Ionization 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 contains a predetermined amount of an acid-stable mass spectrometry ionization control protein. Also described is the use of the elution buffer in detecting and quantitating analytes, for example, by mass spectrometry.

Description

[Technical Field]

[0001] The present invention relates to elution buffers containing predetermined amounts of acid-stable ionization controls for use in mass spectrometry, kits containing such buffers, and methods for making such buffers and kits. [Background technology]

[0002] background Protein profiling by mass spectrometry has important clinical utility in in vitro diagnostics; however, analytical reproducibility remains a potential problem, and peak intensities and m / z values ​​can vary significantly between experiments.

[0003] Current approaches to control analytical variability and reproducibility to enable routine use in the in vitro diagnosis of human disease include automated sample processing, a wide range of prefractionation strategies, immunocapture, prestructured target surfaces, standardized matrix (co)crystallization, improved MALDI-TOF mass spectrometry (MS) instrument components, internal standard peptides, quality control samples, replicate measurements, normalization, and peak detection algorithms (Albrethsen, J., Clin Chem 2007; 53(5): 852-858).

[0004] However, these methods cannot accurately reflect spotting, crystallization, and ionization because they are subject to other factors in addition to crystal formation between the matrix and the sample.

[0005] Previous attempts to control inter- and intra-experimental variability in MALDI-TOF MS have typically utilized internal calibration peptides with comparable physicochemical properties to the protein of interest, spiked into samples at various concentrations, before comparing the ion intensities of the calibration peptide and analyte. However, a problem with this approach is the introduction of variability in peak intensities. Combining controls with iterative algorithms and / or performing replicate analyses has been proposed as a possible solution to correct for some analytical variation over time and improve the reproducibility of protein profiling by MALDI-TOF MS (Albrethsen, J., Clin Chem 2007; 53(5): 852-858).

[0006] For example, one study utilized a method in which a synthetic peptide with the same primary sequence as a specific analyte was spiked into a blood sample (Yi, J et al., Methods Mol Biol 2011; 728: 161-75).

[0007] The use of an internal control in matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis has also been shown to improve the sensitivity of measuring bacterial concentrations in samples. Addition of cytochrome C as an internal control reduced signal intensity by 20–30% in samples containing high concentrations of bacteria, but improved signal intensity for some low concentrations of bacteria. In this case, the protein was spiked into the matrix and premixed at a matrix-to-analyte ratio of 2:1 (Gantt, SL et al., J Am Soc Mass Spectrom 1999; 10(11):1131-7).

[0008] In another example, the effect of ion suppression in atmospheric pressure matrix-assisted laser desorption / ionization mass spectrometry was investigated by spiking all fractions of 384 Prespotted AnchorChips with an angiotensin II analog as an internal standard (Li, G et al., Rapid Commun Mass Spectrom 2019; 33(4): 327-335). Signal intensities were then normalized according to the control, followed by peak clustering analysis. Lower intensity peaks were as reproducible as higher intensity peaks.

[0009] Other attempts to adjust for sample-to-sample variability in quantification include post-analytical adjustment by outlier removal and baseline removal by intensity scaling (Neubert et al., J Proteome Res 2008; 7(6) 2270-9).

[0010] One example of an in vitro diagnostic use in which mass spectrometry has significant utility relates to the many proliferative disorders involving antibody-producing cells.

[0011] Antibody molecules (also known as immunoglobulins) have dyad symmetry and typically consist of two identical heavy chains and two identical light chains, each containing a variable and constant region. The variable regions of the heavy and light chains combine to form the antigen-binding site, and thus both chains contribute to the antigen-binding specificity of the antibody molecule. The basic tetrameric structure of an antibody contains two heavy chains covalently linked by disulfide bonds. Each heavy chain is then linked to a light chain, again via a disulfide bond. This creates an essentially "Y"-shaped molecule.

[0012] Many such proliferative disorders involve proliferation of plasma cells to form monoclonal tumors of the same plasma cells, which result in the production of large amounts of the same immunoglobulin, known as monoclonal gammopathy.

