WO2010086386A1

WO2010086386A1 - Protein quantification methods and use thereof for candidate biomarker validation

Info

Publication number: WO2010086386A1
Application number: PCT/EP2010/051024
Authority: WO
Inventors: Wouter Laroy; Katleen Verleysen
Original assignee: Pronota N.V.
Priority date: 2009-01-30
Filing date: 2010-01-28
Publication date: 2010-08-05

Abstract

The invention is in the field of proteomics. Methods and systems for high throughput quantification of proteins of interest, such as for example candidate biomarkers, from complex biological samples are provided. In particular, the invention uses sample pre-enrichment on the protein or peptide level, followed by separation and MS/MS MRM assay.

Description

Protein quantification methods and use thereof for candidate biomarker validation

FIELD OF THE INVENTION

The invention is in the field of proteomics. Methods and systems for high throughput quantification of proteins of interest, such as for example candidate biomarkers, from complex biological samples are provided.

BACKGROUND OF THE INVENTION

Biomarkers represent biological indicators that signal a changed physiological state, such as due to a disease or a therapeutic intervention. Biomarker discovery usually involves comparing proteomes expressed in distinct physiological states, and identifying proteins whose incidence or expression levels consistently differ between said physiological states.

While biomarker-discovery platforms commonly screen only a few samples representing each physiological state, the diagnostic relevance of identified candidate biomarkers needs to be extensively validated in statistically adequate sample groups, as well as checked for the specificity of the candidate biomarker across different diseases, which may include several hundreds or even thousands of samples. Hence, candidate biomarker verification/validation can require quantification thereof in large sample sets.

When antibodies for a candidate biomarker are available, basic protein-biochemistry methods such as western blots may be performed. Yet, such methods tend to suffer from low- throughput and inadequate sensitivity and reliability of quantification. Availability of antibodies may also allow to design an ELISA quantification assay for a candidate biomarker. However, the development of ELISA and other immunoassays is very time consuming and cost intensive and thus ill-suited for quantification of early candidate biomarkers. Also, most ELISA systems are not amenable to multiplexing. In addition, these approaches are hampered when antibodies against a candidate biomarker are not available and need to be generated first. Consequently, there exists a need in the art for further and improved methods and systems that can reliably quantify proteins even in relatively complex samples (such as for instance blood), with adequate sensitivity, specificity, high throughput and possibility of multiplexing.

SUMMARY OF THE INVENTION

The present invention realises considerable improvements in quantification of proteins from complex biological samples, allowing for lower detection limits (i.e., increased sensitivity), extended dynamic ranges and/or improved accuracy or precision of quantification, while ensuring high throughput, possibility of extensive multiplexing (i.e., quantification of more analytes in one sample) and the advantageous use of accessible mass spectrometry-based technologies. The methods and systems of the invention can achieve reliable quantification of desired proteins from complex protein mixtures, which makes them particularly suitable for quantification of (candidate) biomarkers in relevant biological samples, such as, e.g., serum or plasma. Moreover, the possibility to use the present methods in a high throughput mode allows for evaluation of a relatively large number of samples, as may be required to qualify and verify newly identified biomarker candidates. In addition, lower detection limits and/or the option of multiplexing may allow to depart from lower volumes of starting samples, thereby reducing the stress on sample donors, allowing more tests per sample, and/or allowing to downscale the volumes of other reagents, handling or cost.

Accordingly, in an aspect (herein referred to as aspect "A1") the invention provides a method for quantification of a protein of interest (POI) in a protein mixture (PM), comprising:

(a) enriching the protein of interest (POI) from the protein mixture (PM) to obtain a protein mixture enriched for said protein of interest (POI), or separating the protein mixture (PM) into fractions of proteins using a first separation process and selecting a fraction of proteins containing the protein of interest (POI); (b) proteolysing said protein mixture enriched for the protein of interest (POI), or said fraction of proteins containing the protein of interest (POI), to obtain a protein peptide mixture (PPM) comprising a reference peptide derived from the protein of interest (POI) by said proteolysing; (c) providing in said protein peptide mixture (PPM) a known quantity of a mass-labelled standard peptide having the same amino acid sequence as the reference peptide;

(d) separating the protein peptide mixture of (c) using a further separation process;

(e) subjecting the separated protein peptide mixture of (d) to: mass spectrometry (MS) and comparing the signal intensity for the reference peptide with the signal intensity for the standard peptide, or tandem mass spectrometry multiple reaction monitoring (i.e., MS/MS MRM) and comparing the signal intensity for one or more daughter fragment ions derived from the reference peptide with the signal intensity for one more corresponding daughter fragment ions derived from the standard peptide; and (f) calculating the quantity of the protein of interest (POI) in the protein mixture (PM). In a preferred option, aspect A1 may comprise steps as defined in claim 1.

It shall be understood that the process steps of aspect A1 may be carried out in any order compatible with overall performance of the method, such as, by means of a preferred example and not limitation, in the specific order (a) to (f) set out above. It shall be further understood that step (a) of aspect A1 covers various setups. For example, said step (a) may involve a single enrichment step, such as, e.g., using one capture agent or a combination of two or more different capture agents for the protein of interest (POI). In an alternative, said step (a) may involve two or more successive enrichment steps, e.g., using the same or different capture agents. Likewise, the separation process of step (a) of aspect A1 may be single-dimensional, i.e., wherein the protein mixture (PM) is subjected to a single separation step; or it may be multidimensional. As generally used herein, in a "multidimensional" separation process a sample of analytes (e.g., proteins or peptides) is subjected to a sequence of two or more separation steps ("dimensions"), each of which acts upon all or a part of analytes separated in a previous separation step, wherein any two analytes resolved in a given separation step remain resolved in subsequent separation steps, and wherein the distinct separation steps resolve analytes on the basis of different physical and/or chemical properties.

To realise a multidimensional separation of analytes herein, particularly of proteins or peptides, fraction(s) of interest from a given separation step may be resolved in a subsequent separation step. To obtain best resolution of analyte fractions, particularly protein or peptide fractions, from a given separation step in a subsequent separation step, the conditions in said steps are preferably orthogonal, such that the analytes, such as proteins or peptides, not resolved (i.e., recovered in same fraction) in one step will be resolved in a further step.

Typically, where the protein separation step (a) of aspect A1 is multidimensional, it may involve 4 separation steps or less, preferably 3 separation steps or less, more preferably, it may be three-dimensional (3D) or two-dimensional (2D). In an embodiment, the stages of the separation process may be coupled in an in-line system.

It shall also be understood that step (a) of aspect A1 may involve a combination of one or more steps for enriching the protein of interest (POI) with one or more separation steps for recovering the protein of interest (POI) in a particular fraction, which enrichment and separation steps may be combined in any suitable order, such as, for example, enrichment of protein of interest (POI) followed by separation of so-enriched composition, or separation followed by enrichment of protein of interest (POI) from a particular fraction.

In another aspect (herein referred to as aspect "A2"), the invention provides a method for quantification of a protein of interest (POI) in a protein mixture (PM), comprising:

(a) proteolysing said protein mixture (PM) to obtain a protein peptide mixture (PPM) comprising a reference peptide derived from the protein of interest (POI) by said proteolysing;

(b) providing in said protein peptide mixture (PPM) a known quantity of a mass-labelled standard peptide having the same amino acid sequence as the reference peptide; (c) enriching the reference peptide and the standard peptide from the protein peptide mixture of (b) to obtain a protein peptide mixture enriched for said reference peptide and said standard peptide, or separating the protein peptide mixture of (b) into fractions of peptides using a first separation process and selecting a fraction of peptides containing the reference peptide and the standard peptide;

(d) separating the protein peptide mixture of (c) enriched for the reference peptide and the standard peptide, or the fraction of peptides of (c) containing the reference peptide and the standard peptide, using a further separation process;

(e) subjecting the separated peptides of (d) to: mass spectrometry (MS) and comparing the signal intensity for the reference peptide with the signal intensity for the standard peptide, or tandem mass spectrometry multiple reaction monitoring (MS/MS MRM) and comparing the signal intensity for one or more daughter fragment ions derived from the reference peptide with the signal intensity for one more corresponding daughter fragment ions derived from the standard peptide; and

(f) calculating the quantity of the protein of interest (POI) in the protein mixture (PM). In a preferred option, aspect A2 may comprise steps as defined in claim 2. It shall be understood that the process steps of aspect A2 may be carried out in any order compatible with overall performance of the method, such as, by means of a preferred example and not limitation, in the specific order (a) to (f) set out above. In another alternative, in aspect A2 step (c) may precede step (b). Thereby, the protein peptide mixture (PPM) of (a) is enriched or separated according to step (c) to obtain, respectively, a protein peptide mixture enriched for the reference peptide or a fraction of peptides containing the reference peptide; and subsequently in step (b) a known quantity of the mass-labelled standard peptide is provided in said enriched peptide mixture or peptide fraction obtained in step (c).

It shall be further understood that step (c) of aspect A2 covers various setups. For example, said step (c) may involve a single enrichment step, such as, e.g., using one capture agent or a combination of two or more different capture agents for the reference peptide and, where applicable, the standard peptide. In an alternative, said step (c) may involve two or more successive enrichment steps, e.g., using the same or different capture agents.

Likewise, the separation process of step (c) of aspect A2 may be single-dimensional or it may be multidimensional, as explained above. Typically, where the peptide separation step (c) of aspect A2 is multidimensional, it may involve 4 separation steps or less, preferably 3 separation steps or less, more preferably, it may be three-dimensional (3D) or two- dimensional (2D). In an embodiment, the stages of the separation process may be coupled in an in-line system.

It shall also be understood that step (c) of aspect A2 may involve a combination of one or more steps for enriching the reference peptide and where applicable the standard peptide, with one or more separation steps for recovering the reference peptide and where applicable the standard peptide in a particular fraction. Said enrichment and separation steps may be combined in any suitable order, such as, for example, enrichment followed by separation of so-enriched composition, or separation followed by enrichment of from a particular fraction. In addition, the invention also foresees a suitable combination of approaches as disclosed in aspects A1 and A2. In particular, the invention also contemplates a method which comprises the enrichment and/or separation steps at the protein level, as defined in step (a) of aspect A1 , as well as the enrichment and/or separation steps at the peptide level, as defined in step (c) of aspect A2. For example, a method is provided comprising step (a) of aspect A1 followed by steps (a) to (f) of aspect A2, wherein in step (a) of aspect A2 the term protein mixture (PM) would in this case denote the protein mixture enriched for the protein of interest (POI), or the fraction of proteins containing the protein of interest (POI), as produced in step (a) of aspect A1.

