxycholate, urea, thiourea and sodium dodecyl sulfate. A tendency of acidic polypeptides to bind to phosphoaffinity materials non-selectively can be reduced by methyl esterification of the polypeptide sample (Ficarro et al, 2002; Brill et al, 2004).

A sample can be fractionated prior to use in a method of the invention if desired. Well known fractionation methods such as immunoprecipitation, 1 -D gel electrophoresis, 2-D gel electrophoresis, electroblotting, liquid chromatography, electrochromatography, dialysis, two- phase polymer separations and solid phase extraction can be used for sample fractionation. Various methods for fractionating a fluid sample or cell extract are well known to those skilled in the art, including subcellular fractionation or chromatographic techniques such as ion exchange, hydrophobic and reverse phase, size exclusion, affinity, hydrophobic charge-induction chromatography, and the like (Ausubel et al., supra, 1999; Scopes, Protein Purification: Principles and Practice, third edition, Springer-Verlag, New York (1993); Burton and Harding, J. Chromatoqr. A 814:71 -81 (1998)).

A sample can be labeled with a tag prior to use in a method of the invention. Examples of tags include detectable moieties, such a luminescent moieties, fluorescent moieties, radioactive moieties and the like; purification tags such as polyhistidine, flag, myc and GST tags; polynucleotide tags, aptamers, protein nucleic acids; biological tags such as phage; antibody and antibody-like tags; reactive organic molecule or peptide mass tags (e.g., iTRAQ, SPITC ect) or other mass tags such as particles of defined size, for example, metal beads and nanoparticle tags, and the like.

As already contemplated the present separation and isolation methods may include a subsequent step of further enriching or purifying the eluate comprising mono-phosphorylated peptides; this will ultimately improve the detection limits of the final analysis. A number of naturally occurring mineral oxides, such as goethite (a-FeOOH), gibbsite (a-AI(OH)3), bayerite (13-AI(OH)3), boehmite (y-AI(OH)3), ilmenite (FeTiO3), ilmenorutile (Fex(Nb, Ta) 2x. 4Ti1 -xO2), pseudorutile (Fe2Ti3Og), futile (TiO2), brookite (TiO2), pseudobrookite (Fe2TiOs), geikielite (MgTiO3), pyrophanite (MnTiO3), ecandrewsite (Zn, Fe, Mn)TiO3, melanostibite (Mn (Sb, Fe)O3), armalcolite (Mg, Fe)Ti2O5, srilankite (Ti, Zr)02 and anatase (TiO2), can be used in a phosphoaffinity material for this subsequent selective binding to phosphomolecules, i.e. to purify the mono-phosphorylated peptides eluted or released from the metal ions. In general, these and other inorganic metals, when hydrated, present a surface that is covered with a layer of a metal oxide, hydroxide or oxohydroxidehydroxyl groups which contribute to their overall physicochemical properties, including their ability to adsorb phosphorylated molecules. In an embodiment, a phosphoaffinity material useful in the methods and commercial packages of the invention contain a hydrated metal oxide selected from the group of aluminum oxide, titanium oxide, yttrium iron garnet, yttrium aluminum garnet, yttrium gallium garnet, ferric oxide, gallium oxide, yttrium oxide, vanadium oxide, zirconium oxide, iron titanate, iron aluminate, calcium titanate, sodium titanate, zirconium titanium aluminate, goethite, gibbsite, bayerite, boehmite, ilmenite, ilmenorutile, pseudorutile, rutile, brookite, pseudobrookite, geikielite, pyrophanite, ecandrewsite, melanostibite, armalcolite, srilankite and anatase. In specific embodiments, the hydrated metal oxides are yttrium oxide, yttrium iron garnet and titanium dioxide.

A phosphoaffinity material selected for use in a method or commercial package of the invention for isolating and/or detecting a mono-phosphorylated peptide is capable of binding to any phosphomolecule. It should be mentioned that the present invention is not limited to the separation of mono- from multiply-phosphorylated peptides, but is applicable to the separation of any mono-phosphorylated compound from multiply-phosphorylated compounds. A phosphomolecule can be a macromolecule, such as a polypeptide and polynucleotide, as well as a small molecule, such as an amino acid and nucleotide. Non-limiting examples of molecules that can contain a phosphorylated moiety include an amino acid, a peptide, a polypeptide, a nucleotide, a polynucleotide, a lipid, glycan and a carbohydrate. A phosphorylated moiety present on a phosphorylated polypeptide, such as a protein or peptide, can be phosphoserine, phosphothreonine, phosphotyrosine, 1 -phosphohistidine, 3-phosphohistidine, phosphoaspartic acid, phosphoglutamic acid, N-phospholysine, delta-O- phosphohydroxylysine, N- phosphoarginine, thiophosphorylation, phosphocysteine, pyridoxal phosphate Schiff base conjugated to the e-amino group oflysine, N-acetylglucosamine 1 -phosphate modified serine, mannose 6-phosphate present in asparagine-linked oligosaccharides or O-pantetheine phosphorylated serine. Phosphomolecules isolated and/or detected using a method of the invention include molecules containing one or more phosphomimetic groups. Non-limiting examples of phosphomimetic groups include O-boranophosphopeptides and O- dithiophosphopeptides, derivatized on tyrosine, serine, or threonine residues, phosporamide, H- phosphonate, alkylphosphonate, phosphorothiolate, phosphodithiolate and phosphorofluoridate. Selective binding means that the phosphoaffinity material binds to one or more phosphomolecules but does not substantially bind to non-phosphomolecules.

