[0001] The invention relates, in embodiments, to a method for calibrating a mass spectrometer for measuring intact macromolecules, such as proteins, protein complexes, viral capsules, nucleic acids, or other macromolecules or nanoparticles. The invention relates as well to a method of measuring material quantities in such a mass spectrometer and to a device therefore. The invention is particularly suitable for MALDI/TOF mass spectrometers and for detectors that can detect single molecules resolving their kinetic energy, such as superconducting junctions. Among others, the invention provides a Sl-traceable measurement of the level of clinically relevant macromolecular analytes in serum, such as ferritin, antibodies, large proteins, nanoparticles, or other.

Related art

[0002] Several techniques for the measurement of macromolecules in clinical samples. Immunoassays, like the known ELISA assay, are widely used but present several limitations. In many cases, it is difficult to find an antibody with a selective affinity for a specific ligand. Another limitation lies in the fact that these methods have access only to the quasi-surface of the target molecule. [0003] The measurement of ferritin in serum exemplifies these difficulties. The ELISA assay is not very selective for ferritin and cannot discriminate Iron-loaded ferritin from ferritin that is not filled with iron (apoferritin).

[0004] Another limitation of many biochemistry assays is that it is difficult to calibrate reliably the measurement in a Sl-traceable way. [0005] MALDI/TOF is a mass spectrometric technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation in a time-of-flight mass spectrometer. MALDI/TOF spectrometers conventionally use microchannel plates or other devices sensitive to secondary electrons for measuring the time of arrival of the molecular ions. Since the ionization probability when hitting the microchannel plate to produce secondary electrons for slow molecules is vanishingly low, their use is limited, in practice, to the molecular masses below some tens of kilodaltons. [0006] Superconducting tunnel junctions can detect non-ionizing impacts and have been used in MALDI/TOF to detect molecules in the megadalton range and above, as disclosed by:

1. L. D. Plath, A. Ozdemir, A. A. Aksenov and M. E. Bier, Anal. chem. 2015, 87, 8985-8993 "Determination of Iron Content and Dispersity of Intact Ferritin by Superconducting Tunnel Junction Cryodetection

Mass Spectrometry"

2. D. Twerenbold, J.-L. Vuilleumier, D. Gerber, A. Tadsen, B. van den Brandt and P. M. Gillevet, Appl. Phys. Lett. L996, 68, 3503-3505

3. US Patent 5,640,010, D.Twerenbold " Mass spectrometer for macromolecules with cryogenic particle detectors"

[0007] No matter the nature of the detector used to determine the time of arrival, there is a need of a method to calibrate a MALDI/TOF spectrometer in a reliable and Sl-traceable way, and of the corresponding device. Short description of the invention

[0008] An object of the present invention is the provision of a device and of a method for the Sl-traceable quantitative measurement of the amount of macromolecules and for the Sl-traceable calibration of the mass spectrometric detection of macromolecules.

[0009] This object is attained by the object of independent claims and in particular by a calibration method including measuring a mass distribution of intact macromolecules from a reference solution of the analyte with the mass spectrometer, providing an extract solution from the reference solution containing a component of the macromolecule, providing an isotope-diluted solution of the component by mixing the extract solution with an amount of an isotopically-enriched solution of the component; measuring the isotope abundance distribution of the isotope- diluted solution, measuring, based on the isotope abundance distribution of the isotope-diluted solution and on the mass distribution of intact macromolecules, a calibration coefficient linking the amount or the concentration (relative to volume) or content (relative to mass) of the analyte to integrated area of a region in the mass distribution.

[0010] The object of the invention includes also a measuring method and a calibrated mass spectrometer.

[0011] Dependent claims introduce important and advantageous features that are not, however, essential for the invention, notably: the use of a MALDI/TOF spectrometer, possibly with a cryogenic detector sensitive to the kinetic energy of the individual macromolecules, the use of a double isotope-dilution method to measure the amount of substance content or concentration of the component or the isotope abundance ratio in the isotopically-enriched solution, the molecular mass of the macromolecules above 100 kDa, 400 kDa, or 800 kDa, the nature of the analyte and its component that may be is iron-loaded ferritin, respectively iron, or an antibody, respectively sulphur, the use of a recombinant pure substance for the loaded under controlled conditions to provide the iron-loaded ferritin reference.

Figures

[0012] Embodiment(s) of the invention will now be disclosed with reference to the figures that illustrate schematically: · figure 1 a method according to an aspect of the invention.

• figure 2, the production of the reference substance, exemplified by the embodiment of the iron-loaded ferritin,

• figure 3, the measurement of the mass distribution of intact macromolecules, using the example of the iron-loaded ferritin, · figure 4, the Sl-traceable measurement of the amount of analyte, illustrated by the embodiment of the iron-loaded ferritins using a double isotopic dilution method.

Detailed description

[0013] The present disclosure will describe in detail embodiments of the present invention that concern the measurement of the material quantity of iron-loaded ferritin, with the of the amount of substance quantity of iron, as a component of the target analyte, by isotope dilution. Although this is an important and advantageous use case, the invention is not limited to this application and includes other variants.

[0014] A possible variant of the invention is the calibration of a mass spectrometer for the measurement of antibodies. In this case, iron is not present in the target analyte, and another component is selected, for example sulphur. [0015] As shown in figures 1 and 3. A biological sample material 18, for example serum, is analysed to measure the material quantity of iron-loaded ferritin (ILF for brevity). After an optional purification 14 the sample is mixed with a suitable matrix 38 (good results have been obtained with a dried matrix of sinapinic acid or picolinic acid dissolved in a mixture of water, ethanol, and trifluoroacetic acid, but other choices are possible), dried on a target plate 53, and inserted in the vacuum chamber 51 of a MALDI-TOF spectrometer 50. The target plate 53 may have a plurality of wells for different samples that can be analysed in succession and may include the biologic sample 18 as well as reference solutions and isotope- diluted references, as it will be disclosed in the following.