In any of above aspects A1 and A2, MS/MS MRM within step (e) may typically comprise:

(ea) subjecting the separated peptides of (d) to a first mass spectrometry selection configured to select for

(eaa) peptide ions having mass-to-charge (m/z) ratio about equal to the expected m/z ratio of the reference peptide ion, and

(eab) peptide ions having m/z ratio about equal to the expected m/z ratio of the standard peptide ion; (eb) subjecting the peptide ions selected in (eaa) and in (eab) to fragmentation to produce daughter fragment ions there from;

(ec) subjecting the daughter fragment ions produced in (eb) to a second mass spectrometry selection configured to select for

(eca) fragment ions having m/z ratio about equal to the expected m/z ratio of said one or more daughter fragment ions derived from the reference peptide, and

(ecb) fragment ions having m/z ratio about equal to the expected m/z ratio of said one or more corresponding daughter fragment ions derived from the standard peptide;

(ed) detecting the fragment ions selected in (eca) and in (ecb), whereby signal intensity is obtained for said detected fragment ions.

In any of above aspects A1 and A2, MS within step (e) may typically comprise:

(ea) subjecting the separated peptides of (d) to a mass spectrometry selection configured to select for (eaa) peptide ions having mass-to-charge (m/z) ratio about equal to the expected m/z ratio of the reference peptide ion, and

(eab) peptide ions having m/z ratio about equal to the expected m/z ratio of the standard peptide ion; (ee) detecting the peptide ions selected in (eaa) and in (eab), whereby signal intensity is obtained for said detected peptide ions.

By virtue of further explanation and without limitation, the above aspects A1 or A2 may be seen as encompassing two stages.

The first stage, which generally involves steps (a) to (c), uses advantageous enrichment and/or fractionation strategies - applied on the protein mixture (PM), or on the protein peptide mixture (PPM) obtained from said protein mixture (PM) by proteolysis - to arrive at a peptide composition of considerably reduced complexity, which peptide composition contains the reference peptide and the corresponding mass-labelled standard peptide.

The peptide composition obtained in steps (a) to (c) is next supplied to the second stage, generally involving steps (d) to (f), wherein the peptide composition proceeds through a sequence of selection windows configured to select for and detect the reference peptide and the corresponding standard peptide or desired analytes derived there from, thereby allowing quantification of said reference and standard peptides.

In particular, the separation step (d) provides a first selection window that selects for peptides which display a separation behaviour expected of the reference and standard peptides.

In an embodiment the peptides separated in step (d) may be collected in fractions and the fraction expected to contain the reference and standard peptides may be subjected to the MS or MS/MS MRM. In this embodiment a selection window as above is thus provided by choosing the particular peptide fraction for further analysis. More commonly, the separation process of step (d) may be configured "in-line" with the downstream MS or MS/MS MRM, such that peptides separated in step (d) are directly fed for analysis in said MS or MS/MS MRM. In this configuration, a selection window as above is thus provided by information on relevant elution parameter(s) (e.g., retention or elution time) characterising that subpart of peptides, forwarded in-line from the separation of step (d) to the MS or MS/MS MRM, which contains the reference and standard peptides. An in-line configuration between the separation step (d) and the MS or MS/MS MRM is considerably less labour intensive, thus allowing for increased throughout and improved multiplexing, i.e., in particular wherein reference and standard peptides for different proteins of interest may be detected in subparts of peptides resolved in step (d) which enter the MS or MS/MS MRM at different elution times.

The first MS step (ea) provides a second selection window that selects for peptide ions which display mass-to-charge (m/z) ratio expected of the reference and standard peptide ions; and in case of MS/MS MRM the second MS step (ec) provides a third selection window that selects for peptide fragment ions which display m/z ratio expected of one or more particular daughter fragment ions originating from the reference and standard peptide ions.

Analytes (such as peptide ions or daughter fragment ions) that have passed through said selection windows, and which are therefore deemed as originating or derived from the reference and the standard peptides, are quantitatively detected and this information is used to infer the quantity of the protein of interest (POI) present in the starting protein mixture (PM). It shall be appreciated that the invention is also directed to a device, system or platform that is able to carry out the methods of the invention, in particular comprising the peptide separation step (d) and the MS or MS/MS MRM step (e), and optionally and preferably the preceding protein or peptide enrichment or fractionation steps. Preferably, the system may be configured to perform any two or more or all above steps "in-line", i.e., by directly feeding desired analytes from a previous step to the subsequent step.

These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +1-5% or less, more preferably +/-1 % or less, and still more preferably +/- 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

When referring to a group of members or entities throughout this specification, "substantially all" means 70% or more, e.g., 75% or more, preferably 80% or more, e.g., 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably at least 96%, at least 97%, at least 98%, at least 99% or even 100% of said members or entities.

All documents cited in the present specification are hereby incorporated by reference in their entirety.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. When specific terms are defined in connection with a particular aspect or embodiment, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments, unless otherwise defined.

Analysed samples

The term "protein" as used herein refers to naturally or recombinantly produced macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term thus encompasses monomeric proteins, as well as protein dimers (hetero- as well as homo-dimers) and protein multimers (hetero- as well as homo-multimers). Further, the term also encompasses proteins that carry one or more co- or post-expression modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. In addition, the term includes nascent protein chains as well as partly or wholly folded proteins, misfolded proteins, partly or wholly unfolded or denatured proteins, and may also cover coalesced or aggregated proteins, in particular where the latter are amenable to proteolysis. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native protein, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts, preferably naturally-occurring protein parts, that ensue from further processing of said full-length proteins.

"Protein mixture" or "PM" generally refer to a mixture of two or more different proteins, e.g., a composition comprising said two or more different proteins. For example, a mixture of proteins to be analysed herein may include more than about 10, more than about 50, more than about 100, more than about 500, or even more than about 1000 or more than about 5000 different proteins.

An exemplary complex protein mixture may involve, without limitation, all or a fraction of proteins present in a biological sample or part thereof. The terms "biological sample" or "sample" as used herein generally refer to material, in a non-purified or purified form, obtained from a biological source. By means of example and not limitation, samples may be obtained from: viruses, e.g., viruses of prokaryotic or eukaryotic hosts; prokaryotic cells, e.g., bacteria or archea, e.g., free-living or planktonic prokaryotes or colonies or bio-films comprising prokaryotes; eukaryotic cells or organelles thereof, including eukaryotic cells obtained from in vivo or in situ or cultured in vitro; eukaryotic tissues or organisms, e.g., cell-containing or cell- free samples from eukaryotic tissues or organisms; eukaryotes may comprise protists, e.g., protozoa or algae, fungi, e.g., yeasts or molds, plants and animals, e.g., mammals, humans or non-human mammals. Biological sample may thus encompass, for instance, a cell, tissue, organism, or extracts thereof. A biological sample may be preferably removed from its biological source, e.g., from an animal such as mammal, human or non-human mammal, by suitable methods, such as, without limitation, collection or drawing of urine, saliva, sputum, semen, milk, mucus, sweat, faeces, etc., drawing of blood, cerebrospinal fluid, interstitial fluid, optic fluid (vitreous) or synovial fluid, or by tissue biopsy, resection, etc. A biological sample may be further subdivided to isolate or enrich for parts thereof to be used for obtaining proteins for analysing in the invention. By means of example and not limitation, diverse tissue types may be separated from each other; specific cell types or cell phenotypes may be isolated from a sample, e.g., using FACS sorting, antibody panning, laser-capture dissection, etc.; cells may be separated from interstitial fluid, e.g., blood cells may be separated from blood plasma or serum; or the like. The sample can be applied to the methods of the invention directly or can be processed, extracted or purified to varying degrees before being used.

In particularly useful examples, a sample may be derived from a healthy subject or from a subject suffering from a condition, disorder, disease or infection, and/or from a subject exposed to a treatment of interest, etc. For example, without limitation, the subject may be a healthy animal, e.g., human or non-human mammal, or an animal, e.g., human or non-human mammal, who has cancer, an inflammatory disease, autoimmune disease, metabolic disease,

CNS disease, ocular disease, cardiac disease, pulmonary disease, hepatic disease, gastrointestinal disease, neurodegenerative disease, genetic disease, infectious disease or viral infection, or other ailment(s).

Due to convenient collection and high incidence of relevant biomarkers, particularly useful samples from animals, for example humans and non-human mammals, may include body fluids such as listed above, more preferably blood-derived.

Preferably, protein mixtures derived from biological samples may be treated to deplete highly abundant proteins there from, in order to increase the performance of the present methods.

By means of example, mammalian such as human serum or plasma samples may include abundant proteins, inter alia albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin and fibrinogen, which may preferably be so-depleted from the samples. Methods and systems for removal of abundant proteins are known, such as, e.g., immuno-affinity depletion, and frequently commercially available, e.g., Multiple Affinity Removal System (MARS-7, MARS-

14) from Agilent Technologies (Santa Clara, California) (in any formats, such as, e.g., LC, spin, batch formats, etc.).

Protein of interest (POI)

"Protein of interest" or "POI" as used herein generally denotes any protein the quantification of which in a protein mixture (PM) is desired. In a particularly useful application, the protein of interest (POI) may be a protein known or suspected of being a biomarker, such as, e.g., a protein recognised as a candidate biomarker in proteomics biomarker-discovery assays, or identified using in silico prediction, on basis of available literature, biological knowledge, etc.

Proteolysis The present methods determine the quantity of a protein of interest (POI) in a protein mixture (PM) via MS or MS/MS MRM detection and quantification of one or more reference peptides derived from said protein of interest (POI) by proteolysis. Hence, an analysed protein mixture (PM) or a portion thereof is proteolysed to yield a protein peptide mixture comprising said one or more reference peptides.