A sample or phosphoaffinity material used in a method or commercial package of the invention can be attached to a support. As used herein, the term "support" means a solid or semi-solid material onto which a metal ion, sample or phosphomolecule can be deposited, attached, immobilized, entrapped, captured or coated, or which can be functionalized to include a metal ion, sample or phosphomolecule. A support can be a natural or synthetic material, and can be an organic or inorganic material, such as a polymer, resin, metal or glass. Suitable supports are known in the art and illustratively include an agarose, such as is commercially available as Sepharose; a cellulose, illustratively including a carboxymethyl cellulose; a dextran, such as is commercially available as Sephadex; a polyacrylamide; a polystyrene; a polyethylene glycol; a resin; a silicate; divinylbenzene; methacrylate; polymethacrylate; glass; ceramics; paper; metals; metalloids; polyacryloylmorpholide; polyamide; poly(tetrafluoroethylene); polyethylene; polypropylene; poly(4-methylbutene); poly(ethylene terephthalate); rayon; nylon; polyvinyl butyrate); polyvinylidene difluoride (PVDF); silicones; polyformaldehyde; cellulose acetate; cotton; wool; dextran; Trisacryl; hydroxyalkyl methacrylate, poly(vinylacetate- co-ethylene), oxirane acrylate, polyethylene, polypropylene, poly (vinyl chloride), poly (methyl methacrylate), phenol resin, poly (vinylidene difluoride), poly (ethylene terephthalate), polyvinylpyrrolidone, polycarbonate, starch, nitrocellulose; mixtures thereof, and the like. A support useful in a method of the invention can have a variety of physical formats, which can include for example, a membrane column a hollow, solid, semi-solid, pore or cavity containing particle such as a bead, a gel, a fiber, including a fiber optic material, a sheet, a matrix and sample receptacle. Non-limiting examples of sample receptacles include sample wells, tubes, capillaries, vials and any other vessel, groove or indentation capable of holding a sample, including those containing membranes, filters, matrices and the like. A sample receptacle can be contained on a multi-sample platform, such as a microplate, slide, microfluidics device, array substrate, mass spectrometry sample plate, and the like. A particle to which a phosphoaffinity material is attached can have a variety of sizes, including particles that remain suspended in a solution of desired viscosity, as well as particles that readily precipitate in a solution of desired viscosity. In particular embodiments, a particle support or phosphoaffinity material particle such as a crystal have diameters of between about 1 nm and 1 um. The term "phosphoafffinity particle" means a phosphoafffinity material in particle form. The term encompasses particles coated with a phosphoaffinity material as well as particles made of a phosphoaffinity material, such as a crystal or other solid form. The term "phosphoaffinity sheet" means a phosphoaffinity material in flat form, such as a paper, membrane, filter, and the like. A phosphoaffinity material can be part of or incorporated into a device, such as for example, a spin-column, microcolumn pipette tip, multi-well, microwell strip, multi well microplate and magnetic separator. A support can also contain a ferromagnetic or paramagnetic substance, for example, when magnetic separation procedures are employed.

If desired, a support can include a tag, such as a tag useful for detection and/or purification. A support also can be an inherent characteristic of a hydrated metal oxide, such as a metal oxide particle, crystal or other solid form. For use as a phosphoaffinity material in column, bed or surface form, the support can have characteristics such as uniform porous network and chemical and/or biological inertness.

A variety of procedures can be used for attaching or depositing a metal ion onto a support for preparing a phosphoaffinity material useful in a method or commercial package of the invention. For example, the metal ion can be deposited on the support through liquid-phase deposition, chemical bath deposition, successive ion layer adsorption and reaction (SILAR), electroless deposition, reactive sputtering, reactive evaporation, spray pyrolysis, track-etching, anodic oxidation, cold-press molding, chemical vapor deposition, or sol-gel processing. The deposited metal oxide can be crystalline, nanocrystalline, poorly crystallized or amorphous. In some embodiments, a crystalline layer is subsequently hydroxylated to render it suitable for binding phosphorylated molecules, and the crystalline layer can be hydroxylated by incubation in an aqueous- based medium for a period of time, such as, for example, one hour to several months.

In an embodiment, a metal oxide is attached to a support at about ambient temperature and in an aqueous-based medium. In this embodiment, an organic support material is generally employed. Non- limiting examples of organic support materials include cellulose, cotton, wool, dextran, agarose, polyacrylamide, Trisacryl, hydroxyalkyl methacrylate, poly(vinylacetate-co- ethylene), oxirane acrylate, polyethylene, polypropylene, poly (vinyl chloride), poly (methyl methacrylate), phenol resin, poly (vinylidene difluoride), poly (ethylene terephthalate), polyvinylpyrrolidone, polycarbonate and starch. Deposition can be achieved on an ion-by-ion or particle attachment basis.

