Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS.

OBJECTIVES: Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. METHODS: Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. RESULTS: Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. CONCLUSIONS: A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement.

BACKGROUND: Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin. METHODS: An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93). RESULTS: By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively). CONCLUSIONS: Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

Supramolecular Copolymerization as a Strategy to Control the Stability of Self-Assembled Nanofibers.

A major challenge in supramolecular polymerization is controlling the stability of the polymers formed, that is, controlling the rate of monomer exchange in the equilibrium between monomer and polymer. The exchange dynamics of supramolecular polymers based on benzene-1,3,5-tricarboxamide (BTA) can be regulated by copolymerizing molecules with dendronized (dBTA) and linear (nBTA) ethylene glycol-based water-soluble side chains. Whereas nBTAs form long nanofibers in water, dBTAs do not polymerize, forming instead small spherical aggregates. The copolymerization of the two BTAs results in long nanofibers. The exchange dynamics of both the BTA monomers in the copolymer are significantly slowed down in the mixed systems, leading to a more stable copolymer, while the morphology and spectroscopic signature of the copolymers are identical to that of nBTA homopolymer. This copolymerization is the supramolecular counterpart of styrene/ maleic anhydride copolymerization.

Generation of gas-phase ions from charged clusters: an important ionization step causing suppression of matrix and analyte ions in matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: Ionization in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a very complicated process. It has been reported that quaternary ammonium salts show extremely strong matrix and analyte suppression effects which cannot satisfactorily be explained by charge transfer reactions. Further investigation of the reasons causing these effects can be useful to improve our understanding of the MALDI process. METHODS: The dried-droplet and modified thin-layer methods were used as sample preparation methods. In the dried-droplet method, analytes were co-crystallized with matrix, whereas in the modified thin-layer method analytes were deposited on the surface of matrix crystals. Model compounds, tetrabutylammonium iodide ([N(Bu)4 ]I), cesium iodide (CsI), trihexylamine (THA) and polyethylene glycol 600 (PEG 600), were selected as the test analytes given their ability to generate exclusively pre-formed ions, protonated ions and metal ion adducts respectively in MALDI. RESULTS: The strong matrix suppression effect (MSE) observed using the dried-droplet method might disappear using the modified thin-layer method, which suggests that the incorporation of analytes in matrix crystals contributes to the MSE. By depositing analytes on the matrix surface instead of incorporating in the matrix crystals, the competition for evaporation/ionization from charged matrix/analyte clusters could be weakened resulting in reduced MSE. Further supporting evidence for this inference was found by studying the analyte suppression effect using the same two sample deposition methods. CONCLUSIONS: By comparing differences between the mass spectra obtained via the two sample preparation methods, we present evidence suggesting that the generation of gas-phase ions from charged matrix/analyte clusters may induce significant suppression of matrix and analyte ions. The results suggest that the generation of gas-phase ions from charged matrix/analyte clusters is an important ionization step in MALDI-MS. Copyright © 2016 John Wiley & Sons, Ltd.

Therapeutic drug monitoring of infliximab: performance evaluation of three commercial ELISA kits.

BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab (IFX, Remicade®) can aid to optimize therapy efficacy. Many assays are available for this purpose. However, a reference standard is lacking. Therefore, we evaluated the analytical performance, agreement and clinically relevant differences of three commercially available IFX ELISA kits on an automated processing system. METHODS: The kits of Theradiag (Lisa Tracker Infliximab), Progenika (Promonitor IFX) and apDia (Infliximab ELISA) were implemented on an automated processing system. Imprecision was determined by triplicate measurements of patient samples on five days. Agreement was evaluated by analysis of 30 patient samples and four spiked samples by the selected ELISA kits and the in-house IFX ELISA of Sanquin Diagnostics (Amsterdam, The Netherlands). Therapeutic consequences were evaluated by dividing patients into four treatment groups using cut-off levels of 1, 3 and 7 µg/mL and determining assay concordance. RESULTS: Within-run and between-run imprecision were acceptable (≤12% and ≤17%, respectively) within the quantification range of the selected ELISA kits. The apDia assay had the best precision and agreement to target values. Statistically significant differences were found between all assays except between Sanquin Diagnostics and the Lisa Tracker assay. The Promonitor assay measured the lowest IFX concentrations, the apDia assay the highest. When patients were classified in four treatment categories, 70% concordance was achieved. CONCLUSIONS: Although all assays are suitable for TDM, significant differences were observed in both imprecision and agreement. Therapeutic consequences were acceptable when patients were divided in treatment categories, but this could be improved by assay standardization.