The term "protein peptide" as used herein generally refers to fragments of a protein derived by proteolysis of said protein or of any one or more of its polypeptide chains, into two or more fragments. While the term encompasses peptides of any sizes and molecular weights, protein peptide mixtures preferred in the invention may have average and/or median peptide lengths of less than about 100 amino acids, e.g., less than about 90 amino acids, less than about 80 amino acids, more preferably less than about 70 amino acids or less than about 60 amino acids, even more preferably less than about 50 amino acids, e.g., particularly preferably less than about 40 amino acids or less than about 30 amino acids. Also, protein peptide mixtures preferred in the invention may have average and/or median peptide lengths of at least about 5 amino acids, preferably at least about 10 amino acids, even more preferably at least about 15 amino acids, e.g., at least about 20 amino acids. Hence, protein peptide mixtures preferred in the invention may have average and/or median peptide lengths of between about 5 and about 100 amino acids, preferably between about 10 and about 50 amino acids, e.g., between about 10 and about 40 amino acids or between about 10 and about 30 amino acids. Such peptide sizes may be particularly amenable to MS analysis.

The term "proteolysis" as used herein in relation to a protein refers to cleavage, preferably enzymatic or chemical cleavage, of one or more peptide bonds within said protein or within any one or more of its polypeptide chains. Proteolysis of a protein mixture denotes proteolysis of proteins constituting said protein mixture. Advantageously, protein mixtures may be fragmented so as to yield protein peptide mixtures having the preferred average or median peptide lengths as detailed above. It shall be appreciated that the present methods may in principle also be employed to quantify peptides of interest from peptide mixtures found as such in relevant biological samples. For example, it is known that urine comprises, besides proteins, a very complex peptide mixture resulting from proteolytic degradation of proteins in the body and elimination of the resulting peptides via the kidneys. Yet another illustration is the mixture of peptides present in the cerebrospinal fluid. Accordingly, in embodiments the present methods may employ peptide mixtures obtained from biological samples without further proteolysis in vitro. For example, such methods may be as defined in aspect A2 wherein step (a) would be absent. To ensure adequate representation of a protein of interest (POI) by reference peptide(s) derived there from, the proteolysis should desirably occur at the same peptide bonds in substantially all individual molecules of the protein of interest (POI). This can be advantageously achieved using a proteolysing agent having known sequence specificity, in particular an agent that cleaves preferentially at peptide bonds N-terminally or C-terminally adjacent to one or more specific amino acid residue types (denoted as X¹... X^π). The term "cleaves preferentially at" means that the proteolysis occurs substantially only at the recited peptide bond(s). Preferably, less than 10% of peptide bonds other than the recited ones would be cleaved, e.g., < 7%, more preferably < 5%, e.g., < 4%, 3% or < 2%, most preferably <1 %, e.g., < 0.5%, < 0.1 %, or < 0.01%.

Preferably, a protein mixture (PM) will be proteolysed at substantially all recited peptide bonds. Hence, the proteolysis may occur substantially quantitatively at peptide bonds N- terminally or C-terminally adjacent to residues of the one or more types X¹... X^π.

To achieve protein peptide mixtures (PPM) displaying preferred average and/or median peptide lengths, the protein mixture (PM) may be advantageously proteolysed adjacent to a relatively small number of different amino acid residue types X¹... X^π, such as at peptide bonds adjacent to 5 or less amino acid residue types (i.e., n<5), more preferably n<4, even more preferably n<3, still more preferably n<2, or at peptide bonds adjacent to only 1 amino acid residue type (i.e., n=1 ). For example, proteolysis may be effected by suitable physical, chemical and/or enzymatic agents, more preferably chemical and/or enzymatic agents, even more preferably enzymatic agents, e.g., proteinases, preferably endoproteinases. Preferably, the proteolysis may be achieved by one or more, preferably one, endoproteinase, i.e., a protease cleaving internally within a protein or polypeptide chain. A non-limiting list of suitable endoproteinases includes serine proteinases (EC 3.4.21 ), threonine proteinases (EC 3.4.25), cysteine proteinases (EC 3.4.22), aspartic acid proteinases (EC 3.4.23), metalloproteinases (EC 3.4.24) and glutamic acid proteinases.

By means of preferred examples and not limitation, proteolysis may be achieved using trypsin, chymotrypsin, elastase, Lysobacter enzymogenes endoproteinase Lys-C, Staphylococcus aureus endoproteinase GIu-C (endopeptidase V8) or Clostridium histolyticum endoproteinase Arg-C (clostripain). The invention encompasses the use of any further known or yet to be identified enzymes; a skilled person can choose suitable protease(s) on the basis of their cleavage specificity and frequency to achieve desired protein peptide mixtures.

In a preferred embodiment, the proteolysis may be effected by endopeptidases of the trypsin type (EC 3.4.21.4), preferably trypsin, such as, without limitation, preparations of trypsin from bovine pancreas, human pancreas, porcine pancreas, recombinant trypsin, Lys-acetylated trypsin, trypsin in solution, trypsin immobilised to a solid support, microwave-assisted trypsinisation, etc. Trypsin is particularly useful, inter alia due to high specificity (C-terminally adjacent to Arg and Lys except where the next residue is Pro) and efficiency of cleavage. The invention also contemplates the use of any trypsin-like protease, i.e., with a similar specificity to that of trypsin.

In other embodiments, chemical reagents may be used for proteolysis. For example, CNBr can cleave proteins at Met; BNPS-skatole can cleave at Trp.

The conditions for treatment, e.g., protein concentration, enzyme or chemical reagent concentration, pH, buffer, temperature, time, can be determined by the skilled person depending on the enzyme or chemical reagent employed.

Pre-treatments

Optionally and advantageously, protein mixtures (PM) and/or protein peptide mixtures (PPM) obtained there from may be chemically and/or enzymatically pre-treated, particularly to protect selected moieties. For example, a protein mixture (PM) or protein peptide mixture (PPM) may be reacted with one or more modifying reagents, simultaneously or sequentially in any suitable order, which reagents may preferably fall into the following classes: modifiers of primary amines, particularly modifiers of α-NH₂ groups and/or Lys ε-NH₂ groups; and/or modifiers of cysteine residues. After treatment with one or more modifying reagents, the sample may optionally be purified using known techniques, such as solvent evaporation, washing, filtration, chromatographic techniques, etc.

Suitable blocking reagents, as well as methods and conditions for attaching and detaching protecting groups will be clear to the skilled person and are generally described in standard handbooks of organic chemistry, such as "Protecting Groups", P. Kocienski, Thieme Medical Publishers, 2000; Greene and Wuts, "Protective groups in organic synthesis", 3rd edition, Wiley and Sons, 1999; incorporated herein by reference in its entirety. For example, Cys -SH groups in a protein mixture (PM) or protein peptide mixture (PPM) may be protected to avoid their reactivity, in particular oxidation. Typically, the protein mixture (PM) or protein peptide mixture (PPM) is first treated with a reducing agent known per se, such as, e.g., β-mercaptoethanol, dithiothreitol (DTT), dithioerythritol (DTE) or suitable trialkylphosphine inter alia tris(2-carboxyethyl)phosphine (TCEP), to quantitatively reduce any oxidised -SH groups, e.g., disulphide bridges. The -SH groups are subsequently protected with a blocking reagent that reacts selectively with Cys side chains and presents a non- reactive substituent for subsequent conditions. By means of example and not limitation, -SH groups may be converted to acetamide derivatives by treatment with iodoacetamide in denaturing buffers (e.g., guanidium ion- or urea-containing buffers). Other blocking reagents, such as N-substituted maleimides (e.g., N-ethylmaleimide), acrylamide, N-substituted acrylamide or 2-vinylpyridine, may alternatively be used.

For example, primary amino groups such as α-NH₂ groups and/or side chain primary amino groups including Lys ε-NH₂ groups, in the protein mixture (PM) or protein peptide mixture (PPM) may need to be protected to block their reactivity, using a suitable reagent that reacts selectively with the desired primary amino groups and presents a non-reactive substituent for subsequent conditions. The reagent may be generally substituted once or twice on each so- modified primary amine (i.e., -NH₂ gives -NHZ or -NZ₂, where Z is the substituent introduced by said reagent). Primary amines may be protected inter alia by acylation, more preferably acetylation, using reagents known perse, such as, e.g., using acetyl N-hydroxysuccinimide. Other suitable NH₂- modifying reagents have been extensively described in the art, for example, in Regnier et al. 2006 (Proteomics 6: 3968-3979). During modification of primary amines with acyl such as acetyl, the acyl moiety may be occasionally also introduced on the -OH group of Ser, Thr and/or Tyr. Such ester bonds are preferably subsequently broken by alkali hydrolysis at conditions that do not effect the acylation of the amino groups.

Reference peptide

As noted, the quantity of a protein of interest (POI) in a protein mixture (PM) is determined via MS or MS/MS MRM detection and quantification of one or more reference peptides derived from said protein of interest (POI). "Reference peptide" as used herein denotes a peptide derived by proteolysis from a protein of interest (POI), wherein said peptide is unique to said protein of interest (POI), i.e., wherein a peptide otherwise identical to said reference peptide is not derived by said proteolysis from any other protein of the studied protein mixture (PM). Hence, a reference peptide uniquely represents the protein of interest (POI) from which it was derived; and detecting the reference peptide, or an analyte directly derived there from, can provide information selectively about the protein of interest (POI).

For the sake of clarity, "reference peptide" as used herein denotes the peptide as found in and derivable by proteolysis from the protein of interest (POI), whereas "synthetic reference peptide" refers to a synthetic counterpart of said reference peptide. In general, synthetic peptides can be conveniently produced via peptide synthesis but can also be made recombinantly. Peptide synthesis can for instance be performed using standard liquid- or solid-phase peptide synthesis technologies (see, e.g., Methods in Molecular Biology, vol. 35: "Peptide Synthesis Protocols", by Pennington & Dunn, eds., Humana Press 1994, ISBN 0896032736; or Methods in Enzymology, vol. 289: "Solid-Phase Peptide Synthesis", by Fields, ed., Academic Press 1997, ISBN 0121821900). Recombinant peptide production can be obtained with a multitude of vectors and hosts as widely available in the art.