Functionalization of the organic support, such as with sulfonate, hydroxyl or carboxyl groups can aid in depositing the metal ion. In one embodiment, the metal ion is deposited on to a support of cellulose or modified cellulose. Therefore, in an embodiment, an organic support used in a method or commercial package of the invention is functionalized with organic groups, while in other embodiments, the organic support is functionalized with sulfonate, hydroxyl or carboxyl groups.

In an embodiment, a metal ion is deposited or attached to an inorganic support. Exemplary incorganic supports include ceramic, metal, glass, alumina, silica, zirconia, a ferromagnetic material and a paramagnetic material. More durable porous ceramic-based supports, such as alumina, permit derivatization with metal ions using harsher conditions. Ceramic membranes can be useful for certain biomedical applications because they are generally inert towards various harsh chemicals (strong acids and organic solvents) and high temperatures.

The methods described herein are carried out under conditions that allow a phosphomolecule to bind a phosphoaffnity material to form a phosphomolecule phosphoaffinity material complex. A phosphomolecule generally will bind to a phosphoaffinity material under typical protein interaction assay conditions. Such conditions are well known to those skilled in the art and generally include roughly physiologically salt levels, a buffering agent, and a temperature in the range of 4-37 degrees C. For a chosen phosphoafffinity material, a sample can be adjusted or placed into a solution or environment to have a specified characteristic such as a specified pH, salt concentration, surfactant property, viscosity and the like. The ability of a phosphomolecule to bind selectively to a phosphoafffinity material can be improved, enhanced and/or stabilized in the presence of sample ingredients such as inorganic salts, alcohols, detergents and surfactants, if desired. In an embodiment of a method of the invention, a sample contacted with a phosphoaffinity material in the presence of a detergent. In a specific embodiment, the detergent is an ionic detergent such as SDS. A variety of detergents can be used when contacting a sample with a phosphoaffinity material. The detergent can be anion, cationic, zwitterionic or non-ionic. Those skilled in the art will be able to select a suitable detergent for use with a particular sample and phosphoaffinity material.

In particular embodiments, the phosphoaffinity material includes a support. Exemplary supports include membranes, particles, matrices, spin- columns, microcolumn pipette tips, multi-well microwell strips, and multi- well microplates Specific examples of phosphoaffinity materials include filtration devices including membranes and filters containing one or more porous or semi-porous metal ion surfaces and/or coatings; filtration devices containing filters, particles and/or membranes that contain or incorporate metal ions as a coating on fiber surfaces, entrapped within the membrane's polymeric matrix or pores or presented as a layer on top of the membrane; and filtration devices configured as spin columns, microcolumn pipette tips, multi-well strips, and/or multi-well microplates.

EXPERIMENTAL

Model proteins and peptide mixture

The development and optimization of the presented method were performed using a peptide mixture originating from tryptic digestions of 12 standard proteins. Transferrin (human) was a gift from ACE Biosciences A/S. Serum albumin (bovine), b-lactoglobulin (bovine), carbonic anhydrase (bovine), b-casein (bovine), a-casein (bovine), ovalbumin (chicken), ribonuclease B (bovine pancreas), alcohol dehydrogenase (baker yeast), myoglobin (whale skeletal muscle), lysozyme (chicken) and alpha amylase (Bacillus sp.) were from Sigma. Each protein was dissolved in 50 mM ammonium bicarbonate, pH 7.8, 10 mM dithiotreitol (DTT) (Sigma®) and incubated at 37 ⁰C for 1 h. After reduction, 20 mM iodoacetamide (Sigma®) was added and the sample was incubated at room temperature for 1 h. The reaction was quenched with 10 mM DTT and the proteins were subsequently digested using trypsin (1 -2% wt/wt, modified trypsin, Promega) at 37 ⁰C for 12 h. Total protein from human mesenchymal stem cells

Human mesenchymal stem cells (hTERT20) were grown in T75 flasks in MEM (EARLES) Media w/o Phenol Red, with Glutamax-I (GibcoTM) containing 1 % Penicillin/Streptomycin (GibcoTM) and 10% Foetal Bovine Serum (GibcoTM) at 37⁰C until they reached 90% confluence. The confluent cells were washed once with PBS buffer (37⁰C) and 5 ml media (37⁰C) was added to cover the cells. Phosphatase inhibitor cocktail 1 and 2 (Sigma) (50 μl of each) was added to the media.. The cells were incubated with the phosphatase inhibitors for 30 min at 37⁰C. After washing with icecold PBS buffer, the cells were harvested using Cell Dissociation Buffer.

After harvesting the cells, the cell pellet was resuspended in 1 .5 μl_ lysis buffer (7M Urea (SIGMA®), 2M Thiourea (MERCK), 1 % N-octyl glycoside (Sigma®); 4OmM Tris (Sigma®), 300U Benzonase). The cells were then sonicated 3 times 15 sec on ice (interval etc. details) and incubated at -80 degrees for 30 min. After incubation, 20 mM dithiotreitol (DTT) was added. The sample was incubated at room temperature for 35 min. Then 40 mM lodoacetamide was added followed by incubation for 35 min at room temperature in the dark. For protein precipitation 14 ml. icecold acetone was added to the solution and it was incubated at -20 ⁰C for 20 min. The proteins were pelleted by centrifugation at 6000 g for 10 min at -4 ⁰C and the pellet was dried and stored at -20 ⁰C until further use.