[0013] Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% of cancer deaths in the UK, respectively. Multiple myeloma is the second most common hematologic malignancy after non-Hodgkin's lymphoma. In Caucasian populations, its incidence rate is approximately 40 cases per million per year. Traditionally, the diagnosis of multiple myeloma is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine, and associated organ or tissue damage such as hypercalcemia, renal failure, anemia, or bone lesions. While normal bone marrow plasma cell content is approximately 1%, in multiple myeloma the content is typically greater than 10%, often greater than 30%, and sometimes greater than 90%.

[0014] AL amyloidosis is a protein conformational disorder characterized by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typically, these patients present with cardiac or renal failure, but peripheral nerves and other organs may also be involved.

[0015] Numerous other diseases can be identified by the presence of monoclonal immunoglobulins in a patient's bloodstream or even urine. These include plasmacytoma and extramedullary plasmacytoma (a plasma cell tumor that arises outside the bone marrow and can occur in any organ). When present, the monoclonal protein is typically IgA. Multiple solitary plasmacytomas can occur with or without evidence of multiple myeloma. Waldenström's macroglobulinemia is a low-grade lymphoproliferative disorder associated with the production of monoclonal IgM. There are approximately 1,500 new cases per year in the United States and 300 new cases in the United Kingdom. Quantitation of serum IgM is important for both diagnosis and monitoring. B-cell non-Hodgkin's lymphoma accounts for approximately 2.6% of all cancer deaths in the United Kingdom, and monoclonal immunoglobulins were identified in the serum of approximately 10–15% of patients using standard electrophoresis techniques. In B-cell chronic lymphocytic leukemia, monoclonal proteins have been identified by free light chain immunoassay.

[0016] Additionally, there is a condition called MGUS, which stands for monoclonal gammopathy of undetermined significance. This term describes the unexpected presence of intact monoclonal immunoglobulins in individuals without underlying conditions such as multiple myeloma, AL amyloidosis, or Waldenström's macroglobulinemia. MGUS can be found in up to 1% of the population over 50 years of age, 3% of those over 70 years of age, and 10% of those over 80 years of age. Most of these are IgG- or IgM-related, but more rarely, they can be IgA-related or biclonal. Most individuals with MGUS die from unrelated illnesses, but MGUS can also transform into malignant monoclonal gammopathy.

[0017] In at least some cases of the diseases mentioned above, there are abnormal concentrations of monoclonal immunoglobulins or free light chains. When the disease causes abnormal replication of plasma cells, this cell type often produces more immunoglobulins, and the "monoclones" multiply and appear in the blood.

[0018] A highly sensitive assay capable of separately detecting free kappa and free lambda light chains has been developed. This method uses polyclonal antibodies specific for either free kappa or free lambda light chains. In WO97 / 17372, increasing the likelihood of such antibodies was also discussed as one of various possible specificities. The document discloses a method for tolerizing animals to enable the production of more specific desired antibodies than could be produced by conventional techniques. The free light chain assay uses antibodies to bind to free lambda or free kappa light chains. The concentration of free light chains is measured by nephelometry or turbidimetry.

[0019] Characterization of the amount or type of free light chain (FLC), heavy chain or subclass, or light chain type bound to a heavy chain class or subclass is important in a wide range of diseases, including other immune diseases, including B-cell disorders such as multiple myeloma and B-cell disorders such as monoclonal gammopathy (of which multiple myeloma is an example), and both hypergammaglobulinemias and hypogammaglobulinemias.

[0020] WO2015 / 154052, incorporated herein in its entirety, discloses a method for detecting immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof using MS. A sample containing immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof is immunopurified and subjected to mass spectrometry to obtain a mass spectrum of the sample. This can be used to detect monoclonal proteins in samples from patients. It can also be used for fingerprinting, isotyping, and identifying monoclonal antibodies.

[0021] MS can be used, for example, to separate lambda and kappa chains in a sample by mass and charge. It can also be used to detect heavy and light chain components of immunoglobulins, for example, by reducing disulfide bonds between the heavy and light chains using a reducing agent. MS is also described in WO2015 / 131169, the entire contents of which are incorporated herein.

[0022] Purification of immunoglobulins in samples in diagnostic procedures typically uses antibodies against whole antibodies and / or free light chains, such as anti-IgG, anti-IgA, anti-IgM, anti-IgD, anti-IgE, anti-total kappa, anti-total lambda, or anti-free light chain antibodies, e.g., anti-free kappa (κ) or anti-free lambda (λ) light chain antibodies. It is important to have calibrators to ensure that the purification and detection steps are performed accurately.