The complete genome sequence has been determined for a number of organisms including inter alia man and mice, and extensive gene and protein sequence collections have been generated for an even greater number of organisms. Hence, uniqueness of the primary amino acid sequence of any peptide derivable from a given protein of interest (POI) using a cleavage agent of known sequence specificity (such as, e.g., trypsin) can be readily tested in silico via publicly available sequence databases and sequence alignment programs.

Preferably, a reference peptide chosen to represent a given protein of interest (POI) may be a proteotypic peptide, i.e., a peptide that can be substantially repeatedly and consistently detected from proteolytic (such as, e.g., tryptic) digests of said protein of interest (POI) using a given MS ionisation / mass analyser configuration. Candidate proteotypic peptides for a certain protein of interest (POI) may be located empirically by querying publicly available proteomics data collections, such as inter alia Peptide Atlas (Desiere et al. 2006 Nucleic Acids Res 34: D655-8), GPM (Craig et al. 2004. J Proteome Res 3: 1234-42), SBEAMS

(Marzolf et al. 2006. BMC Bioinformatics 7: 286) or PRIDE (Jones et al. 2006. Nucleic Acids

Res 34: D659-63), for peptides consistently detected and reported for said protein of interest (POI) in particular MS settings. Alternatively or in addition, candidate proteotypic peptides may be predicted in silico, for example using the computational algorithms described in Mallick et al. 2007 (Nat Biotechnol 25: 125-31 ).

Also preferably, a reference peptide chosen to represent a given protein of interest (POI) does not carry or is not amenable to carry (e.g., does not include any amino acids able to carry) any post-translational modifications. The presence or absence of such modifications might cause inconsistent behaviour of said peptide in one or more steps of the present methods, and thereby complicate quantification of said peptide.

As well preferably, a reference peptide chosen to represent a given protein of interest (POI) may have a predicted mono-isotopic weight between 400 Da and 5000 Da, more preferably between 500 Da and 4500 Da, even more preferably between 600 Da and 4000 Da, which may be particularly amenable for MS analysis.

Further, to facilitate synthesis of synthetic counterparts of reference peptides, as well as of the corresponding standard peptides, further selection criteria concerning the chemical composition of such peptides may be considered, as known in the art, such as, e.g., lack of easily modified amino acids, lack of unstable amino acid combinations, overall hydrophobicity, etc.

Further, where the protein of interest (POI) is a candidate biomarker recognised as such using a proteomic biomarker-discovery platform, a reference peptide chosen to represent the protein of interest (POI) in the present quantification methods may be preferably distinct from the peptide of said protein of interest (POI) detected in said proteomic screen. This can ensure greater independence of the quantification provided by the present methods, especially in the context of biomarker qualification and verification studies. Alternatively, a reference peptide chosen to represent the protein of interest (POI) may correspond to the peptide of the protein of interest (POI) detected in such proteomic screen (such as, for example, an N-terminal peptide or a specific post-translational modified peptide, etc.).

Preferably, the present methods may employ only one reference peptide to represent a given protein of interest (POI). This may advantageously increase throughput and allow combining (multiplexing) the quantification of more than one proteins of interest (POI's) in one sample. Otherwise, in particular when quantification on the basis of a single reference peptide would display inadequate measurement variation, the methods may use two or more (preferably between two and four, more preferably two or three, even more preferably two) distinct reference peptides to represent a protein of interest (POI). Results obtained for said two or more distinct reference peptides may then be averaged or otherwise combined, providing a more reliable quantification of the corresponding protein of interest (POI).

To select a reference peptide most appropriately representing a protein of interest (POI), the inventors found it valuable to experimentally test at least 2, preferably between 2 and 7, and more preferably 3, 4 or 5, candidate peptides predicted as promising proteotypic peptides for each protein of interest (POI).

Said experimental testing may preferably employ a synthetic counterpart of such candidate reference peptide; and/or may employ a synthetic, mass-labelled standard peptide corresponding to the candidate reference peptide. Such experimental testing may at first be performed by loading said synthetic reference peptide and/or the corresponding standard peptide as sole analyte(s). Acceptable candidates may next be tested on an increasingly qualitatively complex and/or quantitatively represented background of unrelated peptides (e.g., peptides from a proteolytic digest of a biological sample, such as inter alia serum). For example, the synthetic reference peptide and/or the corresponding standard peptide may be subjected to the separation process of step (d), such as preferably liquid chromatography separation, to select peptides having advantageous behaviour therein; such as, e.g., substantially quantitative recovery, narrow elution window, reproducible retention time, retention time distinct from one or more other peptides with which multiplexing may be desired, and/or elution in a fraction of comparably reduced background complexity, etc.

Also, the synthetic reference peptide and the corresponding standard peptide may be subjected to MS analysis / detection under conditions analogous to the first MS selection of step (ea), to choose peptides having advantageous behaviour therein; such as, e.g., good likelihood of ionisation based on available database knowledge, proficient and reproducible experimental ionisation, narrow and consistent m/z window, extended dynamic range characterised by a linear relationship between the amount of loaded peptide and the detected signal intensity, m/z window encompassing comparably low number of unrelated peptides, and/or where applicable proficient and reproducible fragmentation and other advantageous properties of daughter fragment ions as discussed elsewhere in this specification. Standard peptide

As noted, the present methods quantify a given protein of interest (POI) in MS by selecting for and detecting the signal intensity of one or more reference peptides representative of said protein of interest (POI); or in MS/MS MRM by selecting for one or more reference peptides representative of said protein of interest (POI) and then selecting for and detecting the signal intensity of one or more daughter fragment ions representative of said one or more reference peptides.

In order to convert the so-obtained signal intensity value(s) to the quantity of the measured reference peptide, the present methods simultaneously measure a known quantity of a standard peptide, which is identical to the reference peptide except that it includes a distinguishable mass label.

Hence, "standard peptide" as used herein refers to a synthetic peptide having amino acid sequence identical to a corresponding reference peptide, and comprising a mass-altering label sufficient to distinguish the so-labelled standard peptide from its cognate reference peptide in conventional mass spectrometers.

Preferably, a mass-altering label may involve the presence of a distinct stable isotope in one or more amino acids of the standard peptide vis-a-vis its corresponding reference peptide. Examples of pairs of distinguishable stable isotopes include H and D, ¹²C and ¹³C, ¹⁴N and ¹⁵N or ¹⁶O and ¹⁸O. Usually, proteins of biological samples analysed in the present invention may substantially only contain common isotopes having high prevalence in nature, such as for example H, ¹²C, ¹⁴N and ¹⁶O. In such case, a standard peptide may be labelled with one or more uncommon isotopes having low prevalence in nature, such as for instance D, ¹³C, ¹⁵N and/or ¹⁸O. It is also conceivable that in cases where the proteins of a biological sample would include one or more uncommon isotopes, the standard peptide may be labelled with the respective common isotope(s).

Peptides including one or more distinct isotopes are chemically alike, separate chromatographically and electrophoretically in the same manner and also ionise and fragment in the same way. However, in a suitable mass analyser such peptides and optionally select fragmentation ions thereof will display distinguishable m/z ratios and can thus be discriminated. Isotopically-labelled synthetic peptides may be obtained inter alia by synthesising or recombinantly producing such peptides using one or more isotopically-labelled amino acid substrates, or by chemically or enzymatically modifying unlabelled peptides to introduce thereto one or more distinct isotopes. By means of example and not limitation, D-labelled peptides may be synthesised or recombinantly produced in the presence of commercially available deuterated L-methionine CH3-S-CD₂CD₂-CH(NH₂)-COOH or deuterated arginine H₂NC(=NH)-NH-(CD₂)₃-CD(NH₂)-COOH. It shall be appreciated that any amino acid of which deuterated or ¹⁵N- or ¹³C-containing forms exist may be considered for synthesis or recombinant production of labelled peptides. In another non-limiting example, a peptide may be treated with trypsin in H₂ ¹⁶O or H₂ ¹⁸O, leading to incorporation of two oxygens (¹⁶O or ¹⁸O, respectively) at the COOH-termini of said peptide (e.g., US 2006/105415).

As noted above, standard peptide equivalent to a given reference peptide may be included (i.e., as stated in step (c) of aspect A1 , or step (b) of aspect A2) within the analysed sample at various times, such as, e.g., before or after proteolysis, before or after enrichment, or before or after fractionation, etc. Advantageously, the standard peptide may be added after an analysed protein mixture has been proteolysed and prior to subjecting the resultant protein peptide mixture to any downstream processes (such as, e.g., prior to the peptide enrichment or fractionation step (c) of aspect A2; and prior to the peptide separation step (d) of aspects A1 and A2). This can ensure that the reference peptide and the standard peptide are processed substantially equally and minimises quantification errors that could otherwise arise from loss of only the reference peptides in such downstream steps.

Separation process of step (d)

As explained, to specifically quantify a given protein of interest (POI), the present methods include consecutive selection windows - particularly, the peptide separation step (d) and the MS or MS/MS MRM step (e) - configured to select for peptides, peptide ions and where applicable daughter fragment ions that behave as would be expected of the reference and standard peptides, ions thereof and select daughter fragment ions thereof, respectively.

Hence, in step (d) of above aspects, the peptide composition obtained in steps (a) through (c) is resolved and provided for downstream processing. Preferably, the separation process of step (d) may be one in which the reference peptide and the corresponding mass-labelled standard peptide show identical separation behaviour. This ensures comparable recovery of the reference and standard peptides in step (d), and thus improve the reliability of quantification.

It shall be appreciated that the separation process of step (d) may employ any separation mechanism compatible with downstream analysis of so-separated peptides, in particularly using MS or MS/MS.

Preferably, the separation process of step (d) may involve chromatography, which can provide proficient and reproducible resolution of peptide mixtures for downstream analysis.

As used herein, the term "chromatography" encompasses methods for separating chemical substances, referred to as such and vastly available in the art. In a preferred approach, chromatography refers to a process in which a mixture of chemical substances (analytes) carried by a moving stream of liquid or gas ("mobile phase") is separated into components as a result of differential distribution of the analytes, as they flow around or over a stationary liquid or solid phase ("stationary phase"), between said mobile phase and said stationary phase. The stationary phase may be usually a finely divided solid, a sheet of filter material, or a thin film of a liquid on the surface of a solid, or the like. Chromatography is also widely applicable for the separation of chemical compounds of biological origin, such as, e.g., amino acids, proteins, fragments of proteins or peptides, etc.