Proteolytic digestion of proteins from human mesenchymal stem cells

A total of 2 mg of the dried precipitated protein from human mesenchymal stem cells (weight out) was redissolved in 6M urea, 2M thiourea to a concentration of 50μg/μL. The proteins were subsequently incubated with 1 μg endoproteinase Lys-C (Lysyl Endopeptidase®, WAKO) per 50 μg protein at room temperature for 3 hours. The endoproteinase Lys-C digested sample was diluted five times with 50 mM NH₄HCO₃ (SIGMA®) and 1 μg chemically modified Trypsin (Promega) was added per 50 μg protein and the sample was incubated at room temperature for 18 hours.

Immobilized Metal ion Affinity Chromatography (IMAC)

The procedure is here described for the optimization using 1 pmol of peptide mixture. The numbers in the brackets illustrate the volumes used for 120 μg of the peptide mixtures from the human mesenchymal stem cells. For each experiment 7μL (50 μL) of iron coated PHOS- select™ metal chelate beads (Sigma®) were used. The beads were washed twice in loading buffer (0.1% trifluoroacetic acid (TFA) (protein sequencer grade, Applied Biosystems), 50% acetonitrile (HPLC grade, Bie & Berntsen A/S) as described in (Oda et al., 2004-5). The beads were incubated with 30 μl_ (150 μl_) loading buffer and ^"I pmol peptide mixture (120 μg human mesenchymal stem cell peptide mixture). The beads were shaken in a Thermomixer (Eppendorf) for 30 min at 20 ⁰C. After incubation, the beads were packed in the restricted end of a P200 GELoader tip (Eppendorf) by application of air pressure forming an IMAC micro-column. For the complex peptide mixture from the human mesenchymal stem cells the IMAC beads were packed in 200 μl_ GELoader tips (Eppendorf). The flow-through was collected in an Eppendorf tube for further analysis. The IMAC column was washed using 20 μl (40μL) loading buffer, which was pooled with the flow-through. The mono-phosphorylated peptides were eluted from the IMAC column using 10μl (50 μL) 1% TFA, 20% acetonitrile and the multiply phosphorylated peptides were subsequently eluted from the micro-column using 40 μl (70 μL) ammonia water, pH 1 1 (10 μL 25% ammonia solution (MERCK) in 490 mL UHQ water). The flow-through and the eluted peptides were dried by lyophilization.

Titanium Dioxide (TiO2) Purification

After lyophilization the flow through and wash from the IMAC chromatography was enriched for phosphopeptides by titanium dioxide (TiO₂) chromatography. TiO₂ beads were obtained from a disassembled TiO2 column (1350L250W046 Titansphere, 5 mm, 250 _ 4.6 mm, GL sciences Inc.) A TiO₂ micro-column was prepared by stamping out a small plug of C8 material from a 3 M EmporeTM C8 extraction disk (3M Bioanalytical Technologies) using a HPLC syringe needle (Syringe for HPLC loading (P/N 038250, N25/500-LC PKT 5, SGE), and placing the plug in the constricted end of a P10 tip (Larsen et al., 2005; Thingholm et al., 2006). The TiO2 beads (suspended in 100% acetonitrile) were packed in the P10 tip, where the C8 material prevented the beads from leaking. The micro-column was packed by the application of air pressure. Buffers used for loading or washing of the micro-columns contained 80% acetonitrile to prevent non-specific binding to the C8 membrane and the TiO₂ beads. The lyophilized sample was resuspended in 2 μl 4M urea. 3 μl 1 % SDS (Larsen et al 2007) was added and the sample was diluted five times in loading buffer (1 M glycolic acid (Fluka) in 80% acetonitrile, 5% trifluoroacetic acid (TFA) (ALDRICH)) and loaded onto a TiO2 micro-column of ~5 mm (For the more complex samples 2 TiO₂ micro-columns were used per sample). The TiO2 micro-column was washed with 5 μl loading buffer and subsequently with 30 μl wash buffer (80% acetonitrile, 5% TFA). The phosphopeptides bound to the TiO2 micro-columns were eluted using 50 μl ammonium water (pH 1 1 ) followed by elution using -0.5 μl 30% acetonitrile to elute phosphopeptide bound to the C8 disk. The eluent was acidified by adding 5 μl 100% formic acid (Aldrich) prior to the desalting step. Desalting the TiO₂ eluates using R3 columns prior to Mass Spectrometry

The POROS Oligo R3 Reversed Phase resin (PerSeptive Biosystems) was dissolved in 70% Acetonitrile (HPLC grade, Fisher Scientific). The R3 beads were loaded onto constricted GELoader tips and gentle air pressure was used to pack the beads to gain R3 micro-columns of ~3 mm (Gobom, et al., 1999). Each acidified sample was loaded onto a R3 column. The R3 micro-columns were subsequently washed with 30 μl 0.1% TFA and the phosphopeptides were eluted directly onto the MALDI target using 1 μl 2,5-dihydroxybenzoic acid (DHB, Fluka)(20μg/μl), 50% acetonitrile, 1% phosphoric acid (Merck) for MALDI MS analysis. For LC- ESI MSMS analysis of the mono-phosphorylated peptides originating from the complex sample, the phosphopeptides were desalted in a similar way, however, the phosphorylated peptides were eluted from the Poros R3 column using 30 μl 70% Acetonitrile, 0.1% TFA followed by lyophilization. The phosphopeptides were subsequently resuspended in 0.5 μl 100% formic acid and 10 μl Buffer A (0.5% acetic acid) prior to LC-ESI MSMS analysis.