[0023] WO2017 / 144900 describes a number of controls that utilize either heavier versions of the analyte being detected or monoclonal versions of the analyte being detected, i.e., IgA can be quantified relative to a predetermined amount of heavier IgA kappa.

[0024] This is because different proteins are expected to crystallize at different rates in the mass spectrometry matrix. This means that different amounts of control proteins and analytes will be detected when the matrix is ​​sampled by mass spectrometry. Furthermore, their ionization rates are also expected to differ. This will lead to discrepancies in the apparent amounts of immunoglobulin detected. In addition, analytical reproducibility issues with mass spectrometry-based methods mean that peak intensities can vary significantly between experiments, and mass drift can occur, which can affect the reported m / z values. MALDI-TOF ionization, for example, relies on spot-to-spot variation processes in the crystalline form between the matrix (e.g., HCCA) and the sample.

[0025] The inventors surprisingly discovered that analytical variability in mass spectrometry ionization can be controlled by using independent markers in the acidic elution buffer after immunoprecipitation and before spotting. Summary of the Invention

[0026] overview Provided herein is 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 1-5, more preferably a pH of 1-3, or even more preferably a pH of 1.5-3.0; and the elution buffer contains a predetermined amount of an acid-stable mass spectrometry ionization control protein.

[0027] The elution buffer can be used for elution, for example, to elute an analyte bound to an antibody attached to a substrate, or a target antigen can be attached to a substrate and antigen-specific antibodies or fragments eluted from the target antigen.

[0028] Such elution buffers are used to release analytes bound to analyte-specific antibodies. Including ionization controls in the buffer allows them to be provided by the supplier and reduces user error resulting from the user having to separately measure or prepare the amount of ionization control material to be used.

[0029] Ionization controls can also be used as "lock mass spectral calibrators" in methods of mass spectrometry, including, for example, both MALDI and electrospray mass spectrometry. Such lock mass spectral calibrators can be recalibrated in real time within a spectrum by correcting for m / z shifts resulting from drift in the instrument and MALDI target plate with ions of known m / z values ​​obtained from the ionization controls.

[0030] The sample containing the analyte may be a biological sample such as blood, saliva, serum, plasma, cerebrospinal fluid or urine, more typically blood, serum or plasma.

[0031] The sample may be from a subject exhibiting hypogammaglobulinemia or hypergammaglobulinemia. The subject may have a proliferative disorder associated with antibody-producing cells, such as a monoclonal gammopathy. These include myeloma and primary systemic amyloidosis, plasmacytoma, Waldenstrom's macroglobulinemia, and MGUS.

[0032] The ionization control protein is selected to be matched to the analyte of interest.

[0033] The ionization control protein may be substantially stable in the elution buffer for at least 30 days, more preferably at least 60 days, typically at least 4 months or at least 6 months.

[0034] Long-term storage of inadequate controls can result in physical stability issues, protein precipitation or other effects in the elution buffer, and poor crystallization or ionization on the target, both of which can affect m / z peak height and area.

[0035] Damage to the control itself that results in a change in mass or ionization state can also alter the measured m / z.

[0036] The control protein may be stable, for example, at or below 22° C., or at 4° C. It may be pH, UV or light stable.

[0037] At least one mass spectrometric m / z peak value of the ionization control protein may be substantially stable as described above.

[0038] The ionization control protein can be selected to have at least one mass spectrometry peak with an m / z value that does not substantially overlap with the mass spectrometry peak of the or each given analyte, typically can be selected to ionize consistently, and typically can be selected not to substantially affect the intensity of the mass spectrometry signal.

[0039] Ionization control proteins are used to detect or quantify one or more peaks from at least one predetermined analyte and can be selected to have at least one mass spectrometric m / z peak value within a predetermined mass spectrometric window, or m / z range seen by the mass spectrometer.

[0040] The sample can typically be treated with a reducing agent after elution and before mass spectrometry. This is particularly useful when immunoglobulin light chains in the sample are bound to heavy chains. The use of a reducing agent separates the light chains from the heavy chains, allowing the light chains to be detected separately in a mass spectrometer. The reducing agent can also separate other analyte proteins and, if present, separate subunits.