The separation process of step (d) may preferably use columnar chromatography (i.e., wherein the stationary phase is deposited or packed in a column), preferably liquid chromatography, and yet more preferably HPLC. While particulars of chromatography are well known in the art, for further guidance see, e.g., Meyer M., 1998, ISBN: 047198373X, and "Practical HPLC Methodology and Applications", Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993.

Without limitation, a chromatography separation of step (d) may resolve the component peptides on the basis of their ability or tendency to form certain type(s) of molecular interactions, such as, e.g., dispersive (hydrophobic) interactions, dipole-dipole polar interactions (e.g., hydrogen bonding), dipole-induced dipole polar interactions (e.g., π-π interactions), or ionic interactions. For example, various chromatographic applications allow to resolve peptides based on such properties and may be employed in step (d), including inter alia reversed phase high performance liquid chromatography (RP-HPLC), hydrophobic interaction chromatography (HIC), normal-phase HPLC (NP-HPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEC), and the like. Particularly preferably, the separation process of step (d) may involve reversed phased (RP) chromatography, preferably RP liquid chromatography, even more preferably RP-HPLC, which generally offers excellent and highly reproducible peptide resolution.

Exemplary stationary phases for RP chromatography may include appropriate solid supports (e.g., porous or non-porous silica) functionalised with aliphatic hydrocarbon moieties such as straight, branched and/or alicyclic, saturated or unsaturated aliphatic hydrocarbon moieties of between 2 and 30 carbon atoms (e.g., preferably straight alkyl moieties having between 2 and 30 carbons, such as, e.g., 18 (octadecyl), 8 (octyl), 4 (butyl), 3 (propyl) or 2 (ethyl) carbon atoms).

Exemplary, non-limiting commercially available chromatography columns functionalised with moieties suitable for RP-HPLC are summarised in Table 1 :

Table !

Dionex Corp. (Sunnyvale, California), Phenomenex Corp. (Torrance, California), Agilent Technologies (Santa Clara, California), Waters Corp. (Milford, Massachusetts).

Typically, in RPLC the loading mobile phase is aqueous in nature comprising a (low) percentage of organic modifier (e.g., ACN or methanol). The skilled person will be aware of the percentages of added modifier, the applied flow rates, temperatures, etc. used in RPLC. After loading, the peptides are separated using a solution comprising constant or gradually increasing (gradient) percentages of a water miscible solvent with hydrophobic properties such as acetonitrile (ACN), an alcohol (e.g. methanol, ethanol) or other solvents known in the art of reversed phase separation.

Also preferably, in aspect A2 the separation process of step (d) may be orthogonal with the peptide separation process of step (c), such that peptides co-recovered in the same fraction in step (c) would in general be resolved during the separation process of step (d). In an example, such orthogonality between steps (c) and (d) of aspect A2 may be achieved when said steps resolve the component peptides on the basis of different physical and/or chemical properties.

As explained above, suitable candidate reference and standard peptides may be selected inter alia by testing the separation behaviour of synthetic forms of said peptides, optionally on the background of unrelated peptides, under separation conditions of step (d). Such testing can define the recovery parameters (e.g., retention time peak and spread in chromatography) which characterise the separation of said reference and standard peptides in step (d) and, along with the subsequent MS or MS/MS MRM step, constitute suitable selection windows.

MS or MS/MS MRM

Peptides resolved in step (d) are subsequently subjected to MS or MS/MS MRM.

In both MS and MS/MS MRM setups, the constituent peptides are first ionised using a suitable peptide ionisation technique, such as for example electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI), and so-generated peptide ions are in step (ea) fed to a mass analyser configured to select (such as, e.g., sequentially, and optionally in repeating cycles) for peptide ions having m/z ratio about equal to the expected m/z ratio of the reference peptide ions, and for peptide ions having m/z ratio about equal to the expected m/z ratio of the corresponding standard peptide ions. Any mass analyser capable of such peptide ion selection or filtering may be used herein, such as for example a quadrupole mass analyser, quadrupole ion trap (3D) or linear (2D) quadrupole ion trap analysers. By means of example and not limitation, suitable m/z selection window for a particular peptide ion may be set on the basis of unit resolution, such as for example using peak width of 0.7 +/- 0.1 amu at 50% of maximum peak width or, as another example, using high resolution settings at 0.5 +/- 0.1 amu. In the MS setup, the signal intensity (ion current) for the reference and standard peptide ions selected in step (ea) is then measured and the so-measured values (typically, peak heights and/or areas under the peaks) are compared to a calibration curve that allows to determine the quantity of the reference peptide entering the MS analysis, and to infer there from the quantity of the corresponding protein of interest (POI). Calibration curves may be readily generated, e.g., by loading onto the MS a constant quantity of a synthetic standard peptide along with variable quantities of the corresponding synthetic reference peptide (optionally on the background of unrelated peptides) and obtaining signal intensity readings therefore, as generally known in the art.

In the MS/MS MRM setup, the peptide ions selected in step (ea) are subjected to fragmentation in step (eb) to produce daughter fragment ions there from. "Daughter fragment ions" as used herein generally refer to ions derived by fragmentation of a given parent peptide ion (such as, e.g., a reference peptide ion or a standard peptide ion) at one or more peptide backbone bonds and optionally at one or more amino acid side chain bonds. Parent peptide ion fragmentation can be achieved as known in the art, such as for example using collision- induced dissociation (CID) in tandem mass spectrometry (MS/MS) configurations. Preferably, the present MS/MS MRM step may select for and detect only one daughter fragment ion derived from a given reference peptide ion, and the matching daughter fragment ion derived from the corresponding standard peptide ion. This can advantageously increase throughput. Otherwise, in particular when quantification on the basis of only one fragmentation ion for a given reference peptide ion displays inadequate measurement variation, two or more (preferably between two and four, more preferably two or three, even more preferably two) distinct daughter fragmentation ions may be selected and detected for said reference peptide ion, as well as for the standard peptide ion corresponding thereto. Results obtained for said two or more distinct fragmentation ions may then be averaged or otherwise combined, thereby providing a more reliable quantification of the corresponding protein of interest (POI).

Preferably, a daughter fragment ion may be an y-ion, i.e., a C-terminal ion produced by cleavage of a parent peptide ion at a C(=O)-NH backbone bond. By means of example, when a parent peptide is represented as

I A-C_«HRⁿ-C(=O)-NH-C_«HRⁿ⁺¹-B, where R^π and R^π+1 represent the side chain moieties of respectively an n^th and n+1^th amino acid residues of the parent peptide, and A and B represent respectively the N-terminal and C- terminal remainders of the parent peptide, then an y-ion is produced by cleavage at the C(=O)-NH bond indicated by the arrow and comprising the portion C-terminally to said bond. By virtue of example and not limitation, a potential singly charged y-ion would be NH₃ ⁺- C_αHRⁿ⁺¹-B. The inventors have realised that daughter y-ions tend to ensure particularly sensitive and/or reproducible detection and quantification. To select a daughter fragment ion most appropriately representing a given parent peptide of interest, the inventors found it valuable to experimentally scrutinise at least 2 and optionally up to all fragment ions, preferably y-ions, produced from said parent peptide of interest. This may preferably employ a synthetic parent peptide of interest, such as for example a synthetic candidate reference peptide and/or a corresponding synthetic mass-labelled standard peptide. The tested parent peptide may initially be loaded, fragmented and detected by the MS/MS as a sole analyte, and subsequently on an increasingly qualitatively complex and/or quantitatively represented background of unrelated peptides.

For example, a parent peptide ion may be subjected to MS fragmentation, e.g., by collision induced dissociation (CID), and all or a selection of daughter fragment ions arising there from may be detected by MS to identify fragment ions having advantageous behaviour, such as, e.g., proficient and reproducible incidence upon fragmentation, narrow and consistent m/z windows, extended dynamic range characterised by a linear relationship between the amount of loaded parent peptide and the detected signal intensity of the fragment ion, and/or m/z window encompassing no fragment ions from unrelated peptides, etc.

In addition, once a suitable fragment ion has been selected to represent a particular parent peptide, the fragmentation may be optimised to preferentially and/or reproducibly produce said preferred daughter fragment ion. Fragmentation parameters that may be suitably optimised are in general known by those skilled in the art and may include, without limitation, declustering potential, collision energy, collision cell exit potential, and the like.

The resultant daughter fragment ions are in step (ec) fed to a mass analyser configured to selects for and detect daughter fragment ions having m/z ratio about equal to the expected m/z ratio of one or more particular daughter fragment ions of the reference peptide ion, and daughter fragment ions having m/z ratio about equal to the expected m/z ratio of the corresponding one or more daughter fragment ions of the standard peptide ion. Any mass analyser capable of such peptide ion selection / filtering and detection may be used herein, such as for example a quadrupole mass analyser, quadrupole ion trap (3D) or linear (2D) quadrupole ion trap analysers. By means of example and not limitation, suitable m/z selection window for a particular daughter fragment ion may be set on the basis of unit resolution, such as for example using peak width of 0.7 +/- 0.1 amu at 50% of maximum peak width or, as another example, using high resolution settings at 0.5 +/- 0.1 amu. Various MS and MS/MS systems and configurations may be employed to carry out the above described sequence of peptide ionisation, peptide ion m/z selection, and where applicable peptide ion fragmentation, daughter fragment ion m/z selection and detection. Suitable peptide MS and MS/MS techniques and systems are well-known perse (see, e.g., Methods in Molecular Biology, vol. 146: "Mass Spectrometry of Proteins and Peptides", by Chapman, ed., Humana Press 2000, ISBN 089603609x; Biemann 1990. Methods Enzymol 193: 455-79; or Methods in Enzymology, vol. 402: "Biological Mass Spectrometry", by Burlingame, ed., Academic Press 2005, ISBN 9780121828073) and may be used herein. For example, a particularly suitable MS/MS configuration for use herein may be ESI triple quadrupole MS/MS systems, wherein the first quadrupole may be used to select peptide ions of particular m/z, the second quadrupole may be used as collision cell to achieve peptide ion fragmentation, and the third quadrupole may be used to select for daughter fragment ions of desired m/z before detection.