[0016] In the spectrometer 50, a laser 56, for example a nitrogen UV laser, is aimed at the wells of the target plate where it causes the macromolecules in the sample to be desorbed and ionized. The free molecules are accelerated by high voltage electrodes 52 and travel along the free flight zone to the detector 59 where they are detected. Preferably, the detector 59 comprises an array of superconducting Josephson junctions in a cryostat 57 that can detect individual molecule impacts and measure their kinetic energy and their charge, even if the impact speed is too low for the emission of secondary electrons. The time elapsed between the laser pulse and the impact measured at the detector 59 is a measure of the molecule speed, from which the mass of the molecular ion can be computed.

[0017] Other MS configurations, for example including reflectrons, are possible and included in the scope of the present invention.

[0018] After a sufficient number of molecules have been detected and their masses signals accumulated, the mass spectrum (visible in figure 1) presents a region of interest 33 whose area correspond to the total number of molecules detected, for example iron-loaded ferritin, and other features not necessarily related to the analyte in study, such as the peak 30 that corresponds to apoferritin. Other features that may appear on the spectrum are fragments, or subunit of the analyte, or ions with multiple charges. These features can be discriminated based on the mass (based on the time of flight) and in the case of multiply-charged ions, from their kinetic energy.

[0019] Besides the clinical sample 18, the invention foresees that a reference solution 40 containing the macromolecule analyte is measured and its mass spectrum is taken. This yields a second mass spectrum with similar features.

[0020] According to the invention, the amount of analyte of the reference solution 40 is measured by isotope dilution on a fragment of the macromolecule of low mass, possibly an elemental component, by a second mass spectrometer adapted for the measure of isotope abundance ratios. Thanks to this measurement, a calibration factor a linking the integral of the region of interest 33 with the material amount of the target analyte in the initial solution can be determined.

[0021] Advantageously, as depicted in figure 2, the reference solution 40 is prepared from a pure recombinant macromolecule 20, (in this embodiment, apoferritin) produced by a suitable genetically manipulated host 15. The apoferritin is loaded by iron ions 25 under controlled conditions 29.

[0022] Returning to figure 1, a measured or known amount of reference solution 40 is taken. Extraction (separation, digestion) step 13 provides a solution of an element that can be measured by isotope dilution.

[0023] In the ferritin measurement embodiment, it is advantageous to choose iron as element, because iron has several suitable stable isotopes ( ⁵⁶ Fe and ⁵⁷ Fe notably) to which isotope dilution can be applied, and because the amount of iron bound to serum ferritin is clinically significant. The extract 16 may contain ferrous (Fe ²⁺ ) or ferric (Fe ³⁺ ) ions, or another form of elemental or molecular iron in any oxidation state preferably low molecular species or single ions.

[0024] In other embodiments, where one search a calibration of a mass spectrometer for another macromolecule, iron may be replaced by another suitable component. Sulphur, for example, may be extracted and used in lieu of iron in the determination of the material quantity of antibodies or other proteins, large biomolecules or similar.

[0025] Isotope dilution allows the measurement of the material number quantity of iron n _/¾ in the reference solution 4. Preferably, as shown in figure 4, a double isotope dilution mass spectrometry (reverse IDMS) is used. While reverse IDMS is known in other contexts, it has never been applied to the calibration of mass spectrometers for macromolecules as claimed. The reference solution 40 with isotopic abundance ratio RAN is mixed with an isotopically enhanced solution 17 (the "spike" solution) with isotopic ratio R _S P and the isotopic abundance ratio RAN _S P of the mixture 27 is determined by a high-resolution mass spectrometer specialized for measuring masses close to 56-57 u.

[0026] Another mixture 28 is produced by mixing the isotopically enhanced solution 17 with a reference solution of the component 37, precisely measured, with isotopic abundance ratio RR _S , and the isotopic abundance ratio RRS SP of the mixture 28 is measured in the same way as that of mixture 27. The number quantity of iron in the reference solution 40 is then given by

[0027] Since the isotope abundance ratios RR _S and RAN are both equal to the natural isotopic abundance ratio of iron, the equation above gives the number quantity or amount of iron in the reference solution eliminating the need to measure the quantity of the isotopically-enhanced solution 17. [0028] From , one derives the proportionality coefficient a between the amount of the analyte in the reference solution 40 and the integral 35 of the mass distribution of the reference solution in the mass-region corresponding to iron-loaded ferritin. Since the amount of iron is bound to the ferritin in both cases, the same coefficient links the integral 33 in the mass distribution of the sample 18 with the amount of analyte in the same. The MALDI-TOF mass spectrometer 50 is calibrated for the analyte by a calibration coefficient a.

Reference numbers used in the drawings

[0029]

12 macromolecule analyte, ferritin

13 extraction of a component

14 purification

15 apoferritin produced

16 extract solution

17 isotope-diluted solution

18 biological sample, serum 20 recombinant apoferritin

25 macromolecule component, iron

26 isotope-enhances solution

27 first diluted solution

28 second diluted solution

29 controlled loading of apoferritin

30 apoferritin peak 33 iron-loaded ferritin peak

35 iron-loaded peak in the reference solution

36 natural-isotope reference solution

37 natural-isotope reference solution

38 matrix, e.g. sinapinic acid 40 reference solution 50 MALDI/TOF mass spectrometer 51 vacuum chamber

52 high-voltage electrodes

53 sample target

55 free flight 56 UV laser

57 cryostat

59 superconducting molecule detectors