The invention relates to the determination of the nature and strength of enzymatic activity in blood using mass spectrometric measurement of a profile of the reaction products. The determination of the enzymatic activity can be used for medical diagnostics, for example, and also to check the effectiveness of medication.
The invention provides a method whereby adding probe substances usually not present in blood offers standardized substrates for measuring the enzymatic activity. The probe substances may be added to whole blood, plasma, or serum. The mass spectrometric measurement of the reaction products, after their reversible immobilization on actively binding surfaces of solids, for example, can deliver biomarker patterns of the reaction products which may be indicators for metabolic anomalies or diseases, since these are often accompanied by the formation or activation of characteristic enzymes.
In a recent publication “Differential exoprotease activities confer tumor-specific serum peptidome patterns”, J. Villanueva et al., J. Clin. Invest., 116: 271-284 (2006), it was explained that the digest peptides in blood, which have been known for a long time, are not just useless trash, as had largely been assumed, but that it was possible to identify characteristic patterns therein for the identification of disease-specific enzyme activities. The authors were able to use the digest peptide patterns of endogenous blood proteins which are present in high concentrations, such as fibrinogens or other clotting factors, to distinguish between three different types of cancer as well as healthy control samples. They coined the name “serum peptidome” for the pattern of the peptides present in blood. The paper was held to be so sensational that two famous specialists in the field wrote a long accompanying comment: “Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold”, L. A. Liotta and E. F. Petricoin, J. Clin. Invest., 116: 26-30, 2006.
After broadband extraction by superficially hydrophobic magnetic beads, the digest peptides were measured in a MALDI mass spectrometer, i.e. with ionization by matrix-assisted laser desorption; the peptides extracted from a sample in this way can be recorded in a single mass spectrum. The analysis of these breakdown products shows that the breakdown reactions of the proteins by the enzymes in the blood did not proceed in the same way in all blood samples but rather the nature and speed of the reactions differed depending on the disease. Moreover, the paper confirmed that not all proteins in the blood are broken down. Practically no digest peptides of the most prevalent proteins, i.e. the albumins and globulins, can be found. These high-molecular proteins are protected by their structure in such a way that they resist enzyme attack. The digest peptides could be mostly assigned to the clotting factors such as fibrinogen α or C3f.
Mass spectrometric diagnostics carried out by measuring and evaluating mass spectra obtained from substance mixtures extracted from body fluids is still in its infancy. This is true for both the development of the measurement and evaluation methods as well as for the licensing of the mass spectrometers for diagnostic purposes. The first mass spectrometers which are licensed for medical diagnostics are now coming onto the market. In Europe, the licensing is effected by means of a manufacturer's IVD declaration of conformity (CE), which is strictly monitored by official bodies. The abbreviation IVD stands for “in vitro diagnostics”. In Germany, this licensing is regulated by the Medical Devices Act (MPG), which is based on the European Directive 98/79/EC. Outside Europe there are usually regulations which require licensing by official bodies.
A particularly promising diagnostic method is one which measures protein mixtures extracted from body fluids, especially blood. The mass spectra of the protein mixtures are often simply called “protein profiles”. Blood is routinely taken from a vein in all general practices; the risk from side effects is very low. The risk is even smaller when only one drop of blood is taken from the fingertip or earlobe.
In the protein profiles from the blood, significant over- and underexpressions of specific proteins can be measured, which are reflected in concentrations that are too high or too low. It is also possible to measure chemical changes in proteins, which then appear with a different molecular weight at other points in the mass spectrometric protein profile. Statistically significant changes of this nature are always an indication of a specific stress situation of the body, and in some cases are even characteristic of a specific disease of bodily organs or of a metabolic anomaly. Such proteins which undergo characteristic changes in their concentration or molecular weight as a result of stress are now termed “biomarkers”. The term “biomarker” increasingly refers not just to an individual protein, but rather to a pattern of several proteins or protein derivatives which characteristically change in terms of their concentration ratio to each other. In a wider sense, it does not have to be a protein profile; it can also be a mass spectrum of any extracted substance mixture from which biomarkers can be obtained. Measurements of such biomarkers or biomarker patterns can be used to medically diagnose diseases, metabolic anomalies or the response to medication, and also for many more purposes, from the explanation of breakdown pathways in metabolomics through to pharmacokinetic analyses in the development of new drugs. After all, biomarker patterns are not only to be found in blood, but also in any body fluids including lyzed tissue, even if the focus of attention here is especially on blood samples.
Blood consists mainly of water (over 90%), various types of blood particles, small amounts of salts, several non-protein organic substances, and around seven percent is made up of proteins, of which albumins and globulins form the largest part. The next most prevalent are the so-called clotting factors, above all the fibrinogens. The general term “blood samples” below can mean “whole blood”, “blood serum” or “blood plasma”; the differences are explained in more detail below. The proteins which are of interest here as possible biomarkers are present in the blood samples at concentrations of less than one percent down to 10−10 percent. The proteins present in very low concentrations elude direct measurement if they cannot be especially “fished out” in a substance-specific way or indirectly measured by the effects they cause. One type of such indirect measurement by an enzymatic effect has already been described in detail above.