The MS/MS MRM measures signal intensity (ion current) for the one or more daughter fragment ions for each of reference peptide ion and standard peptide ion. The so-measured values (typically, peak heights and/or areas under the peaks) are then compared to a calibration curve that allows to determine the quantity of the reference peptide entering the

MS/MS MRM analysis, and to infer there from the quantity of the corresponding protein of interest (POI). Calibration curves may be readily generated, e.g., by loading onto the MS/MS MRM a constant quantity of a synthetic standard peptide along with variable quantities of the corresponding synthetic reference peptide (optionally on the background of unrelated peptides) and obtaining signal intensity readings therefore, as generally known in the art.

Protein and/or peptide enrichment

As noted, step (a) of aspect A1 or step (c) of aspect A2 may enrich desired proteins (e.g., in particular a given protein of interest (POI) the quantification of which is intended) or desired peptides (e.g., in particular reference peptide(s) which represent a given protein of interest (POI) and the corresponding standard peptide(s)) from, respectively, protein mixtures (PM) or protein peptide mixtures (PPM), in order to improve the performance of the present quantification methods. "Enrichment for" a desired protein or peptide as used herein can typically involve (i) contacting a composition comprising a desired protein or peptide to be enriched, such as respectively a protein mixture or a protein peptide mixture, with a capture agent capable of specifically binding said desired protein or peptide or a family of desired proteins or peptides; (ii) allowing binding of said capture agent and said desired protein or peptide, thus forming a complex; (iii) isolating said complex, i.e., separating said complex from analytes such as proteins or peptides not bound by the capture agent; and (iv) recovering the desired protein or peptide from the complex, thereby obtaining a protein mixture enriched for said desired protein or a peptide mixture enriched for said desired peptide.

For example, enrichment may yield enriched protein mixtures wherein the desired protein constitutes at least about 30% (w/w), more preferably at least about 50%, even more preferably at least about 80% and most preferably up to about 90% or up to about 95% or even up to about 100% of all proteins. For example, enrichment may yield enriched peptide mixtures wherein the desired peptide constitutes at least about 30% (w/w), more preferably at least about 50%, even more preferably at least about 80% and most preferably up to about 90% or up to about 95% or even up to about 100% of all protein peptides.

Protein or peptide enrichment can inter alia provide any one or more advantages, such as for example: multiplexing using capture agents of different specificities; high degree of enrichment and thus improved detection limits; improved proteolysis of enriched proteins (i.e., due to proteolysis on less complex background); and option of automation.

The term "specifically bind" reflects a situation when a capture agent binds to the desired protein or peptide or a group or family thereof, but substantially not to random, unrelated proteins or peptides. Hence, a capture agent specifically binding to a desired protein or peptide would display little or no binding to other proteins or peptides, including to homologous proteins or peptides, under conditions where said capture agent binds to said desired protein or peptide. Typically such binding may be non-covalent.

Preferably, the capture agent may display high affinity for binding to its cognate protein or peptide. High-affinity binding advantageously allows for substantially quantitative enrichment of the desired protein or peptide, and thus for more reliable quantification.

As used herein, binding between a capture agent and a desired protein or peptide can be considered "high affinity" when the affinity constant (K_A) of such binding is K_A > 1x10⁵ M^"1, more preferably K_A > 1x10⁶ M^"1 such as, e.g., K_A > 1x10⁷ M^"1, yet more preferably K_A > 1x10⁸ M^"1, even more preferably K_A > 1x10⁹ M^~1, e.g., K_A > 1x10¹⁰ M^"1, K_A > 1x10¹¹ M^"1, K_A > 1x10¹² M^"1, K_A > 1x10¹³ M^"1, or K_A > 1x10¹⁴ M^"1, or even higher, wherein K_A = [CA_P]/[CA][P], CA denotes the capture agent, P denotes the desired protein or peptide. Determination of K_A can be carried out by methods known in the art, such as, e.g., using equilibrium dialysis and Scatchard plot analysis.

In a preferred embodiment, the capture agent may be an antibody directed to the desired protein or peptide (e.g., a desired tryptic peptide). Antibody reagents may already be available for many potential proteins of interest, or may be reliably raised for desired proteins or peptides using established techniques. In addition, antibodies provide for very high binding specificities and affinities and thus tend to be excellent capture agents. Moreover, methods of antibody immobilisation on solid supports are well-known, thereby allowing for easy recovery of the antibody-protein or antibody-peptide complexes, e.g., using centrifugation, filtration, magnetic capture or columnar affinity chromatography.

As used herein, the term "antibody" is used in its broadest sense and generally refers to any immunologic binding agent. The term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments. The term "antibody" is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro or in vivo.

In an embodiment, the antibody may be any of IgA, IgD, IgE, IgG, and IgM classes, and preferably IgG class antibody. In an embodiment, the antibody may be a polyclonal antibody, e.g., an antiserum or immunoglobulins purified there from (e.g., affinity-purified). Polyclonal antibodies may recognise more than one epitope and thereby tend to provide for higher avidity.

In another preferred embodiment, the antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies offer the advantages of, e.g., greater selectively and reproducibly targeting a particular antigen and even a particular epitope within the said antigen, as well as inter alia reproducible production and titre. By means of example and not limitation, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. 1975 (Nature 256: 495), or may be made by recombinant DNA methods (e.g., as in US 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624-628) and Marks et al. 1991 (J MoI Biol 222: 581-597), for example.

In a further embodiments, the antibody capture agent may be antibody fragments. "Antibody fragments" comprise a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv and scFv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies. The above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art-established meaning.

The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., Camelus bactήanus and Camelus dromaderius), llama (e.g., Lama paccos, Lama glama or Lama vicugna) or horse. A skilled person will understand that an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen. An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g., glycosylation, etc.). Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art, as are methods to produce recombinant antibodies or fragments thereof (see for example, Harlow and Lane, "Antibodies: A Laboratory Manual", Cold Spring Harbour Laboratory, New York, 1988; Harlow and Lane, "Using Antibodies: A Laboratory Manual", Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447; "Monoclonal Antibodies: A Manual of Techniques", by Zola, ed., CRC Press 1987, ISBN 0849364760; "Monoclonal Antibodies: A Practical Approach", by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: "Antibody Engineering: Methods and Protocols", Lo, ed., Humana Press 2004, ISBN 1588290921 ).

In another preferred embodiment, the capture agent may be an aptamer directed to the desired protein or peptide (e.g., a desired tryptic peptide). The term "aptamer" as used herein refers to single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo-DNA/RNA or any analogue thereof, that can specifically bind to a target molecule such as a protein or peptide, more typically to a peptide. Advantageously, aptamers can display fairly high specificity and affinity (e.g., K_A in the order 1x10⁹ M^"1) for their targets.

Aptamer production is described inter alia in US 5,270,163; Ellington & Szostak 1990 (Nature 346: 818-822); Tuerk & Gold 1990 (Science 249: 505-510); or "The Aptamer Handbook: Functional Oligonucleotides and Their Applications", by Klussmann, ed., Wiley-VCH 2006, ISBN 3527310592, incorporated by reference herein.

The ability to select aptamers in vitro allows a rapid isolation of extremely rare oligonucleotides that have high specificity and affinity for a desired protein or peptide. Advantageously, aptamers can be isolated for virtually any protein or peptide targets, even ones that are toxic or display low immunogenicity. Moreover, once a suitable aptamer has been selected, it can be synthesised at will since its production does not depend on a biological system. A further advantage of aptamers is their stability under a wide range of buffer conditions, resistance to harsh conditions without loss of activity, and possibility of further modifications (e.g., to increase resistance to nucleases, etc.)

Preferably, capture agents such as defined above may be immobilised on a suitable solid support thereby allowing for easy recovery of the capture agent-protein or capture agent- peptide complexes, e.g., using solid phase extraction, columnar affinity chromatography, centrifugation, filtration, magnetic capture or the like. The requirements for solid supports for use in separation methods are generally known in the art, being solid materials that are structurally stable and chemically inert under conditions of separation and which exhibit low or no non-specific interactions with analytes. Solid supports may need to allow for the immobilisation thereon of one or more moieties such as capture agents. Such methods for immobilisation and optionally the choice of spacers or linkers therefore, are well known in the field; see, e.g., Immobilized Affinity Ligand Techniques, Hermanson, G. T. et a/, Academic Press, INC, 1992; Combinatorial Chemistry, Eds: Bannwarth, Willi, Hinzen, Berthold, Wiley-VCH. Solid supports may be made from organic or inorganic materials or hybrid organic/inorganic materials, and may be polymer-based materials. Non-limiting examples of solid supports include ones prepared from a native polymer, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc.; ones prepared from a synthetic polymer or copolymer, e.g., styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, etc.; or ones prepared from an inorganic polymer, such as, e.g., silica. A solid support can be in the form of, e.g., beads, pellets, resin, small particles, a membrane, a frit, a sintered cake, pillars in microfabricated structures or a monolith or any other form desirable for use. Depending on the type of separation, solid supports may be comprised in a chromatography column as a chromatography matrix, in a phase extraction cartridge (SPE), in a magnetic bead, in a centrifugable or filterable bead or in any other known format suitable for separations.

After capture and recovery, the desired protein or peptide may be eluted from capture agents using suitable conditions known per se, such as, e.g., altered pH, increased ionic strength, or the like.

Optionally, the resultant composition enriched for the desired protein or peptide may be concentrated to allow loading of a substantial quantity of the enriched protein or peptide in downstream processing such as the separation step (d). Concentrating may involve, e.g., drying such as reverse-phase or vacuum drying.

Protein and/or peptide fractionation

To improve the performance of the instant quantification methods, step (a) of aspect A1 or step (c) of aspect A2 may decrease the complexity of the analysed samples by fractionating the protein mixture (PM) or the protein peptide mixture (PPM) and selecting for further analysis a fraction containing, respectively, desired proteins (e.g., a given protein of interest (POI) the quantification of which is intended) or peptides (e.g., in particular reference peptide(s) which represent a given protein of interest (POI) and where applicable the corresponding standard peptide(s)).