MALDI MS

MALDI MS was performed on a Bruker Ultraflex (Bruker Daltonics, Bremen, Germany). All spectra were obtained in positive reflector ion mode. The matrix used was 2.5- dehydroxybenzoic acid (DHB) (20 mg/mL) in 50% acetonitrile, 0.1% TFA/1% phosphoric acid. The spectra were processed using either bruker flexanalysis software or MoverZ software.

Nano-liquid chromatography tandem mass spectrometry (nano-LC-MS)

The nano-LC-MS experiments were performed using a 7T LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany). After the desalting step the samples were transferred to a 96 well sample plate. The sample was applied onto an EASY nano-LC system (Proxeon A/S, Denmark). The peptides were concentrated on a 1.5 cm pre-column (75 μm inner diameter, 360 μm outer diameter, ReproSil - Pur C18 AQ 3 μm (Dr. Maisch, Germany)) and eluted at 200 nl/min by an increasing concentration of acetonitrile (1 %/min gradient) onto a 8 cm analytical column (50 μm inner diameter, 360 μm outer diameter, ReproSil - Pur C18 AQ 3 μm A 2-step linear gradient eluting by increasing the level of Buffer B (0,5% acetic acid, 80% acetonitrile) from 0% to 100% over a period of 100 minutes.

The instrument was operated in a data-dependent mode automatically switching between MS, MS2 and neutral loss-dependent MS3 acquisition. The MS3 acquisition was set to automatically select and fragment the fragment ion originating from the loss of phosphoric acid from the parent ions when analyzing the mono-phosphorylated fraction of the un-separated fraction from TiO₂ chromatography. For the analysis of multiply phosphorylated peptides the MS3 acquisition was automatically set to select and fragment the fragment ion originating from the loss of a minimum of 2 phosphate groups from the parent ion.

Database searching using an in-house MASCOT server

The MS^π data were processed (smoothing, background subtraction and centroiding) using the program DTASuperCharge. The processed files were subsequently searched against the human sequence library in the IPI protein sequence database using an in-house Mascot server (version 2.1 ) (Matrix science Ltd., London, UK).

The search was performed choosing Trypsin as enzyme. Carbamidomethyl(C) was chosen as fixed modification. As variable modification, Oxidation(M), N-Acetyl (Protein), Phospho(STY) and IntactPhospho(STY) were chosen. The data were searched with a peptide mass tolerance of ± 30 ppm and a fragment mass tolerance of ± 0.6 Da. Maximum 1 missed cleavages were allowed.

Manual evaluation

A peptide identified by Mascot was accepted, if it had a peptide score above 20. The merged files can be found in supplementary data.

Representative Examples of the present invention

The separation was tested and optimized using a mixture of tryptic digests from 12 different standard proteins including three phosphoproteins (i.e. peptide mixture, see "Experimental").

Figure 1 schematically shows how the separation of mono- and multiply-phosphorylated peptides is carried out. Specifically, an aliquot of the tryptic peptide mixture (1 pmol) was batch- incubated with 7 μl of iron-coated PHOS-select IMAC beads in 30 μL 0.1% TFA, 50% acetonitrile for 30 min. After incubation, the IMAC beads were packed in the constricted end of a GELoader tip. The IMAC micro-column was washed using the loading buffer. A "gradient" of decreasing pH using increasing amount of TFA, was generated and used to elute peptides stepwise off the IMAC column. If the mono-phosphorylated peptides were bound weaker to the IMAC resin, they would be expected to elute at a higher pH than the multiply phosphorylated peptides. The IMAC micro-column was eluted stepwise using 20% acetonitrile and increasing concentrations of TFA (0.2%, 0.5%, 1.0%, 1 .5% and 2.0%) followed by an elution using ammonia water (pH 1 1 ). The IMAC eluents were lyophilized and resuspended in 0.5 μl 100% Formic acid and 9.5 μl_ water. Each IMAC eluents were subsequently desalted and concentrated on reversed-phase micro-columns and eluted directly onto a MALDI target using 2,5-dihydroxybenzoic acid, DHB (20 μg/μl DHB, 50% acetonitrile, 1% phosphoric acid). The MALDI TOF MS analysis of the eluents is shown Figure 2.

From the initial experiments the present inventors found that the IMAC beads showed a higher selectivity towards phosphopeptides when using 0.1 % TFA in the loading buffer compared to the traditional acetic acid (data not showed). However, the inventors observed a lower capacity when using 0.1 % TFA compared to acidic acid. Therefore, the flow-through from the IMAC column was further purified using TiO₂ chromatography. Here almost only mono-phosphorylated were observed indicating a lower capacity for IMAC for mono-phosphorylated peptides in the optimized loading buffer. This also showed that in order to achieve an efficient enrichment of the mono-phosphorylated peptides TiO₂ chromatography of the IMAC flow-through is needed.