[0041] Decoupling can be achieved by treating the whole immunoglobulin with a reducing agent, such as DTT (2,3-dihydroxybutane-1,4-dithiol), DTE (2,3-dihydroxybutane-1,4-dithiol), thioglycolate, cysteine, sulfite, bisulfite, sulfide, bisulfide, TCEP (tris(2-carboxyethyl)phosphine), 2-mercaptoethanol, and salt forms thereof. In some embodiments, the reduction step is carried out at elevated temperatures, e.g., in the range of about 30°C to about 65°C, such as about 55°C, to denature the protein.

[0042] The decoupling step is typically performed after immunopurification or other enrichment of immunoglobulins in a sample, or as part of an elution step after immunopurification of a sample.

[0043] The antibodies used in immunopurification may be intact antibodies or fragments thereof, such as Fab, F(ab) and F(ab'). 2 The antibody or fragment thereof may be cross-linked, for example, as described in WO2017144903, which is incorporated herein in its entirety.

[0044] Any acidic buffer (pH 1 to 5, more preferably pH 1 to 3, or pH 1.5 to 3) can be used as long as it does not interfere with the ionization step of a mass spectrometer such as MALDI-TOF.

[0045] Elution buffers may contain organic acids such as citric acid, acetic acid, formic acid, uric acid, propionic acid, and inorganic acids such as hydrochloric acid. Acidic buffers or solutions containing salts should be avoided, especially at higher concentrations, as these may interfere with ionization or crystallization.

[0046] For example, the elution buffer of the present invention may comprise: (a) 5% v / v acetic acid in water; (b) 0.1 M glycine pH 2.0–3.0 or 0.2 M glycine pH 2–6 The elution buffer may comprise an elution buffer selected from:

[0047] The buffer containing 5% acetic acid preferably has a pH of approximately 2.

[0048] The elution buffer may contain 1 to 100 ng / μl, more preferably 1 to 10 ng / μl, of an ionization control protein.

[0049] The reducing agent may be used in combination with the elution buffer and may further comprise tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol, or cysteine. The reducing agent may be pre-weighed or provided to provide a final concentration ranging from 10 to 100 mM, or more preferably approximately 20 mM.

[0050] The ionization control protein may contain at least 30 amino acids, or at least 50 amino acids, and / or may have a mass of at least 3 kDa, or for eluting one or more predetermined antibodies or fragments from the target antigen kDa.

[0051] Ionization control proteins advantageously have different mass ranges, or ion gates, or have multiple charge states to allow for use within the assay window of the analyte. Ionization control proteins or peptides can be natural or synthetic.

[0052] Proteins suitable for use as ionization controls may include aprotinin, α1-acid glycoprotein, β2-glycoprotein, or prealbumin (also known as transthyretin). More preferably, the ionization control may include aprotinin or transthyretin. Aprotinin is a serine protease inhibitor derived from bovine pancreas. It is readily available from commercial sources as both a pure protein and a pharmaceutical; TRASYLOL (CAS No. 9087-70-1, molar mass 6511.5 Da, UniProtKB Accession No. P00974, isoelectric point pH 10.5). It is stable at elevated temperatures in neutral or acidic media. Transthyretin (TTR, prealbumin, or TBPA) is a transport protein found in serum and cerebrospinal fluid, transporting the thyroid hormones thyroxine and retinol-binding protein bound to retinol. It is a 55 kDa homotetramer or dimer with a quaternary structure. The human protein has the UniProtKB accession number P02766.

[0053] However, other substantially acid-stable proteins can be used if a different mass range (ion gate) or one or more protein charge states are suitable for use in a particular m / z assay window. For example, in the m / z 5-30 kDa assay window, α1-acid glycoprotein (+1-21560), β2-glycoprotein I (+1-36255), or prealbumin monomer (+1-13760) would be suitable.

[0054] Targeting larger mass windows, such as dual ion gating approaches, or wide mass windows is possible through the use of additional plates in the instrument and reversal of the gate.

[0055] Also provided herein is a kit for use in the analysis of one or more analytes by mass spectrometry, comprising an elution buffer as described above and one or more analyte-specific antibodies or fragments thereof specific for one or more predetermined analytes.

