RESUMO
Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.
RESUMO
The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top-down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4BC185∗ proteoform is unable to associate with the plasma membrane, resulting in a decrease in mitogen-activated protein kinase signaling pathway activation. Collectively, our study provides a reference set of functionally distinct KRAS proteoforms and the colorectal cancer contexts in which they are present.
Assuntos
Neoplasias Colorretais , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Humanos , Neoplasias Colorretais/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Proteômica , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
Identification of proteoforms, the different forms of a protein, is important to understand biological processes. A proteoform family is the set of different proteoforms from the same gene. We previously developed the software program Proteoform Suite, which constructs proteoform families and identifies proteoforms by intact-mass analysis. Here, we have applied this approach to top-down proteomic data acquired at the National High Magnetic Field Laboratory 21 tesla Fourier transform ion cyclotron resonance mass spectrometer (data available on the MassIVE platform with identifier MSV000085978). We explored the ability to construct proteoform families and identify proteoforms from the high mass accuracy data that this instrument provides for a complex cell lysate sample from the MCF-7 human breast cancer cell line. There were 2830 observed experimental proteforms, of which 932 were identified, 44 were ambiguous, and 1854 were unidentified. Of the 932 unique identified proteoforms, 766 were identified by top-down MS2 analysis at 1% false discovery rate (FDR) using TDPortal, and 166 were additional intact-mass identifications (â¼4.7% calculated global FDR) made using Proteoform Suite. We recently published a proteoform level schema to represent ambiguity in proteoform identifications. We implemented this proteoform level classification in Proteoform Suite for intact-mass identifications, which enables users to determine the ambiguity levels and sources of ambiguity for each intact-mass proteoform identification.
Assuntos
Ciclotrons , Proteômica , Análise de Fourier , Humanos , Espectrometria de Massas , SoftwareRESUMO
Proton-transfer reactions (PTRs) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to "concentrate", in the gas phase, nearly all the signals from an intact protein charge state envelope into a single charge state, improving the signal-to-noise ratio (S/N) by 10× or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides a tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call "PTR-MS1-PIP", generates a pseudo-MS1 spectrum derived from ions that are exposed to the PTR reagent and PIP waveforms but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in the spectral S/N of intact proteins. This permits the observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the PTR rate without compromise to the parking efficiency. We demonstrate that this process, called "rapid park", can improve reaction rates by 5-10× and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS1 and rapid park provides a 10-fold reduction in ion injection time, improving the rate of tandem MS sequencing.
Assuntos
Proteínas , Prótons , Indicadores e Reagentes , Íons , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.
Assuntos
Mieloma Múltiplo , Proteínas do Mieloma , Proteômica/métodos , Anticorpos Monoclonais , Humanos , Imunoeletroforese , Espectrometria de Massas , Mieloma Múltiplo/diagnósticoRESUMO
Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.
Assuntos
Resinas Acrílicas , Eletroforese em Gel de Poliacrilamida , Espectrometria de MassasRESUMO
Fourier transform mass spectrometers routinely provide high mass resolution, mass measurement accuracy, and mass spectral dynamic range. In this work, we utilize 21 T Fourier transform ion cyclotron resonance (FT-ICR) to analyze product ions derived from the application of multiple dissociation techniques and/or multiple precursor ions within a single transient acquisition. This ion loading technique, which we call, "chimeric ion loading", saves valuable acquisition time, decreases sample consumption, and improves top-down protein sequence coverage. In the analysis of MCF7 cell lysate, we show collision-induced dissociation (CID) and electron-transfer dissociation (ETD) on each precursor on a liquid chromatography-mass spectrometry (LC-MS) timescale and improve mean sequence coverage dramatically (CID-only 15% vs chimeric 33%), even during discovery-based acquisition. This approach can also be utilized to multiplex the acquisition of product ion spectra of multiple charge states from a single protein precursor or multiple ETD/proton-transfer reactions (PTR) reaction periods. The analytical utility of chimeric ion loading is demonstrated for top-down proteomics, but it is also likely to be impactful for tandem mass spectrometry applications in other areas.