From the IMAC micro-column mono-phosphorylated peptides started eluting already at 0.2% TFA whereas no multiply phosphorylated peptides were detected (Figure 2A). However, subsequent elution with higher concentrations of TFA indicated that a high level of mono- phosphorylated peptides had remained bound to the IMAC micro-column up to elution using 1.0% TFA (Figure 2B-C). Only low levels of mono-phosphorylated peptides remained bound to the IMAC micro-column after elution using 1.0% TFA (Figure 2D). However, by increasing the concentration of TFA in the elution buffer further, multiply phosphorylated peptides started to elute from the IMAC column (Figure 2D-E). A subsequent elution step using ammonia water (pH 1 1 ) resulted in the elution of mostly multiply phosphorylated peptides, indicating that most of the mono-phosphorylated peptides had already been eluted from the IMAC beads (Figure 2F). Here we should also take into account that mono-phosphorylated peptides ionize much better than multiply phosphorylated peptides.

Based on the initial experiments, a multistage phosphopeptide separation strategy was developed, where the peptide mixture is incubated with IMAC material in 0.1 % TFA/50 % Acetonitrile. After binding to IMAC the mono-phosphorylated peptides were eluted using 1 % TFA/20% acetonitrile. After elution of the monophosphorylated peptides, the multiply phosphorylated peptides were eluted from the IMAC material using ammonia water pH 1 1 . For optimal recovery of phosphopeptides the flowthrough from the IMAC separation was further enriched for phosphopepitdes using TiO₂ chromatography. The multistage strategy is illustrated below using 1 pmol peptide mixture and 7 μl of iron-coated PHOS-select IMAC beads. The mono-phosphorylated peptides were eluted from the IMAC micro-column using 10 μl 1% TFA in 20% acetonitrile and the multiply phosphorylated peptides were subsequently eluted using 50 μl ammonia water (pH 1 1 ). Both IMAC eluents were desalted and concentrated by reversed-phase chromatography and subsequently analyzed using MALDI TOF MS (Figure 3A-B). Since we expected a high level of mono-phosphorylated peptides to be present in the flow through from IMAC due to the limited capacity of IMAC, the flow through was further enriched for phosphopeptides using TiO₂ chromatography. The IMAC flow through was lyophilized, resuspended in 5% TFA, 1 M glycolic acid, 80% acetontrile and subsequently loaded onto a TiO₂ micro-column (~ 3 mm). The phosphopeptides were eluted from the TiO₂ micro-column, desalted and concentrated by reversed-phase chromatography prior to MALDI TOF MS analysis. The MALDI Spectrum of the eluted phosphopetides are shown in Figure 3C. As observed only mono-phosphorylated peptides were found in the flow- through from the IMAC separation. This clearly shows that combining IMAC with TiO₂ chromatography for phosphopeptide enrichment results in a more complete coverage of the phosphoproteome.

Development of a modified pdMS? method for multiply phosphorylated peptides

Tandem MS fragmentation of phosphorylated peptides commonly results in the loss of phosphoric acid as the dominant fragmentation pathway and is in some cases the only fragmentation observed. To obtain information on the peptide sequence pdMS³ was developed, where the fragment ion signal originating from the loss of phosphoric acid was subsequently selected for a second round of fragmentation. This tandem MS strategy has recently been applied to large scale phosphoproteomics. However, for multiply phosphorylated peptides the subsequent fragmentation of the ion corresponding to the loss of phosphoric acid results in a loss of a second phosphoric acid and will in most cases not provide adequate sequence information to identify the peptide. Initial phosphopeptide identification from an aliquot (50%) of the multiply phosphorylated peptides isolated by the above strategy from 120 μg of proteins from human mesenchymal stem cells (hMSC) was analyzed using traditional pdMS3 on an LTQ-FT instrument. Manual interpretation of the peptide fragment ion spectra, which had not provided any identifications in the database searches revealed predominantly signals from the loss of additional phosphoric acid (data not shown). In order to increase the peptide sequence information and thereby the identification rate we designed a pdMS3 experiment, where the instrument was set to select the fragment ion that had lost a minimum of two phosphoric acids for a second round of CID, thus ignoring the fragment ion originating from the loss of the first phosphoric acid. The remaining 50% of the purified multiply phosphorylated peptides from the hMSC was analyzed using the optimized multi-pdMS3. When using the multi-pdMS3 the present inventors identified multiply phosphorylated peptides, which is a significant increase, when compared to the normal pdMS3 method.