Such protein or peptide sample fractionation can inter alia provide any one or more advantages, such as, e.g., relatively short development time; generic applicability to proteins and peptides even where capture agents do not exist or are problematic to derive, and thus particular usefulness for biomarker assays; relatively low cost; and possibility of automation and in-line arrangements with the separation step (d).

Without limitation, step (a) of aspect A1 or step (c) of aspect A2 may resolve the component proteins or peptides, respectively, on the basis of physical and/or chemical properties chosen from inter alia net charge, electrophoretic mobility (EPM), isoelectric point (pi), molecular size and/or ability or tendency to form certain type(s) of molecular interactions, such as, e.g., dispersive (hydrophobic) interactions, dipole-dipole polar interactions (e.g., hydrogen bonding), dipole-induced dipole polar interactions (e.g., π-π interactions) or ionic interactions.

For example numerous chromatographic and electrophoretic applications are known per se that can resolve proteins or peptides on the basis of the above described properties, including inter alia RP-HPLC, hydrophobic interaction chromatography (HIC), normal-phase HPLC (NP- HPLC), hydrophilic interaction liquid chromatography (HILIC), chromatofocusing, size exclusion chromatography (SEC), ion exchange chromatography (IEC), affinity chromatography (AC), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), free flow electrophoresis (FFE), isoelectric focusing (IEF) including capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary, IPG-IEF (immobilized pH gradient - IEF = IEF in a strip) and the like.

In a preferred embodiment, in step (a) of aspect A1 , proteins of a protein mixture (PM) may be separated by ion exchange chromatography (IEC), such as cation (CEC) or anion (AEC) exchange chromatography, more particularly strong cation exchange (SCX) chromatography or strong anion exchange (SAX) chromatography. SAX may be particularly suited for protein resolution. Here, proteins are resolved on the basis of their net charge and ability to form ionic interactions under the separation conditions. Advantageously, the inventors have realised that IEC can yield a sensible number of protein fractions of suitably reduced complexity.

The term "strong cation exchange" or "SCX" chromatography refers to cation exchange chromatography (preferably columnar chromatography or optionally solid phase extraction techniques inter alia SCX using solid phase extraction cartridges, magnetic or centrifugable SCX beads, etc.) using a stationary phase that maintains constant net negative charge in the range of pH about 2-12, preferably about 1-14, or even substantially irrespective of pH. For example, SCX stationary phase may include solid support functionalised with strong acidic groups, such as preferably sulphonic acid groups. A non-limiting example hereof may be, e.g., wide-pore silica packing with a bonded coating of hydrophilic polymer, e.g., poly(2- sulfoethyl aspartamide); see, e.g., Crimmins et al. 1988 (J Chromatogr 443: 63-71 ). Typically, elution of solutes in SCX chromatography can be achieved by increasing ionic strength, e.g., with salt solutions, such as, e.g., NaCI, KCI or (NhU)₂SO₄ gradients. Commercially available SCX columns may be used in the present method, such as without limitation ones summarised in Table 2:

Table 2

The term "strong anion exchange" or "SAX" chromatography generally refers to anion exchange chromatography (preferably columnar chromatography or solid phase extraction techniques inter alia SAX using solid phase extraction cartridges, magnetic or centrifugable SAX beads, etc.), using a stationary phase that maintains constant net positive charge in the range of pH about 2-12, preferably about 1-14, or even substantially irrespective of pH. Non- limiting example of SAX stationary phase include solid supports functionalised with quaternary ammonium groups, such as inter alia - CH₂CH₂N⁺(CH₂CH₃)₂CH₂CH(OH)CH3. In another preferred embodiment, in step (c) of aspect A2, peptides of a protein peptide mixture (PM) may be separated by IEC, such as CEC or AEC, more particularly SCX chromatography or SAX chromatography, yet more preferably SCX chromatography. Here, peptides are resolved on the basis of their net charge and ability to form ionic interactions under the separation conditions. Advantageously, the inventors have realised that IEC and in particular SCX chromatography can yield a sensible number of peptide fractions of suitably reduced complexity.

In another preferred embodiment, in step (c) of aspect A2, peptides of a protein peptide mixture (PM) may be separated by chromatography, such as HPLC, using stationary phases functionalised with aromatic moieties (e.g., preferably phenyl) optionally substituted with one or more electron-withdrawing or electron-donating moieties. The inventors have realised that phenyl-based separations may produce particularly satisfactory fractionation. Commercially available phenyl-based columns may be used, such as without limitation ones summarised in Table 3:

Table 3.

In a further particularly preferred embodiment, in step (a) of aspect A1 , proteins of a protein mixture (PM) may be separated using free flow electrophoresis (FFE).

Depending on the separation mode, FFE may resolve proteins on the basis of their isoelectric point (pi) (e.g., in isoelectric focusing, IEF mode) or electrophoretic mobility (EPM) (e.g., in zone electrophoresis, ZE; isotachophoresis, ITP; or field step electrophoresis, FSE modes). The inventors have realised that FFE, particularly in IEF mode, can yield a sensible number of protein fractions of suitably reduced complexity. Additional advantages of FFE may include any one or more of: ability to more easily predict the protein fraction which contains the protein of interest based on predicted pi for the latter (i.e., in IEF mode); excellent resolution, reproducibility and recovery of proteins in FFE separations; and shorter development times than many other separation techniques. By means of example and not limitation, apparatuses, reagents and manuals for performing FFE are commercially-available from inter alia Becton Dickinson (BD); related technologies such as off-gels are available from inter alia Agilent Technologies; IEF chips are available from inter alia Protein Forest (Waltham, Massachusetts).

In another particularly preferred embodiment, in step (c) of aspect A2, peptides of a protein peptide mixture (PM) may be separated using FFE on the basis of pi or EPM, more preferably pi (i.e., IEF mode).

The inventors have realised that FFE can yield a sensible number of peptide fractions of suitably reduced complexity. Additional advantages of FFE may include any one or more of: ability to more easily predict the peptide fraction which contains the peptide of interest based on predicted pi for the latter (i.e., in IEF mode); excellent resolution, reproducibility and recovery of peptides in FFE separations; the possibility to study peptide retention behaviour. As noted in the Summary section, in embodiments the protein or peptide fractionation may be multidimensional, such as, e.g., 2-D, 3-D or 4-D, preferably 2-D or 3-D. Such multidimensional separation may involve one or more protein or peptide fractionation methods as described here above. In the above fractionation of protein or peptide mixtures, the width of the fraction selected for downstream processing in steps (d) etc. may be suitably chosen. For example, the fraction may be selected such as to contain less than about 30%, more preferably less than about 20%, even more preferably less than about 10%, less than about 5%, still more preferably less than about 2%, less than about 1 %, or even less than about 0.5% or less than about 0.1 % of the proteins or peptides present in the starting mixtures, such as to achieve advantageous decrease in complexity.

Where fractionation is performed on peptide mixture level, the particular fraction in which the desired peptide is expected may be tested using a synthetic version of said peptide.

Multiplexing As can be appreciated, the present methods may allow to quantify more than one, i.e., two or more, different proteins of interest (POI) from one same sample, using preferably one and optionally more than one (preferably at most two, such as to ensure throughput) reference peptides for any of said proteins of interest (POI). For example, the methods may allow to quantify between 2 and about 500 different proteins from the same sample, or between about 5 and about 200, or between about 10 and about 100, or between about 2 and about 50 or between about 5 and about 30 different proteins from the same sample.

As can be appreciated, such multiplexing may require enrichment for more than one desired proteins or peptides in steps (a) to (c) of the methods, and/or collection of more than one fractions in said steps (a) to (c). In addition, such multiplexing may involve setting multiple m/z selection windows in the MS or MS/MS MRM process. Such modifications are well within the reach of a skilled person armed with the teachings of the present specification.

Biomarker quantification

The invention can quantify one or more proteins of interest even from relatively complex protein mixtures, and is thus particularly well-suited for inter alia quantification of known or suspected (i.e., candidate) biomarkers in biological samples, such as for example serum. As used herein "biomarker" refers to a protein or polypeptide which is differentially present in samples from subjects having a genotype or phenotype of interest and/or who have been exposed to a condition of interest (herein "query samples"), as compared to equivalent samples from control subjects not having said genotype or phenotype and/or not having been exposed to said condition (herein "control samples"). Samples can be as disclosed above and may be broadly applied to compare for instance subcellular fractions, cells, tissues, biological fluids (e.g., nipple aspiration fluid, saliva, sperm, cerebrospinal fluid, urine, blood, serum, plasma, synovial fluid), organs and/or complete organisms.

Particularly relevant phenotypes may be pathological conditions in patients, such as, e.g., cancer, an inflammatory disease, autoimmune disease, metabolic disease, CNS disease, ocular disease, cardiac disease, pulmonary disease, hepatic disease, gastrointestinal disease, neurodegenerative disease, genetic disease, infectious disease or viral infection; visa-vis the absence thereof in healthy controls. Other comparisons may be envisaged between samples from, e.g., stressed vs. non-stressed conditions/subjects, drug-treated vs. non drug- treated conditions/subjects, benign vs. malignant diseases, adherent vs. non-adherent conditions, infected vs. uninfected conditions/subjects, transformed vs. untransformed cells or tissues, different stages of development, conditions of overexpression vs. normal expression of one or more genes, conditions of silencing or knock-out vs. normal expression of one or more genes, and so on. The phrase "differentially present" refers to a demonstrable, preferably statistically significant, difference in the quantity and/or frequency of a protein or polypeptide in a query sample group as compared to a control sample group. For example, a biomarker may be a protein which is present at an elevated level or at a decreased level in query samples compared to control samples. A biomarker may also be a protein which is detected at a higher frequency or at a lower frequency in query samples compared to control samples.

For example, a protein may be denoted as differentially present between two samples or between two sample groups if the protein's quantity in one sample or one sample group is at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900% or at least about 1000% of its quantity in the other sample or the other sample group; or if the protein is detectable in one sample or one sample group but not detectable in the other sample or the other sample group. Also, a protein may be denoted as differentially present between two sample groups if the frequency of detecting the protein in one group of samples is at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900% or at least about 1000% of the frequency of detecting the protein in the other group of samples; or if the protein is detectable at a given frequency in one group of samples but is not detected in the second group of samples.