Assuntos
Proteínas de Neoplasias/análise , Proteômica , Análise de Fourier , Humanos , Células MCF-7 , Espectrometria de Massas em Tandem , Células Tumorais CultivadasRESUMO
Stored waveform inverse Fourier transform (SWIFT) is a versatile method to generate complex isolation/ejection waveforms for precursor isolation prior to tandem mass spectrometry experiments. Here, we report ultrahigh resolving power ion isolation by SWIFT on a 21 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Individual histone proteoforms are isolated (0.6 m/z isolation window) with near 100% efficiency using a 52 ms SWIFT isolation, followed by in-cell fragmentation by ultraviolet photodissociation (UVPD). Ion isolation resolving power of 175â¯000 (m/Δm) is demonstrated by isolation of individual peaks at a spacing of 0.0034 Da at m/z 597 from a complex mixture of Canadian bitumen. An individual m/z ion, which corresponds to a single elemental composition, from a complex mixture is isolated and fragmented by infrared multiphoton dissociation (IRMPD). Theoretical and experimental considerations that limit achievable ion isolation resolving power are discussed.
Assuntos
Ciclotrons , Análise de Fourier , Espectrometria de Massas/instrumentação , Sequência de Aminoácidos , Histonas , Proteômica , Razão Sinal-RuídoRESUMO
A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.
Assuntos
Aminoácidos/análise , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteoma/análise , Proteômica/métodos , Animais , Biologia Computacional , Eletroforese Capilar , Humanos , Linguagens de Programação , Reprodutibilidade dos Testes , SoftwareRESUMO
The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is â¼51%, indicating the urgent need for better diagnosis methods and treatment plans. Here, we describe highly specific and sensitive top-down and middle-down MS/MS methods owning the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages with 21 telsa FT-ICR MS/MS. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by database-aided de novo MS/MS and 100% matched the gene sequencing result, except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. The monoclonal immunoglobulin heavy chains were also classified into specific germline sequences based on the present method. This work represents the first application of top/middle-down MS/MS sequencing of endogenous human monoclonal immunoglobulins with polyclonal immunoglobulins background.
Assuntos
Amiloidose/classificação , Cadeias Leves de Imunoglobulina/sangue , Mieloma Múltiplo/classificação , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Amiloidose/diagnóstico , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão , Análise de Fourier , Humanos , Cadeias Leves de Imunoglobulina/química , Imunoglobulinas/isolamento & purificação , Imunoglobulinas/metabolismo , Mieloma Múltiplo/diagnóstico , Paraproteinemias/classificação , Paraproteinemias/diagnósticoRESUMO
BACKGROUND: Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS: We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance-tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and ß-thalassemia diagnosis. RESULTS: We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and ß subunits (δ/ß) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose ß-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS: We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and ß-thalassemia diagnosis (MS1).
Assuntos
Análise de Fourier , Hemoglobinopatias/sangue , Hemoglobinas/química , Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Talassemia beta/sangue , Sequência de Aminoácidos , Ciclotrons , Variação Genética , Hemoglobinopatias/genética , Humanos , Sensibilidade e Especificidade , Análise de Sequência de Proteína/métodos , alfa-Globinas/química , Globinas beta/química , Talassemia beta/genética , Globinas delta/químicaRESUMO
Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617-624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/Elite(TM) capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.
Assuntos
Histonas/metabolismo , Proteoma/análise , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Butiratos/química , Variação Genética , Células HeLa , Histonas/genética , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.