[0056] The analyte or antigen-specific antibody may be a protein or peptide, more preferably a serum protein or peptide.

[0057] Antigen-specific antibodies include anti-streptolysin O, anti-tetanus toxoid immunoglobulin, Haemophilus influenzae-specific immunoglobulin, diphtheria toxoid-specific immunoglobulin, Streptococcus pneumoniae-specific immunoglobulin, Salmonella typhi-specific immunoglobulin, or Varicella zoster virus-specific immunoglobulin.

[0058] When the analyte is a serum protein, the serum protein may include one or more complement proteins, for example, the serum protein may include one or more complement protein components such as C1, C2, C3, C4, or components thereof, e.g., components C3a, C3b, C3c.

[0059] The serum protein may include immunoglobulins or fragments thereof, albumin, β2-microglobulin, α1-microglobulin, cystatin C, microalbumin, α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, antistreptolysin O, anti-tetanus toxoid immunoglobulin, apolipoprotein A, apolipoprotein B, ceruloplasmin, C-reactive protein, haptoglobin, prealbumin, rheumatoid factor, or total serum protein transferrin.

[0060] The analyte may be a monoclonal antibody, such as a therapeutic monoclonal antibody. The analyte-specific antibodies that may be included in the kit may be one or more of anti-IgA, anti-IgG, anti-IgM, 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 antibodies; more preferably anti-IgG, anti-IgA, anti-IgM, anti-kappa, and / or anti-lambda specific antibodies.

[0061] The antibody or fragment thereof specific for one or more predetermined analytes may further be bound to a substrate; for example, the antibody or fragment thereof may be bound to a latex bead. The target antigen may also be bound to a substrate such as a latex bead.

[0062] The kit further includes a predetermined amount of a control sample.

[0063] The kit may include one or more of a sample dilution buffer, an immunocapture reagent or beads, a wash buffer, an elution buffer including an optional reducing agent, a mass spectrometry matrix, a mass spectrometry matrix solvent, a MALDI target, and a mass spectrometry calibrator.

[0064] The reducing agent of the kit may include tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol, or cysteine, and may be as defined above.

[0065] The reducing agent is preferably pre-weighed or otherwise provided to provide a final concentration in the range of 10-100 mM, or more preferably approximately 20 mM.

[0066] The kit may additionally include a standard serum protein control, for example, the kit may include an antibody that is an anti-human specific antibody.

[0067] Also provided herein is a method for detecting and quantifying an analyte, comprising immunopurifying the analyte of interest, eluting the analyte with an elution buffer according to the present invention, and detecting the analyte and ionization control protein by mass spectrometry.

[0068] The method is not limited to any particular method of mass spectrometry; however, the method of mass spectrometry may include liquid chromatography mass spectrometry (LC-MS) or MALDI-TOF mass spectrometry. More preferably, the method of mass spectrometry may include MALDI-TOF mass spectrometry.

[0069] The immunoassay to which the present invention is applied has three main steps: 1) immunocapture of the analyte, 2) elution of the analyte, 3) optional reduction of the analyte, and 4) spotting of the analyte onto the MALDI-TOF target plate.

[0070] The present invention provides that an ionization control protein may be included in the reagent used in step 2), so that the control and analyte are combined before step 3) and advantageously spotted together in step 4. This is important because the ionization control is used to control for variability in step 3 and subsequent ionization in the MALDI-TOF mass spectrometer.

[0071] The method may further provide for the use of a kit according to the invention for use in mass spectrometric analysis of one or more analytes comprising an elution buffer according to any one of the previous claims and one or more analyte-specific antibodies or fragments thereof specific for one or more predetermined analytes,

[0072] Also provided herein is a method for producing an elution buffer according to the present invention, wherein the elution buffer is for eluting one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof; The elution buffer has a pH of 1 to 6, more preferably a pH of 2 to 6, even more preferably a pH of 1 to 4, or even more preferably a pH of 1.5 to 3.0; and the elution buffer contains a predetermined amount of an acid-stable mass spectrometry ionization control protein.

[0073] The method for producing an elution buffer according to the present invention comprises: (a) the method of identifying the specimen; (b) identifying the m / z of at least one peak in the ionization control by comparing it with the m / z of one or more expected peaks in the analyte; (c) a method for identifying ionization control proteins with m / z range and acid stability; Includes.

