IONISATION CONTROL

Abstract

An elution buffer for eluting one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof or for eluting one or more predetermined antibodies or fragments from a target antigen, wherein: the elution buffer has a pH of 1 to 5; and the elution buffer comprises a predetermined amount of an acid stable mass spectrometry ionisation control protein. The use of the elution buffer in the detection and quantifying of analytes, for example by mass spectrometry is also described.

Claims

1. An elution buffer for eluting one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof or for eluting one or more predetermined antibodies or fragments from a target antigen, wherein: the elution buffer has a pH of 1 to 5; and the elution buffer comprises a predetermined amount of an acid stable mass spectrometry ionisation control protein.

2. An elution buffer according to claim 1, wherein the ionisation control protein is substantially stable in the elution buffer for at least 30 days.

3. An elution buffer according to claim 1, wherein at least one mass spectrometry m/z peak value of the ionisation control protein is substantially stable for at least 30 days.

4. An elution buffer according to claim 1, wherein the ionisation control protein is selected to have at least one mass spectrometry peak having an m/z value which does not substantially overlap with a mass spectrometry peak of the or each predetermined analyte.

5. An elution buffer according to claim 4, wherein the ionisation control protein is selected to have at least one mass spectrometry m/z peak value within a predetermined mass spectrometry window used for the detection or quantification of one or more peaks from the at least one predetermined analyte.

6. An elution buffer according to claim 1 comprising an elution buffer selected from: (a) 5% v/v acetic acid in water; (b) 0.1 glycine, pH 2.0-3.0, or 0.2 M glycine pH 2-6.

7. An elution buffer according to claim 1 comprising 0.5 to 100 ng of ionisation control protein.

8. An elution buffer according to claim 1, wherein the ionisation control protein comprises at least 30 amino acids.

9. An elution buffer according to claim 1, wherein the ionisation control protein has a mass of at least 3 kDa.

10. An elution buffer according to claim 1, wherein the ionisation control protein is selected from aprotinin, β2 glycoprotein, transthyretin and α1 acid glycoprotein.

11. A kit for use in the analysis by mass spectrometry of one or more analytes comprising an elution buffer according to claim 1 and one or more analyte specific antibodies or fragments thereof specific for the one or more predetermined analytes.

12. A kit according to claim 11, wherein the analyte is a protein or peptide.

13. A kit according to claim 11, wherein the analyte or antigen specific antibody is a serum protein or peptide.

14. A kit according to claim 11, wherein the serum protein is a complement protein, an immunoglobulin or fragment thereof, albumin, β2 microglobulin, α1 microglobulin, cystatin C, a microalbumin, α1 acid glycoprotein, α1 antitrypsin, α2-macroglobin, anti-streptolysin-O, anti-tetanus toxoid immunoglobulin, apolipoprotein A, apolipoprotein B, caeruloplasmin, C-reactive protein, haptoglobin, prealbumin, rheumatoid factor, total serum protein transferrin, Haemophilia influenzae-specific immunoglobulin, diptheria toxoid-specific immunoglobulin, Streptococcus pneumoniae specific immunoglobulin, Salmonella typhi-specific immunoglobulin or Varicella zoster virus-specific immunoglobulin.

15. A kit according to claim 14, wherein the analyte specific antibodies are anti-IgA, anti-IgG, anti-IgD, anti-IgD, anti-IgE, anti-total light chain, anti-free light chain, anti-lambda light chain, anti-kappa light chain, anti-lambda free light chain, anti-kappa free light chain, anti-heavy chain subclass, anti-heavy chain class-light chain type or anti-heavy chain subclass-light chain type specific.

16. A kit according to claim 11, wherein the antibodies or fragments thereof are bound to a substrate.

17. A kit according to claim 11 comprising anti-IgG, anti-IgA, anti-IgM, anti-kappa and/or anti-lambda-specific antibodies.