Influence of the solvent and the enantiomeric purity on the transition between different supramolecular polymers.

The self-assembly of two enantiomerically pure hexa(oligo(p-phenylene vinylene))-substituted benzenes having 24 stereocenters was studied in pure methylcyclohexane (MCH) and in a mixture of MCH/toluene (4:1). Irrespective of the solvent a cooperative supramolecular polymerization mechanism was determined for these star-shaped molecules by using temperature-dependent CD and UV/Vis spectroscopy. Quite remarkably, a transition from one helical supramolecular state (A) to a second more thermodynamically stable supramolecular helical assembly (B) was observed. The rate of the A→B transition was strongly dependent on the nature of the solvent; being faster in the solvent mixture than in pure MCH. By using size exclusion chromatography we could relate the increased rate to a decreased stability of the supramolecular A state in the solvent mixture. Next, we mixed the two enantiomerically pure hexa-substituted benzene derivatives in a so-called majority-rules experiment, which lead to the anitcipated chiral amplification in the A state. More importantly it appeared that the A→B transition was significantly hampered in these mixed systems. Furthermore, the absence of chiral amplification in the B state revealed the formation of separated enantiomerically pure assemblies. Therefore, by using a wide variety of spectroscopic and chromatographic techniques we determined the influence of solvent and enantiomeric purity on the transition between different supramolecular states.

Disentangle a Complex MALDI TOF Mass Spectrum of Polyethylene Glycols into Three Separate Spectra via Selective Formation of Protonated Ions and Sodium or Potassium Adducts.

In MALDI TOF MS analysis, complicated mass spectra can usually be recorded for polymers with high affinities to protons and alkali metal ions. For these polymers, protonated ions and sodium and potassium adducts can often be formed concomitantly. By distributing these ions into three separate spectra of protonated ions, sodium adducts, and potassium adducts, significantly simplified spectra can be acquired. Mass spectra consisting of only sodium or potassium adducts can often be obtained by simply adding sodium salt and potassium salt, respectively. We report here a method to selectively generate protonated ions. A polyethylene glycol (PEG) sample with amino end groups was selected as the model polymer and α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. Octadecylamine (ODA) or a mixture of a tetrabutylammonium (TBA) salt and an ammonium salt was used as the co-matrix to inhibit the release of sodium and potassium ions and their related adducts into the MALDI gas phase plume. By depositing the polymer sample on top of a preloaded layer of CHCA with a co-matrix, the generation of Na+ and K+ adducts is suppressed, while [ODA + H]+ and NH4+ released from the preloaded matrix layer can serve as protonation reagents to protonate the polymer molecules via proton transfer reactions. It is clearly demonstrated that disentangling a complex mass spectrum filled densely with various series of ions into three separate spectra, with each one consisting of only one type of ions, allows unambiguous identification of mass peaks and greatly helps the interpretation of MS results.

Quantification of uracil, dihydrouracil, thymine and dihydrothymine for reliable dihydropyrimidine dehydrogenase (DPD) phenotyping critically depend on blood and plasma storage conditions.