Application of the multistage strategy to the analysis of phosphopeptides from hMSC

The strategy for separation of mono-phosphorylated peptides from multiply phosphorylated peptides was applied to a whole protein lysate from human mesenchymal stem cells (hMSC). The cells were cultured to confluence of 90%. At the time of harvesting, the cells were incubated with phosphatase inhibitor cocktails from Sigma® for 30 min to preserve the phosphate groups present on non-stimulated hMSCs proteins. After harvesting the cells, the total protein complement was precipitated with icecold acetone and subsequently digested with Lys-C and trypsin. A peptide total of 120 μg of the peptide mixture (measured by weight from the precipitated sample) was then incubated with 50 μl IMAC beads in 150 μl loading buffer for 30 min. After packing the IMAC beads in a P200 GELoader tip, and collecting the flow through, the column was washed using 30 μl 0.1% TFA, 50% acetonitrile. The mono-phosphorylated peptides were eluted using 50 μl 1% TFA, 20% acetonitrile and the multiply phosphorylated peptides were subsequently eluted using 70 μl ammonia water (pH 1 1 ). The eluted multiply phosphorylated peptides (from ammonia water eluent) were lyophilized prior to tandem MS analysis. Due to the high degree of unspecific binding of IMAC beads in especially very complex samples, we expected to find many mono-phosphorylated peptides in the flow through. Initial experiments on complex mixtures using MALDI MS revealed a high number of non- phosphorylated peptides co-eluting from the IMAC beads together with the mono- phosphorylated fraction using 1% TFA. Therefore, both the mono-phosphorylated peptide fraction and the IMAC flow through were lyophilized and subsequently enriched for phosphorylated peptides using TiO₂ chromatography. After TiO₂ chromatography, the fraction with the mono-phosphorylated peptides and the IMAC flow through were desalted and concentrated using reversed-phase chromatography prior to tandem MS. All samples were analyzed using liquid chromatography (LC) - nanoelectrospray (ESI) tandem mass spectrometry on a LTQ-FT ICR. The mono-phosphorylated fraction and the IMAC flow through were analyzed using a standard neutral loss (NL) directed MS³ method, where the detection of a neutral loss originating from the loss of phosphoric acid from a phosphopeptide due to gas phase beta- elimination automatically triggered a subsequent fragmentation of this species. The multi- phosphorylated fraction was analyzed using the optimized method for multi-phosphorylated (especially di-phosphorylated) peptides that is described above.

In the mono-phosphopeptide fraction enriched by IMAC and TiO₂, 244 phosphopeptides were identified. Of these 94.7% were mono-phosphorylated, 4.5% were multi-phosphorylated and 0.8% were non-phosphorylated (Figure 4A). In the multi-phosphopeptide fraction recovered from the IMAC resin, 263 phosphopeptides were identified of which 67.3% were multi- phosphorylated, 31.2% were mono-phosphorylated and 1.5% were non-phosphorylated (Figure 4A). By combining the phosphopeptides identified from each fraction with the phosphopeptides identified from the flow-through from the IMAC column after TiO₂ enrichment, a total of 515 unique phosphopeptides were identified from 120 μg total hMSC protein. Of these, 183 were multi-phosphorylated peptides. In contrast, only 327 unique phosphopeptides were identified from the same amount of starting material when using an optimized TiO₂ protocol. Of these, 56 were multi-phosphorylated peptides (Figure 4B).

In this study 183 unique multiply phosphorylated peptides were identified from 120μg of total protein lysate using this optimized multistage strategy. According to the present inventors this is the highest number of multiply phosphorylated peptides and the highest number of phosphorylated peptides in general identified from such low amount of starting material. This clearly indicates that fractionation of the mono-phosphorylated peptides from the multiply phosphorylated peptides, when combined with optimal pdMS3 setups, significantly increase the number of identified phosphorylated peptides in a single experiment.

Thus, as the above examples illustrate the present invention also provides a new method for large scale phosphoproteome analysis in which the mono-phosphorylated peptides are separated from the multiply phosphorylated peptides prior to tandem MS by combined IMAC and TiO₂ chromatography. The mono-phosphoryated peptides are eluted off the IMAC material with 1% TFA and the multiply phosphorylated peptides are subsequently eluted using ammonia water (pH 1 1 ). Accordingly the combination of IMAC with TiO₂ chromatography significantly reduces the level of non-phosphorylated peptides in both the IMAC flow-through and the mono- phosphorylated fraction. The separation of the two species optimizes the subsequent pdMS3 experiment to favor analysis of either mono-phosphorylated or multiply phosphorylated peptides. As evidenced with the above example when applying this new method the number of detected multiply-phosphorylated peptides dramatically increases.

Claims

1. A method for separation of multiply phosphorylated peptides from mono-phosphorylated peptides in a sample comprising the steps:

• releasing or eluting the mono-phosphorylated peptides from the phosphoaffinity environment by treating with a solution having a pH of from about 0.7 to about 1.0; and

• releasing or eluting the multiply-phosphorylated peptides from the phosphoaffinity environment by treating with an IMAC elution buffer, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent with high affinity for the metal ions onto which the phosphomolecules are bound.

2. The method of claim 1 further comprising removing unbound sample components from the phosphomolecule-phosphoaffinity material complex prior to eluting by washing with an organic solvent and/or water.

3. The method of claim 1 or 2, wherein the method further comprises a step for purifying the eluted or released mono-phosphorylated peptides.