[0074] A computer-implemented method comprising inputting an analyte, comparing one or more m / z peaks of the analyte with the m / z peaks of a plurality of candidate ionization control proteins having acid stability, and outputting the identification of one or more ionization control proteins having the m / z range and acid stability for the analyte. [Brief explanation of the drawings]

[0075] DESCRIPTION OF THE DRAWINGS The invention will now be described by way of example only with reference to the following figures. [Figure 1] Figure 1 shows an example of a MALDI-TOF mass spectrum showing the mass distribution of an analyte (kappa light chain (k)) after elution with acetic acid containing aprotinin. A single peak of aprotinin (+1 charge) was observed, which did not interfere with the peak of the analyte (kappa light chain). The ion charge state is indicated in parentheses. [Figure 2]Figure 2 shows that the signal of the relative ionization control protein is stable in the presence and absence of analyte. The MALDI-TOF mass spectrum of aprotinin obtained in the absence of analyte (black line) is not significantly different from the spectrum of aprotinin containing kappa light chains from normal human serum (NHS) (gray line and inset). [Figure 3] Figure 3 shows that aprotinin is stable in 5% acetic acid. Kappa light chains were periodically eluted with aprotinin stored at 22°C in 5% acetic acid. MALDI-TOF mass spectra were acquired at each time point, and the peak areas (±standard deviation) of aprotinin and kappa light chain (+2) were measured. No signal degradation was observed for either protein over an 8-week period. [Figure 4] Figure 4 shows that the analyte signal relative to aprotinin as an ionization control remained stable over time. Aprotinin in 5% acetic acid was stored at 22°C and used periodically to elute the kappa light chain. MALDI-TOF mass spectra were acquired at each time point, and the peak area ratios (±standard deviation) of aprotinin and kappa light chain (+2) were measured. No apparent changes in the peak area ratios were observed over an 8-week period. [Figure 5a] Figure 5 shows transthyretin (TTR) as a MALDI-TOF ionization control. MALDI-TOF mass spectra show the mass distribution of TTR after elution with acetic acid, with or without mixing with polyclonal IgG (Figures 5A and 5B). TTR peaks are observed at m / z 13827 and 6914 and do not interfere with any lambda or kappa polyclonal light chain peaks. The signal intensity of the TTR ionization control peak remains unchanged in the presence or absence of analyte (B). The TTR signal peak also does not overlap with aprotinin (C) or (larger mass) glycosylated kappa free light chain (D). Ion charge states are indicated in parentheses. [Figure 5b]Figure 5 shows transthyretin (TTR) as a MALDI-TOF ionization control. MALDI-TOF mass spectra show the mass distribution of TTR after elution with acetic acid, with or without mixing with polyclonal IgG (Figures 5A and 5B). TTR peaks are observed at m / z 13827 and 6914 and do not interfere with any lambda or kappa polyclonal light chain peaks. The signal intensity of the TTR ionization control peak remains unchanged in the presence or absence of analyte (B). The TTR signal peak also does not overlap with aprotinin (C) or (larger mass) glycosylated kappa free light chain (D). Ion charge states are indicated in parentheses. [Figure 5c] Figure 5 shows transthyretin (TTR) as a MALDI-TOF ionization control. MALDI-TOF mass spectra show the mass distribution of TTR after elution with acetic acid, with or without mixing with polyclonal IgG (Figures 5A and 5B). TTR peaks are observed at m / z 13827 and 6914 and do not interfere with any lambda or kappa polyclonal light chain peaks. The signal intensity of the TTR ionization control peak remains unchanged in the presence or absence of analyte (B). The TTR signal peak also does not overlap with aprotinin (C) or (larger mass) glycosylated kappa free light chain (D). Ion charge states are indicated in parentheses. [Figure 5d] Figure 5 shows transthyretin (TTR) as a MALDI-TOF ionization control. MALDI-TOF mass spectra show the mass distribution of TTR after elution with acetic acid, with or without mixing with polyclonal IgG (Figures 5A and 5B). TTR peaks are observed at m / z 13827 and 6914 and do not interfere with any lambda or kappa polyclonal light chain peaks. The signal intensity of the TTR ionization control peak remains unchanged in the presence or absence of analyte (B). The TTR signal peak also does not overlap with aprotinin (C) or (larger mass) glycosylated kappa free light chain (D). Ion charge states are indicated in parentheses.