Establishing dihydropyrimidine dehydrogenase (DPD) activity is highly important in determining the correct starting dose of fluoropyrimidines such as 5-fluorouracil and capecitabine. The concentration ratio of endogenous uracil with its metabolite dihydrouracil (DHU) is a well-known parameter that is linked to DPD activity. Concentration ratios such as thymine over its DPD-converted metabolite dihydrothymine (DHT) is less described and may serve as an alternative diagnostic biomarker for DPD activity. In this study, we describe the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of uracil, DHU, thymine, and DHT in human plasma. In addition, stability experiments were performed. Uracil and thymine were quantified up to 80.0 ng/mL and DHU and DHT up to 800 ng/mL. Intra- and inter-assay precision were maximum 8.0 % and 7.6 %. respectively. Also, recovery was adequate and significant matrix-effects and carry-over were excluded. Stability experiments showed that uracil concentrations increased with 27-52 % when stored for 1 or 2 h at ambient temperatures compared to cold storage. Thymine, DHU, and DHT concentrations remained stable, thymine after 1 h in plasma excluded, showing the DHT:T ratio might be a more robust marker for DPD activity than DHU:U. In conclusion, we present here a novel assay capable of quantifying uracil, thymine, DHU and DHT in a single analytical run. We provide additional data showing increased stability for DHU, thymine and DHT compared to uracil. This assay may be used as a diagnostic test in future studies, establishing the association of these endogenous biomarker concentrations with DPD activity and safety to treatment with fluoropyrimidines. In addition, future research should also be focused on reducing pre-analytical instability. Standardization in this field is essential to set proper reference values and to allow inter-study comparison on clinical outcomes.

Elucidating dynamic behavior of synthetic supramolecular polymers in water by hydrogen/deuterium exchange mass spectrometry.

A comprehensive understanding of the structure, self-assembly mechanism, and dynamics of one-dimensional supramolecular polymers in water is essential for their application as biomaterials. Although a plethora of techniques are available to study the first two properties, there is a paucity in possibilities to study dynamic exchange of monomers between supramolecular polymers in solution. We recently introduced hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize the dynamic nature of synthetic supramolecular polymers with only a minimal perturbation of the chemical structure. To further expand the application of this powerful technique some essential experimental aspects have been reaffirmed and the technique has been applied to a diverse library of assemblies. HDX-MS is widely applicable if there are exchangeable hydrogen atoms protected from direct contact with the solvent and if the monomer concentration is sufficiently high to ensure the presence of supramolecular polymers during dilution. In addition, we demonstrate that the kinetic behavior as probed by HDX-MS is influenced by the internal order within the supramolecular polymers and by the self-assembly mechanism.

Electrospray-ionization mass spectrometry for screening the specificity and stability of single-stranded-DNA templated self-assemblies.

Supramolecular complexes consisting of a single-stranded oligothymine (dTn) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen-bonding unit have been studied with electrospray-ionization mass spectrometry (ESI-MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn. By changing the hydrogen-bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single-stranded-DNA (ssDNA)-guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20-component self-assembled objects. Gas-phase breakdown experiments on single- and multiple-guest-DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI-MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.

Noncovalent synthesis of protein dendrimers.

The covalent synthesis of complex biomolecular systems such as multivalent protein dendrimers often proceeds with low efficiency, thereby making alternative strategies based on noncovalent chemistry of high interest. Here, the synthesis of protein dendrimers using a strong but noncovalent interaction between a peptide and complementary protein is proposed as an efficient strategy to arrive at dendrimers fully functionalized with protein domains. The association of S-peptide to S-protein results in the formation of an active enzyme (ribonuclease S) and therefore serves as an ideal system to explore this synthetic approach. Native chemical ligation was used to couple four S-peptides by means of their C-terminal thioester to a cysteine-functionalized dendritic scaffold, thus yielding a tetravalent S-peptide wedge. A fully functional ribonuclease S tetramer was prepared by addition of four equivalents of S-protein. Biophysical techniques (isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and mass spectrometry) and an enzymatic activity assay were used to verify the formation of the multivalent complex. The noncovalent synthetic strategy presented here provides access to well-defined, dynamic, semisynthetic protein assemblies in high yield and is therefore of interest to the field of nanomedicine as well as biomaterials.