If the blood particles are removed from blood which has just been taken (the “whole blood”) by centrifuging, for example, then the “blood plasma” is obtained, which still contains all the clotting factors, above all the fibrinogens. If it is to be stored for a long period or transported, the blood plasma must be prevented from coagulating by adding anticoagulants. If, on the other hand, the whole blood is coagulated, the fibrinogens are broken down to fibrins through different stages with the assistance of the other clotting factors. These fibrins polymerize and together with the blood corpuscles form the blood clot. If this blood clot is removed by centrifuging, one obtains the “blood serum”, which now contains (almost) no coagulants.
In a good mass spectrometer, a hundred attomols of a protein (60 million molecules) can still provide a measurable signal; but in a protein profile with its background noise, this detection limit is higher because of an unavoidable background noise; it is around a hundred femtomoles.
For a peptide with a molecular weight of 1,000 Daltons, this corresponds to a hundred picograms, and to one nanogram for a protein with a molecular weight of 10,000 Daltons. Thus, in a small drop of blood containing only ten microliters of blood (ten milligrams), proteins down to a concentration of 10−5 percent by weight can be measured if it proves possible to feed all the proteins of interest to the measurement and to measure a protein profile which is not completely overloaded with proteins. Close to the detection limit, however, the accuracy of the measurement is not very good, so direct measurement and evaluation of the protein profiles is today generally limited to the concentration range between 10−1 and 10−3 percent by weight.
The smaller proteins with molecular weights of up to several thousand Daltons, which consist of only a few tens of amino acids, are called peptides; unless otherwise specifically mentioned, they are included here in the term “proteins”, however. The definition line between peptides and proteins is very unclear. The vast majority of peptides in the blood are so-called “digest peptides”, which are created as a result of the continuous enzymatic breakdown of larger proteins, which is sometimes stronger and sometimes weaker, for example the breakdown of fibrinogens, but also of foreign proteins. Until very recently, these digest peptides (the “peptidome”) were considered to be “trash” which contained no information about the state of the body.
In the paper cited above it was demonstrated, however, that characteristic patterns of peptides can be measured in blood, each indicating the activity of particular enzymes. The catalytic activity of these enzymes is principally directed at the fibrinogens and other clotting factors as substrate and breaks these down in various stages in conjunction with endopeptidases and exopeptidases to give digest peptides which, through the action of the exopeptidases, are present in part as digestion ladders of various lengths. In keeping with their task, the clotting factors are neither particularly stable nor particularly protected, and hence easily accessible to enzymatic breakdown. The activities of the enzymes measured indirectly on the basis of the nature and strength of the digest peptides were, in turn, unambiguously assigned to specific types of cancer. It was possible to detect the patterns of these digest peptides not only in the whole blood but also in the blood serum and blood plasma. The enzymes themselves eluded mass spectrometric measurement because of their very low concentration; they only revealed themselves through the reaction products of their catalytic activity. These peptide patterns can thus indeed serve as biomarkers. This indirectly extends the concentration range which is available for biomarker measurements to specific types of enzymes and increases it by several powers of ten for these enzymes.
Endo-peptidases cut proteins at certain enzyme-specific points in the middle of the amino acid chain of the proteins. One example of this is the familiar digest enzyme trypsin, which cuts adjacent to the amino acids lysine and arginine respectively. The motif at which a specific enzyme cuts can consist of a specific amino acid or a short chain of several amino acids. Exopeptidases, on the other hand, break down peptides from the end: one amino acid after the other is removed, generally creating a mixture of digest peptides of different lengths, the difference between them being one amino acid, which enables the sequence of the broken down peptides to be identified in a mass spectrometric measurement by the mass differences. Exopeptidases work from either the C-terminal or the N-terminal end of the peptide, not from both ends at the same time. The mixtures of breakdown products created by exopeptidases are also called “digestion ladders”.
All enzymes have a catalytic effect on other substances, which are termed the “substrate” of the enzyme, and which are modified by the catalytic activity of the enzyme in a way which is characteristic of that enzyme. The enzymes are therefore not used up through their activity, but rather their activity gradually decreases over quite long periods of some days; the activities of other enzymes or even self-digestion also play a part. The half lives of the enzymes' activity are a few days; freezing prevents the activity from diminishing. The speed of the catalytic reactions, and hence the speed of modification of the substrates, varies greatly. “Sluggish enzymes” have a reaction rate of around one substrate molecule per second and enzyme molecule; fast enzymes can exhibit a reaction rate of up to 100,000 substrate molecules per second and enzyme molecule. The fastest known enzyme is catalase, which breaks down hydrogen peroxide, which is toxic to the body. A fast reaction rate requires that sufficient substrate molecules are available, however, and also that diffusion does not cause a restriction. The peptidases, which digest proteins and peptides, have reaction rates of around 100 to 1,000 substrate molecules per second and enzyme molecule.