4. The method of claim 3, wherein the purification is performed by TiO₂-chromatography.

5. The method of any one of the preceding claims, wherein the loading buffer comprises 0.1% TFA and 50% acetonitrile.

6. The method of any one of the preceding claims, wherein the mono-phosphorylated peptides are released or eluted with a solution comprising 1 % TFA and 20% acetonitrile.

7. The method of claim 6, wherein the multiply phosphorylated peptides are subsequently released or eluted from the micro-column with ammonia water, pH 1 1.

8. A method for isolating mono-phosphorylated peptides from a sample, comprising:

• contacting a sample with a phosphoaffinity material comprising metal ions and a support, under conditions wherein phosphomolecules are capable of binding to the phosphoaffinity material to form a phosphomolecule phosphoaffinity material complex in a liquid medium, said metal ions are selected from Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Fe(III), Sc(III), AI(III), Lu(III), Th(III), and Ga(III);

9. The method of claim 8, wherein the solution has a pH of about 1 .

10. The method of claim 9, wherein the solution comprises TFA and acetonitrile.

1 1 . A method for detecting a mono-phosphorylated peptide in a sample, comprising:

• contacting a sample with a phosphoaffinity material comprising a metal ions and a support under conditions wherein the mono-phosphorylated peptide is capable of binding to the phosphoaffinity material to form a phosphomolecule-phosphoaffinity material complex; said metal ions are selected from Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Fe(III), Sc(III), AI(III), Lu(III), Th(III), and Ga(III);

• washing with an organic solvent or water; • eluting the mono-phosphorylated peptides from the phosphomolecule-phosphoaffinity material complex with a solution having a pH of from about 0.7 to about 1 .0; and

12. The method of claim 1 1 , wherein the method further comprises a step of purifying the eluted mono-phosphorylated peptides prior to analyzing the eluent for bound peptides.

13. The method of claim 12, wherein the purification is performed by TiO₂-chromatography.

14. The method of any one of claims 1 1 -13, wherein analyzing the eluent for bound peptides comprises a mode selected from the group of absorbance, transmission, mass measurement, fluorescence intensity, fluorescence polarization, fluorescence resonance energy transfer, time resolved fluorescence, resonance light scattering, surface-enhanced Raman scattering, mass spectrometry, electron paramagnetic resonance, refractive index absorbance, nuclear magnetic resonance, microcalorimetry, Fourier transform infrared spectrometry, atomic spectrometry, surface plasmon resonance, refractive index changes, spectropolarimetry and ellipsometry.

15. The method of any one of claims 1 1 -14, wherein analyzing the eluent is performed by a mass spectrometer.

16. A method for detecting a multiply-phosphorylated peptide in a sample, comprising:

• releasing or eluting the mono-phosphorylated peptides from the phosphoaffinity environment by treating with a solution having a pH of from about 0.7 to about 1.0; • releasing or eluting the multiply-phosphorylated peptides from the phosphoaffinity environment by treating with an IMAC elution buffer, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent with high affinity for the metal ions onto which the phosphomolecules are bound; and

• analyzing the eluent comprising the multiply-phosphorylated peptides, thereby detecting the multiply-phosphorylated peptides in the sample.

17. The method of claim 16, wherein analyzing the eluent comprising the multiply- phosphorylated peptides comprises a mode selected from the group of absorbance, transmission, mass measurement, fluorescence intensity, fluorescence polarization, fluorescence resonance energy transfer, time resolved fluorescence, resonance light scattering, surface-enhanced Raman scattering, mass spectrometry, electron paramagnetic resonance, refractive index absorbance, nuclear magnetic resonance, microcalorimetry, Fourier transform infrared spectrometry, atomic spectrometry, surface plasmon resonance, refractive index changes, spectropolarimetry and ellipsometry.

18. The method of claim 16 or 17, wherein analyzing the eluent is performed by a mass spectrometer.

19. Kit for separating mono-phosphorylated peptides from multiply-phosphorylated peptides, said kit comprising:

• a phosphoaffinity environment obtainable by washing a phosphoaffinity material based on metal ions with a loading buffer, said buffer comprising an ion pairing agent dissolved in an organic solvent and/or water; said metal ions are selected from Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, Fe(III), Sc(III), AI(III), Lu(III), Th(III), and Ga(III);

• first means for releasing or eluting the mono-phosphorylated peptides from the phosphoaffinity environment, said means comprising a solution having a pH of from about 0.7 to about 1.0; and • second means for releasing or eluting the multiply-phosphorylated peptides from the phosphoaffinity environment by treating with an IMAC elution buffer, said elution buffer having higher affinity for the metal ions than the multiply-phosphorylated peptides, such as an alkaline reagent, an acidic solution having a pH of less than 0.7, a sulphuric compound, a phosphoric compound, or a chelating agent with high affinity for the metal ions onto which the phosphomolecules are bound.

20. The kit according to claim 19, wherein said metal ions are Fe(III), said first means is a solution comprising comprising 1 % TFA and 20% acetonitrile, and said second means is ammonia water, pH 1 1.

21 . A hyphenated chromatographic system for separating mono-phosphorylated peptides from multiply-phosphorylated peptides and subsequently purifying the phosphorylated peptides, said system comprising an IMAC column and a TiO2 column, wherein the outlet of the IMAC column is connected to the inlet of the TiO2 column.