Matrix suppression and analyte suppression effects of quaternary ammonium salts in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: an investigation of suppression mechanism.

In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of some quaternary ammonium salts (QASs), very clean spectra of the quaternary ammonium ions were recorded with a strong matrix suppression effect (MSE). The QASs also showed a considerable analyte suppression effect (ASE). It was demonstrated that the MSE and ASE of the QASs can be explained well by the cluster ionization model. According to this model, MALDI ions are formed from charged matrix/analyte clusters. Various analyte ions and matrix ions might coexist in the cluster, and they will compete for the limited number of net charges available. If enough quaternary ammonium ions are present in the cluster, they will take away the net charges, thus resulting in the MSE and ASE. Our results also suggest that 'the cluster ionization model' is not in conflict with 'the theory of ionization via secondary gas-phase reactions'. The initial MALDI ions produced from charged matrix/analyte clusters will collide with other molecules or ions in the MALDI plume. Depending on the properties of the initial ions and the composition of the MALDI plume, secondary gas-phase reactions might result from these collisions. The final ions observed are the combined results of 'cluster ionization' and 'ionization via secondary gas-phase reactions'.

Dehydrogenation of tertiary amines in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of various compounds synthesized in our laboratory, strong [M - H]+ ion peaks were often observed for the molecules with tertiary amino groups. In this work, the MALDI TOF MS behavior of two groups of compounds that incorporate tertiary amino moieties was investigated. One group is bisurea dimethylanilines (BUDMAs) prepared for the study of molecular recognition in thermoplastic elastomers, and the other group is the poly(propylene imine) diaminobutane dendrimers. The results clearly demonstrate the appearance of the [M - H]+ ions. In order to understand the possible mechanisms for the generation of these ions, a series of model compounds, ranging from primary to tertiary amines, were investigated. Unlike the tertiary amines, no [M - H]+ ion peaks were recorded for the primary amines, and only barely detectable ones, if any, for some secondary amines. It appears that the tertiary amino groups play an important role in the formation of these ions. In addition to MALDI TOF MS analysis, these samples were also applied to electrospray ionization (ESI) MS where no [M - H]+ ions were observed. The results indicate that the generation of [M - H]+ ion is due to the unique MALDI conditions and is likely to be formed via dehydrogenation of a protonated tertiary amine resulting in an N=C double bond. The absence of [M - H]+ ion peaks for the primary and secondary amines is probably because upon their formation these ions could easily transfer one proton to the corresponding amines in the MALDI gas-phase plume, yielding neutral imines that cannot be detected by MS.

Mapping preferred sites for fluorescent labeling by combining fluorescence and MS analysis of tryptic CNA35 protein digests.

HPLC-MS analysis of tryptic protein digests in combination with fluorescence detection is presented as a convenient and quantitative method to gain insight into the relative reactivity of lysine side chains. In this scheme (tandem) mass spectrometry was used for identification of the modified residue, whereas fluorescence detection allowed determination of their relative abundance. Our method identified 'labeling hot-spots' at two flexible parts of the collagen-binding protein CNA35, positions that were consistent with all available structural and biochemical data on the collagen-binding properties of CNA35.

Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the pseudo-molecular ions formed by counterion adduction, deprotonation and electron capture, a number of fragment ions were also observed. Charge splitting by fragmentation was found to be a viable route for charge reduction leading to the formation of the observed singly charged fragment ions. Unlike multivalent metal ions, organic ions can rearrange and/or fragment during charge reduction. This fragmentation process will evidently complicate the interpretation of the MALDI MS spectrum. Because MALDI MS is usually considered as a soft ionization technique, the fragment ion peaks can easily be erroneously interpreted as impurities. Therefore, the awareness and understanding of the underlying MALDI-induced fragmentation pathways is essential for a proper interpretation of the corresponding mass spectra. Due to the fragment ions generated during charge reduction, special care should be taken in the MALDI MS analysis of multiply charged ions. In this work, the possible mechanisms by which the organic ions bearing fixed multiple charges fragment are investigated. With an improved understanding of the fragmentation mechanisms, MALDI TOF MS should still be a useful technique for the characterization of organic ions with fixed multiple charges.