Existing biomarker-discovery platforms usually pinpoint potential (i.e., candidate) biomarkers by comprehensively comparing proteomes between a relatively low number of query samples vs. control samples, and locating proteins differentially present between said sample sets.

So-identified candidate biomarkers next need to be validated, i.e., their ability to sensitively and specifically diagnose the phenotype or condition of interest needs to be convincingly established in query vs. control sample groups of statistically adequate size. Biomarker validation may commonly comprise three stages: qualification, verification and specificity tests.

Qualification usually aims to confirm differential presence of a candidate biomarker between the same query vs. control samples as were used in biomarker-discovery, but using an independent quantification method. Hence, biomarker qualification may typically require to quantify a candidate biomarker in relatively few samples, such as, e.g., in about 5 to about 50 query and control samples, more usually in about 7 to about 20 query and control samples, and even more usually in about 10 to about 15 query and control samples.

Once a candidate biomarker has been qualified, the verification stage typically aims to replicate the finding of differential presence of said candidate biomarker in statistically more powerful sets of query vs. control samples. Hence, depending on statistics such as inter alia the observed mean quantities and standard deviations of the candidate biomarker in query vs. control samples, and the desired power and statistical significance of the verification study, biomarker verification may commonly require to quantify the candidate biomarker in a high number of samples, such as, without limitation, in about 50 to about 5000 query and control samples, or in about 100 to about 2000 query and control samples, or in about 200 to about 800 query and control samples, or in about 300 to about 500 query and control samples, or the like. The biomarker verification data may allow to delineate intervals (or cutoff values) for normal values of the biomarker vs. values of the biomarker expected to be diagnostic for the studied phenotype/condition of interest, and to estimate sensitivity and specificity of said intervals or cut-offs for the query and control phenotypes.

A biomarker may be frequently wanted as a diagnostic molecule for a particular pathological condition, and candidate biomarker discovery, qualification and verification thus usually involve testing samples from patients having said pathological condition vs. healthy controls.

To test whether a candidate biomarker is specific (selective) for the pathological condition of interest, the incidence and/or quantity of the candidate biomarker in other diseases, especially in disorders related to the pathological condition of interest, may need to be examined. Consequently, such specificity tests may require to quantify a candidate biomarker in high numbers of subjects, commonly in the order of 10² to 10⁴ subjects, across more than one disease phenotypes.

In addition, detection and quantification of a validated biomarker may be required in clinical settings to diagnose the respective pathology in samples from patients known or suspected to suffer there from. Thus, the protein quantification methods of the invention may be advantageously employed to validate candidate biomarkers, in particular for qualification, verification and/or specificity testing of candidate biomarkers. Most commonly, the present methods may be used to quantify a candidate biomarker within the qualification and verification stages of the validation process. Moreover, the present methods may in principle also be employed to quantify biomarkers in diagnostic methods.

The present invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1 : Design of optimisation of a pilot protein quantification assay

The present LC-MS/MS MRM assay uses an Ultimate 3000™ (Dionex) Nano-LC system integrated with the triple quadrupole MS/MS system 4000 QTRAP™ (Applied Biosystems).

To set up a protein quantification assay, a peptide pair specific to said protein (one pair consisting of two peptides with identical sequence; one is isotopically-labelled leading to a specified mass difference) is synthesised. Typically, 3 to 4 different peptide pairs are synthesised for each protein to be quantified, and synthesis, purification, sequencing and quantification of a peptide pair may usually take only about 3 to 4 weeks. The assay aims to obtain protein quantification at low- to sub- ng/ml levels in serum. To determine current metrics in complex samples, non-human proteins (termed herein the Spike-in Protein Set or SPS) are spiked in human serum. MRM quantification of these proteins can be done based on peptides selected for their absence in the human proteome. Such proteins can be of non-mammalian origin, or can be mammalian homologues of human proteins. The latter option is preferred, as it mimics the natural situation the most. The SPS consists of 3 to 4 such proteins. For each protein, 3 to 4 proteotypic peptides are selected for peptide pair synthesis.

For each of the peptides, optimal conditions are found for MRM quantification. Some aspects that need optimisation are:

- Selection of peptides with best LC and MS properties; Selection of daughter ions of each peptide with best MS properties;

- Optimisation of fragmentation conditions for each peptide transition for optimal signal in MS/MS. When conditions have been optimised, an MRM assay is programmed. Leaving the loading amount of isotopically labelled peptides (herein, standard peptide) constant and that of non- labelled peptides (herein, synthetic reference peptide) variable, calibration curves are generated. This is done in both low and high complexity backgrounds. This provides detection limits, dynamic range, precision and accuracy metrics of the MRM. However, these may not be necessarily representative for the peptide pairs metrics, as sample preparation is not taken into account. Therefore, the SPS is spiked in similar backgrounds, and samples are prepared using:

- Limited depletion of albumin and IgG (samples with serum background) Trypsinisation in solution After trypsinisation, the standard peptide is spiked into the digest, and quantification is done using the calibration curves generated before. This experiment provides the current peptide pair metrics.

The SPS approach is used to verify the success of different sample preparation optimisation steps. Success is determined by: - Improved lower limit of quantification and/or Improved (linear) dynamic range and/or - Acceptable throughput

The sample preparation optimisation steps are tested and implemented into the protein quantification platform when found successful: - Depletion of abundant proteins: the high dynamic range of serum is a problem in both discovery and validation platforms. As MRM looks for specific peptides rather then screen all peptides, depletion can be less thorough in the validation platform. The degree of depletion needed to allow sensitive detection is tested, using different commercially available depletion resins. - Concentration strategies: depletion steps may lead to the dilution of the original sample and thus to lower biomarker concentrations. Concentration strategies, both on the protein and peptide level, are tested. Reversed phase and vacuum drying is considered.

Trypsinisation strategies: trypsinisation can be done in solution or on column. This optimization step may have impact on throughput and reproducibility.

Prefractionation strategies: sample complexity is reduced at the protein or peptide level prior to MRM analysis. Prefractionation can be based on charge (ion exchange), isoelectric point (FFE, isoelectric focusing mode), or other physical and/or chemical properties of peptides.

Claims

1. A method for quantification of a protein of interest (POI) in a protein mixture (PM), comprising:

(a) separating the protein mixture (PM) into fractions of proteins using a first separation process and selecting a fraction of proteins containing the protein of interest (POI);

(b) proteolysing said fraction of proteins containing the protein of interest (POI) to obtain a protein peptide mixture (PPM) comprising a reference peptide derived from the protein of interest (POI) by said proteolysing;

(c) providing in said protein peptide mixture (PPM) a known quantity of a mass-labelled standard peptide having the same amino acid sequence as the reference peptide;

(e) subjecting the separated protein peptide mixture of (d) to:

- mass spectrometry (MS) and comparing the signal intensity for the reference peptide with the signal intensity for the standard peptide, or - tandem mass spectrometry multiple reaction monitoring (i.e., MS/MS MRM) and comparing the signal intensity for one or more daughter fragment ions derived from the reference peptide with the signal intensity for one more corresponding daughter fragment ions derived from the standard peptide; and

(f) calculating the quantity of the protein of interest (POI) in the protein mixture (PM).

2. A method for quantification of a protein of interest (POI) in a protein mixture (PM), comprising:

(b) providing in said protein peptide mixture (PPM) a known quantity of a mass-labelled standard peptide having the same amino acid sequence as the reference peptide;

(c) separating the protein peptide mixture of (b) into fractions of peptides using a first separation process and selecting a fraction of peptides containing the reference peptide and the standard peptide; (d) separating the fraction of peptides of (c) containing the reference peptide and the standard peptide, using a further separation process;

(e) subjecting the separated peptides of (d) to:

- mass spectrometry (MS) and comparing the signal intensity for the reference peptide with the signal intensity for the standard peptide, or

- tandem mass spectrometry multiple reaction monitoring (MS/MS MRM) and comparing the signal intensity for one or more daughter fragment ions derived from the reference peptide with the signal intensity for one more corresponding daughter fragment ions derived from the standard peptide; and (f) calculating the quantity of the protein of interest (POI) in the protein mixture (PM).

3. The method according to claim 2, wherein step (c) precedes step (b), whereby the protein peptide mixture (PPM) of (a) is separated in step (c) into fractions of peptides using a first separation process and a fraction of peptides containing the reference peptide is selected; and then in step (b) a known quantity of the mass-labelled standard peptide is provided in said fraction of peptides obtained in step (c).

4. The method according to claim 1 , wherein in step (a) the protein mixture (PM) is separated using ion exchange chromatography, preferably anion exchange chromatography, more preferably strong anion exchange (SAX) chromatography, or using free flow electrophoresis (FFE), preferably in isoelectric focusing (IEF) mode.

5. The method according to any of claims 2 or 3, wherein in step (c) the protein peptide mixture is separated using ion exchange chromatography, preferably cation exchange chromatography, more preferably strong cation exchange (SCX) chromatography, or using free flow electrophoresis (FFE), preferably in isoelectric focusing (IEF) mode.

6. The method according to any of claims 1 or 4 wherein the separation of step (a) is multidimensional, or the method according to any of claims 2, 3 or 5 wherein the separation of step (c) is multidimensional.

7. The method according to any of claims 1 to 6, wherein step (d) comprises chromatography separation, preferably reversed-phase liquid chromatography (RPLC).

8. The method according to any of claims 1 to 7, wherein step (e) comprises:

(eab) peptide ions having m/z ratio about equal to the expected m/z ratio of the standard peptide ion;

(eb) subjecting the peptide ions selected in (eaa) and in (eab) to fragmentation to produce daughter fragment ions there from; (ec) subjecting the daughter fragment ions produced in (eb) to a second mass spectrometry selection configured to select for

9. The method as defined in any of claims 1 to 8, wherein two or more different proteins of interest (POI) are quantified from the same sample.

10. Use of the method as defined in any of claims 1 to 9 for quantification of biomarkers or candidate biomarkers, particularly for validation of candidate biomarkers, including qualification, verification and/or specificity testing of candidate biomarkers.

11. A device or system configured to perform the method according to any of claims 1 to 10, preferably wherein two or more steps are performed in-line.