[0076] 2ng ml as ionization control for mass spectrometry in 5% acetic acid containing 20mM tris(2-carboxyethyl)phosphine (TCEP) reducing agent -1 An elution buffer containing 100 mM aprotinin was prepared. 5% acetic acid was used to both elute the analytes from the immunocapture beads and simultaneously facilitate the separation of immunoglobulin heavy and light chains. 20 mM TCEP was used as an acid-stable reducing agent to cleave the disulfide bonds holding intact immunoglobulins together.

[0077] A normal human serum sample (NHS) was diluted 1:10 and captured (as described in step 1 above) using paramagnetic microparticles containing an antibody specific for the human kappa immunoglobulin light chain. This was eluted with an acidic buffer solution containing both a reducing agent and aprotinin (as an ionization control). The eluate was then spotted onto a MALDI-TOF target plate sandwiched between MALDI matrix (HCCA) and dried. Mass spectra were acquired in positive ion mode, covering an m / z range of 5,000–30,000, including the singly (+1, m / z 22705), doubly (+2, m / z 11353), and triply (+3, m / z 7569) charged ions of the analyte (human kappa light chain; Table 1).

[0078] [Table 1]

[0079] The signal intensity of aprotinin is clearly shown in Figure 1 as a distinct peak at m / z 6512 that is not interfering with or overlapping any of the three peaks of the analytes. To demonstrate that the aprotinin signal is independent of the presence of the analytes, it was analyzed in the presence (+NHS) and absence (-NHS) of the latter. Figure 2 shows that the ionization control signal intensity of aprotinin is the same in both cases.

[0080] To investigate the stability of the ionization control under acidic conditions, individual 50 ml aliquots of the formulation (2 ng ml -1 A solution of 5% acetic acid (supplemented with aprotinin) was stored at 22 °C. Individual aliquots were removed at regular intervals, replenished with reducing agent (TCEP), and then used to elute the analytes from the anti-kappa microparticles for MALDI-TOF analysis. The mass spectral peak areas obtained for both the kappa analyte and aprotinin are shown in Figure 3. Both peak areas varied during the experiment. This variation is due to known MALDI spot-to-spot sample inconsistencies, but there was no degradation in either the analyte or aprotinin signal over 60 days at 22 °C. Extrapolation indicated that aprotinin is stable under acidic conditions for at least 6 months when stored at 4 °C (using the Arrhenius equation). When the stability data are expressed as the ratio of the analyte signal peak to the ionization control signal peak, the variation is significantly less (Figure 4). This explains the use of aprotinin (as an ionization control) to overcome ionization differences between different MALDI-TOF acquisitions.

[0081] Figure 5 shows another example of an ionization control, transthyretin. MALDI-TOF mass spectra showed 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 (Figures 5A and 5B). TTR monomer peaks were observed at 13827 m / z (+1 charge state) and 6914 m / z (+2 charge state), neither of which interfered with any lambda or kappa polyclonal light chain peaks from IgG. The signal intensity of the TTR ionization control peaks did not change in the presence or absence of analyte (Figure 5B). The TTR signal peaks also did not overlap with those of aprotinin (Figure 5C) or glycosylated kappa free light chain (at higher m / z).

Claims

1. an elution buffer for eluting one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof, comprising an acid-stable ionization control protein or peptide for use in mass spectrometry; wherein the elution buffer has a pH of 1 to 5 and further comprises a reducing agent; the ionization control protein or peptide is stable in the elution buffer for at least 30 days; and the one or more analytes are kappa light chain, lambda light chain, kappa free light chain, or lambda free light chain; and an elution buffer selected such that the ionization control protein or peptide has at least one mass spectrometry peak with an m / z value that does not substantially overlap with the mass spectrometry peak of the or each predetermined analyte.

2. An elution buffer as described in claim 1, wherein the kappa light chains or lambda light chains are separated from their respective heavy chains by a reducing agent.

3. An elution buffer as described in claim 1 or 2, wherein the reducing agent is selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), 2-mercaptoethanol, and cysteine.