18. A kit according to claim 14 comprising a predetermined amount of a control analyte.

19. A kit according to claim 11 comprising one or more of a sample diluent buffer, a reducing agent, a mass spectrometry matrix, a mass spectrometry matrix solvent, a MALDI target and a mass spectrometer mass calibrator.

20. A kit according to claim 11, additionally comprising a standard serum protein control.

21. A method of detecting or quantifying an analyte comprising immunopurifying a predetermined analyte, eluting the analyte with an elution buffer according to claim 1 and detecting the analyte and the ionisation control protein by mass spectrometry.

22. A method according to claim 21 wherein the mass spectrometry is MALDI-TOF.

23. A method according to claim 21 comprising the use of a kit.

24. A method of producing an elution buffer according to claim 1, comprising: (a) identifying the analyte; (b) identifying the m/z of at least one peak for the ionisation control compared to the m/z of one or more expected peaks for the analyte; (c) identifying ionisation control proteins having the m/z range and acid stability.

25. A computer implemented method comprising imputing an analyte, comparing one or more m/z peaks for the analyte with the m/z peak of a plurality of potential ionisation control proteins having acid stability, and outputting the identification of one or more ionisation control proteins having the m/z range and acid stability for the analyte.

26. A method according to claim 25, wherein the computer comprises a computer processor and a computer memory.

27. An apparatus for analysis by mass spectrometry of one or more analytes a method according to claim 21, comprising the use of a computer implemented method comprising imputing an analyte, comparing one or more m/z peaks for the analyte with the m/z peak of a plurality of potential ionisation control proteins having acid stability, and outputting the identification of one or more ionisation control proteins having the m/z range and acid stability for the analyte.

28. An apparatus according to claim 27, comprising a mass spectrometer.

Description

DESCRIPTION OF FIGURES

[0082] The invention will now be described by way of example only with reference to the following figures.

[0083] FIG. 1 is an example of a MALDI-TOF mass spectrum showing the mass distribution of analyte (kappa light chain (k)) following elution with acetic acid containing aprotinin. A single aprotinin peak is observed (+1 charge) which does not interfere with the analyte (kappa light chain) peaks. Ion charge states are given in parentheses.

[0084] FIG. 2 shows that the relative ionisation control protein signal remains stable in the presence and absence of analyte. The MALDI-TOF mass spectrum of aprotinin obtained in the absence of analyte (black line) does not significantly differ from the aprotinin spectrum containing kappa light chains (grey line and inset) derived from normal human serum (NHS).

[0085] FIG. 3 shows that aprotinin remains stable in 5% acetic acid. Kappa light chains were periodically eluted with 5% acetic acid containing aprotinin that had been stored at 22° C. MALDI-TOF mass spectra were acquired at each time point and the aprotinin and kappa light chain (+2) peak areas were determined (±standard deviation). No depreciation in signal was observed for either protein over an 8 week period.

[0086] FIG. 4 shows that the analyte signal relative to aprotinin as an ionisation control stays stable over time. 5% Acetic acid containing aprotinin was stored at 22° C. and used periodically to elute kappa light chains. MALDI-TOF mass spectra were acquired at each time point and the aprotinin and kappa light chain (+2) peak area ratio were determined (±standard deviation). No appreciable change in peak area ratio was observed over an 8 week period.

[0087] FIG. 5 Transthyretin (TTR) as an MALDI-TOF ionisation control. MALDI-TOF mass spectrum showing the mass distribution of TTR following elution with acetic acid, with and without mixing with polyclonal IgG (FIGS. 5A and B). A TTR peak is observed at 13827 m/z and at 6914 m/z neither of which interferes with any of the lambda or kappa polyclonal light chain peaks. The signal intensity of the TTR ionisation control peak is unchanged in the presence or absence of the analyte (B). The TTR signal peaks also do not overlap with those of aprotinin (C), nor a glycosylated kappa free light chain (of greater mass) (D). Ion charge states are given in parentheses.