Assuntos
Neoplasias Colorretais/química , Espectrometria de Massas/métodos , Proteínas de Neoplasias/análise , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Neoplasias Colorretais/patologia , Misturas Complexas/química , Ciclotrons/instrumentação , Análise de Fourier , Humanos , Espectrometria de Massas/instrumentação , Proteômica/instrumentaçãoRESUMO
Previously, we described implementation of a front-end ETD (electron transfer dissociation) source for an Orbitrap instrument (1). This source facilitates multiple fills of the C-trap with product ions from ETD of intact proteins prior to mass analysis. The result is a dramatic enhancement of the observed ion current without the need for time consuming averaging of data from multiple mass measurements. Here we show that ion-ion proton transfer (IIPT) reactions can be used to simplify ETD spectra and to disperse fragment ions over the entire mass range in a controlled manner. We also show that protein derivatization can be employed to selectively enhance the sequence information observed at the N- and C-termini of a protein.
RESUMO
Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion-ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.
Assuntos
Transporte de Elétrons , Espectrometria de Massas por Ionização por Electrospray/métodos , ÍonsRESUMO
ß-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A2. Diagnosis of ß-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/ß ratio can act as a biomarker to identify ß-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/ß ratio as a thalassemia biomarker.
RESUMO
Protein mass measurement by mass spectrometry is complicated by wide isotopic distributions that result from incorporation of heavy isotopes of C, H, N, O, and S, thereby limiting signal-to-noise ratio (SNR) and accurate intact mass determination, particularly for larger proteins [Fenselau et al. Anal. Chem. 1983, 55 (2), 353-356]. Observation of the monoisotopic mass-to-charge ratio (m/z) is the simplest and most accurate way to determine intact protein mass, but as mass increases, the relative abundance of the monoisotopic peak becomes so low that it is often undetectable. Here, we used an isotopically depleted growth medium to culture bacterial cells (Escherichia coli), resulting in isotopically depleted proteins. Isotopically depleted proteins show increased sequence coverage, mass measurement accuracy, and increased S/N of the monoisotopic peak by Fourier transform ion cyclotron resonance mass spectrometry analysis. We then grew Caenorhabditis elegans cells in a medium containing living isotopically depleted E. coli cells, thereby producing the first isotopically depleted eukaryotic proteins. This is the first time isotopic depletion has been implemented for four isotopes (1H, 12C, 14N, and 16O), resulting in the highest degree of depletion ever used for protein analysis and further improving MS analysis.
Assuntos
Caenorhabditis elegans , Escherichia coli , Animais , Escherichia coli/química , Análise de Fourier , Ciclotrons , Proteínas/química , Espectrometria de Massas/métodos , Isótopos , Cromatografia Líquida/métodos , Linhagem CelularRESUMO
N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products.
Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Humanos , Metilação , Metiltransferases/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Especificidade por Substrato/genéticaRESUMO
Hemoglobinopathies are one of the most prevalent genetic disorders, affecting millions throughout the world. These are caused by pathogenic variants in genes that control the production of hemoglobin (Hb) subunits. As the number of known Hb variants has increased, it has become more challenging to obtain unambiguous results from routine chromatographic assays employed in the clinical laboratory. Top-down proteomic analysis of Hb by mass spectrometry is a definitive method to directly characterize the sequences of intact subunits. Here, we apply "chimeric ion loading" to characterize Hb ß subunit variants. In this technique, product ions derived from complementary dissociation techniques are accumulated in a multipole storage device before delivery to a 21 T Fourier-transform ion cyclotron resonance mass spectrometer for simultaneous detection. To further improve the efficiency of identification of Hb variants and localization of the mutation site(s), we developed an R programming script, "Variants Identifier", to search top-down data against a database containing accurate intact mass differences and diagnostic ions from investigated Hb variants. A second R script, "PredictDiag", was developed and employed to determine relevant diagnostic ions for additional Hb variants with known sequences. These two R scripts were successfully applied to the identification of a Hb δ-ß fusion protein and other Hb variants. The combination of chimeric ion loading and the above R scripts enables rapid and reliable interpretation of top-down mass spectrometry data, regardless of activation type, for Hb variant identification.