4. 3. The elution buffer of claim 1, wherein at least one mass spectrometry m / z peak value of the ionization control protein or peptide is stable for at least 30 days.

5. 5. The elution buffer of claim 4, wherein the ionization control protein or peptide is selected to have at least one mass spectrometry m / z peak value within a predetermined mass spectrometry window used for detection or quantitation of one or more peaks from at least one of the predetermined analytes.

6. (a) 5% v / v acetic acid in water; (b) 0.1 M glycine, pH 2.0-3.0, or 0.2 M glycine, pH 2-6 6. The elution buffer of claim 1 , wherein the elution buffer is selected from the group consisting of:

7. 7. The elution buffer of claim 1, comprising 0.5 to 100 ng / μl of an ionization control protein or peptide.

8. 8. The elution buffer of claim 1, wherein the ionization control protein or peptide comprises at least 30 amino acids.

9. 9. The elution buffer of claim 1, wherein the ionization control protein or peptide has a mass of at least 3 kDa.

10. 10. The elution buffer of claim 1, wherein the ionization control protein or peptide is selected from aprotinin, beta-2 glycoprotein, transthyretin, and alpha-1 acid glycoprotein.

11. 11. A kit for use in the analysis of one or more analytes by mass spectrometry, comprising an elution buffer according to any one of claims 1 to 10 and one or more analyte-specific antibodies or fragments thereof.

12. 12. The kit of claim 11, comprising an anti-IgA, anti-IgG, anti-IgM, 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 and / or anti-heavy chain subclass-light chain type specific antibody.

13. The kit of claim 11 or 12, wherein the antibody or fragment thereof is bound to a substrate.

14. 14. The kit of any one of claims 11 to 13, comprising a predetermined amount of a control sample.

15. 15. The kit of any one of claims 11 to 14, comprising one or more of a sample dilution buffer, a reducing agent, a mass spectrometry matrix, a mass spectrometry matrix solvent, a MALDI target, and a mass spectrometer mass calibrator.

16. 16. The kit of any one of claims 11 to 15, further comprising a standard serum protein control.

17. 17. The kit of any one of claims 11 to 16, further comprising a reducing agent.

18. 11. A method for detecting or quantifying an analyte, comprising immunopurifying a predetermined analyte, eluting the analyte with an elution buffer according to any one of claims 1 to 10, and detecting the analyte and the ionization control protein or peptide by mass spectrometry.

19. The method of claim 18, wherein the mass spectrometry is MALDI-TOF.

20. The method of claim 18 or 19, comprising the use of a kit.

21. (a) a method for identifying said analyte; (b) identifying the m / z of at least one peak of the ionization control by comparing it to the m / z of one or more expected peaks of the analyte; (c) A method for identifying an ionization control protein or peptide having said m / z range and acid stability. A method for producing the elution buffer of any one of claims 1 to 10, comprising:

22. 21. The method of any one of claims 18 to 20, further comprising separating the kappa or lambda light chains from the respective heavy chains with a reducing agent.

23. The method of claim 21, further comprising computer-implemented steps of inputting a sample, comparing one or more m / z peaks of the sample with the m / z peaks of multiple candidate ionization control proteins or peptides having acid stability, and outputting identification of one or more ionization control proteins or peptides having the m / z range and acid stability for the sample.

24. 24. The method of claim 23, wherein the computer comprises a computer processor and computer memory.

25. 25. An apparatus comprising a mass spectrometer programmed to carry out the method of any one of claims 18 to 21, 23 and 24.

26. The apparatus of claim 25, wherein the mass spectrometer is selected from MALDI-TOF and liquid chromatography mass spectrometer (LC-MS).

27. An elution buffer for eluting one or more predetermined immunoglobulins or fragments thereof from one or more immunoglobulin-specific antibodies or fragments thereof, or for eluting one or more predetermined antibodies or fragments thereof from target immunoglobulins or fragments thereof, comprising an acid-stable ionization control protein or peptide for use in mass spectrometry, comprising: wherein the elution buffer has a pH of 1 to 5; and wherein said ionization control protein or peptide is substantially stable in said elution buffer for at least 30 days; and An elution buffer wherein the ionization control protein or peptide is selected from aprotinin and transthyretin.

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