[0088] An elution buffer was prepared comprising 2 ng ml.sup.−1 of aprotinin as an ionisation control for mass spectrometry in 5% acetic acid containing 20 mM tris(2-carboxyethyl) phosphine (TCEP) reducing agent. 5% acetic acid was used to both elute the analyte from the immunocapture bead and to simultaneously facilitate separation of immunoglobulin heavy chain and light chain. 20 mM TCEP was used acid an acid stable reducing agent to break the disulphide bonds holding intact immunoglobulins together.

[0089] A normal human serum sample (NHS) was diluted 1:10 and captured, (as per step 1 above), using a paramagnetic microparticle containing antibodies specific for human kappa immunoglobulin light chains. This was eluted with an acidic buffer solution containing both reducing agent and aprotinin (as an ionisation control). The elution was subsequently spotted, in a sandwich with MALDI matrix (HCCA) onto a MALDI-TOF target plate and dried. Mass spectra were acquired in positive ion mode covering the m/z range of 5000 to 30,000 which includes the singly charged (+1, m/z22705), doubly charged (+2, m/z 11353) and triply charged (+3, m/z 7569) ions of the analyte (human kappa light chains; Table 1).

TABLE-US-00001 TABLE 1 Mass spectra acquired in positive ion mode Protein +1 +2 +3 Aprotinin 6512 3257 2171 (assuming (assuming (assuming [M + H].sup.+) [M + 2H].sup.2+) [M + 3H].sup.3+) Transthyretin 13827 6914 Not (assuming (assuming determined [M + H].sup.+) [M + 2H].sup.2+) Polyclonal 22705 11353 7569 kappa LC (assuming (assuming (assuming [M + H].sup.+) [M + 2H].sup.2+) [M + 3H].sup.3+) Polyclonal 23440 11720.5 7814 lambda (assuming (assuming (assuming LC [M + H].sup.+) [M + 2H].sup.2+) [M + 3H].sup.3+)

[0090] The aprotinin intensity signal is clearly seen in FIG. 1 as a distinct peak of m/z 6512 that does not interfere or overlap with any of the three peaks of the analyte. To show that the aprotinin signal is independent of the presence of the analyte, it was analysed in the presence (+NHS) and absence (−NHS) of the latter. FIG. 2 illustrates that the aprotinin ionisation control signal intensity is the same in either case.

[0091] To investigate the stability of the ionisation control in acidic conditions, individual 50 ml aliquots of the formulation (5% acetic acid supplemented with 2 ng ml.sup.−1 aprotinin) were stored at 22° C. Individual aliquots were removed at regular intervals, supplemented with the reducing agent (TCEP) and then used to elute the analyte from an anti-kappa microparticle for MALDI-TOF analysis. The mass-spectrometric peak areas obtained for both the kappa analyte and aprotinin are shown in FIG. 3. The peak areas for both vary during the experiment. This variation is due to known MALDI spot-to-spot sample inconsistency, but there is no degradation in either analyte or aprotinin signal over a 60 day period at 22° C. By extrapolation this indicates that aprotinin is stable in acidic conditions for at least 6 months when stored at 4° C. (using the Arrhenius equation). When the stability data is expressed as a ratio of analyte signal peak to ionisation control signal peak, the variability is considerably minimised (FIG. 4). This illustrates the use of the aprotinin (as an ionisation control) to overcome ionisation differences between different MALDI-TOF acquisitions.

[0092] FIG. 5 shows another example of an ionisation control, transthyretin. MALDI-TOF mass spectrum were produced showing the mass (m/z) distribution of TTR (0.01 mg/ml) in elution buffer in the presence or absence of 0.1 mg/ml polyclonal IgG (FIGS. 5A and B). A TTR monomer peak is observed at 13827 m/z (+1 charge state) and at 6914 m/z (+2 charge state) neither of which interferes with any of the lambda or kappa polyclonal light chain peaks from the IgG. The signal intensity of the TTR ionisation control peak is unchanged in the presence or absence of the analyte (FIG. 5B). The TTR signal peaks also do not overlap with those of aprotinin (FIG. 5C), nor a glycosylated kappa free light chain (of greater m/z) (FIG. 5D).