Double cation adduction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of electron deficient anthraquinone derivatives.

Six anthraquinone derivatives were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Clear (pseudo) molecular ions were observed for all the compounds. Interestingly, for some derivatives, strong ions with double cation adduction were also recorded in the positive mode. It is remarkable that all these ions are singly charged. In this work, possible mechanisms for the double cation adduction were investigated and discussed. It appears that the double cation adduction was due to the electron deficient nature of the derivatives, and formed by taking up two singly charged cations and one electron. Substituents on the anthraquinone ring were found to have a significant effect on the double cation adduction. In contrast, no considerable influence of the acidity of MALDI matrix/solution was observed, even on the double proton adduction. Furthermore, it was demonstrated that double cation adduction might occur in the MALDI gas-phase plume. In addition to the anthraquinones, three more electron deficient compounds of different types, i.e. a perylene bisimide derivative (PB), 3,7-decanoylamino-4,8-dihydrobenzo[1,2-b:4,5-b']dithiophene-4,8-dione (TQ) and 6,6-phenyl C61-butyric acid methyl ester (PCBM), were also analyzed with MALDI TOF MS. The results indicate that the 'abnormal' double cation adduction might be a 'normal' phenomenon in the MALDI TOF MS analysis of many electron deficient compounds.

Dynamic diversity of synthetic supramolecular polymers in water as revealed by hydrogen/deuterium exchange.

Numerous self-assembling molecules have been synthesized aiming at mimicking both the structural and dynamic properties found in living systems. Here we show the application of hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to unravel the nanoscale organization and the structural dynamics of synthetic supramolecular polymers in water. We select benzene-1,3,5-tricarboxamide (BTA) derivatives that self-assemble in H2O to illustrate the strength of this technique for supramolecular polymers. The BTA structure has six exchangeable hydrogen atoms and we follow their exchange as a function of time after diluting the H2O solution with a 100-fold excess of D2O. The kinetic H/D exchange profiles reveal that these supramolecular polymers in water are dynamically diverse; a notion that has previously not been observed using other techniques. In addition, we report that small changes in the molecular structure can be used to control the dynamics of synthetic supramolecular polymers in water.

Radical cation formation in characterization of novel C3-symmetric disks and their precursors by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Four C3-symmetrical tris(dipeptide) disks and their precursors were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The C3-symmetrical disks were based on a benzene-1,3,5-triscarboxamide core extended by oligopeptides with trialkoxyanilide tails. The results indicate that MALDI TOF MS is a powerful and straightforward analytical technique for characterizing C3-symmetrical disks and their precursors. Clear (pseudo)-molecular ion peaks could readily be identified. It is remarkable that strong radical ion signals were observed for all the compounds, including the anilines that were expected to be protonated prior to laser irradiation using acidic MALDI matrixes. Possible mechanisms for radical ion formation were investigated with the employment of radical scavengers, with various matrixes and with direct laser desorption/ionization (LDI). Most likely the radicals are formed by losing one electron from the aniline nitrogen and stabilized by conjugation through the phenyl ring. It appears that direct photo/thermal ionization of analytes is an important route for the radical ion formation of the compounds with trialkoxy aniline/anilide groups.

Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins.

BACKGROUND: Given the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions. METHODS: By parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and ß-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/ß-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared. RESULTS: The α/ß-ratio is a poor marker for thalassemia patients, yet deviant α/ß-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval. CONCLUSIONS: Although subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/ß-ratio as screening parameters.