The number of different types of protein in the blood is extremely high, far above 100,000. Even in the narrow concentration range for the direct measurement of the protein profiles there are many thousands of proteins. A mass spectrum with such a large number of proteins would not enable an individual protein to be identified because the mass spectrometric signals would produce unresolved superimpositions. It is therefore necessary to drastically reduce the number of proteins before measuring a protein profile, yet still provide a large number of proteins for the measurement. This is generally done by means of broadband extractions, which are able to extract proteins which share specific properties from the blood. Such broadband extractions can simultaneously extract several tens to several hundreds of proteins whose concentrations are in the measurable range. The term “broadband extraction” should not be interpreted too narrowly here, however; extractions which, for example, extract only two proteins for a determination of their concentration ratio in a reproducible way should also be understood as being covered by the term broadband extraction.
There are different types of broadband extraction, different with respect to “what” (types of protein extracted) as well as to “how” (extraction mechanism). Reversible immobilization of proteins on suitable actively binding surfaces of solids is the easiest extraction mechanism to work with. It is the only one considered here. The actively binding surfaces of solids are generally produced by stably coating the surfaces of the solids with suitable substances.
The different modes of operation of broadband extractions are distinguished by actively binding surfaces of solids which have different coatings; they extract completely different types of protein out of the blood sample. The proteins can be bound to the surface by means of electric interactions, for example, by stably coating the surfaces with anion or cation exchangers. This extracts proteins with different ionic charges from the blood. Other proteins can be affinely bound via hydrophobic bonds, as occurs in reversed phase chromatography. Other types of protein again can be held on the surface by means of various chelate-type bonds, by substance-specific ligand bonds, and also by customized, protein-specific bonds along the lines of the antigen-antibody bond. This means that a mixture of different types of antibodies stably bound to the surfaces of solids can also extract a protein profile.
Different types of broadband extraction usually produce totally different protein profiles because quite different types of proteins are extracted by reversible immobilization on the surface of the solids in each case. Patterns of characteristic biomarkers can also be composed of signals in different types of protein profiles; they therefore do not need to originate from a single protein profile.
Digest peptides are generally extracted by hydrophobic surfaces. C8 and C18 surfaces, in particular, have proven to be successful here. These surfaces are covalent bonds of alkanes with 8 or 18 carbon atoms (generated by “alkylation”). If the digest peptides have some non-polar amino acids in their chain, then they are reversibly bound here. These hydrophobic coating layers are also called reversed phases. Ferromagnetic particles (magnetic beads) with such coatings are commercially available.
The actively binding surfaces for this broadband extraction can belong to different types of solids. The vessel walls of the sample containers can themselves be actively coated, or actively binding sampling spots can be located on special sample supports. The coatings must be stably, i.e. irreversibly, bound. The samples can be forced through actively binding filter material in the form of felt, open-pored foams or particle-filled cavities. Macroscopic beads or pellets, or suspensions of microparticles or nanoparticles with actively binding surfaces can be added to the liquid sample and later recovered from the sample liquid together with the immobilized extraction substances by filtration, centrifuging or magnetic forces. The extraction substances can be washed in the immobilized state on the surface of the solid and hence freed of all other substances. The immobilized extraction substances can then be released again where and when necessary by suitable eluents.
The above-described indirect measurement of the enzyme activity in blood with the aid of the resulting reaction products is a breakthrough for the diagnostic application of biomarkers, but also has its disadvantages. Alone the changing composition of the blood proteins which can be used for a digestion, and other problems as well, mean that analysis of the digest peptides cannot be standardized very well. It has turned out, for example, that some clotting factors in the blood of patients with certain diseases were not present in measurable quantities at all because they had already been completely digested in the patient's body. In the paper cited, the researchers were able to prove that adding such clotting factors, for example fibrinogen β, leads to a breakdown in seconds. Furthermore, there are always some enzymes in the blood which continuously lead to breakdown products so that it is sometimes difficult to distinguish between these and disease-specific enzymes.
A further problem of carrying out diagnostics with the aid of digest peptides from blood samples is the undesired, continuous change during storage and transportation. Blood is a long-lastingly reactive fluid; the enzymes it contains do not lose their effectiveness when a blood sample is taken. It is not just that proteins are digested or changed in a characteristic way; chemical processes such as oxidation are also frequently enzyme-controlled. Equilibria which have formed in the blood circulation are disturbed in the blood sample taken. Another consideration is clotting, caused by the conversion of fibrinogens into fibrous fibrin, which is also controlled by enzymes such as thrombin. Thrombin is formed by the decomposition of the platelets, which are a type of small blood particle. The speed of change of the proteins in blood depends on many factors. For example, the temperature of the sample is important, as is its state of motion, the oxygen content, and the individual composition of the blood itself. Blood must therefore always be stabilized for transportation or storage. This causes problems when the samples have to be transported from the location where the blood is taken (usually a doctor's practice) to the place where the mass spectrometric measurement is carried out.
Freezing the whole blood sample, the blood serum or the blood plasma at minus 80° C. (−112° F.) has therefore established itself as the best type of stabilization method, but it can only be carried out in a small number of doctor's practices, and usually only in hospitals. Carriers who specialize in refrigerated transportation have to be used to transport the blood over long distances, making the transportation expensive.
The objective of the invention is to provide methods for determining enzyme activities in blood.
USPTO Backgrounds
USPTO Backgrounds