RESUMO
Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography (LC) is the current gold standard for intact protein analysis. However, inherent speed limitations of LC/MS prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI-MS), an ambient ionization MS technology, has recently been established as a platform for high-throughput small molecule analysis. Here, we report the applications of such a system for the analysis of intact proteins commonly performed within the drug discovery process. A wide molecular weight range of proteins 10-150 kDa was detected on the system with improved tolerance to salts and buffers compared to ESI. With high concentrations and model proteins, a sample rate of up to 22 Hz was obtained. For proteins at low concentrations and in buffers used in commonly employed assays, robust data at a sample rate of 1.5 Hz were achieved, which is â¼22× faster than current technologies used for high-throughput ESI-MS-based protein assays. In addition, two multiplexed plate-based high-throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing excessive amounts of salts and buffers without fully compromising productivity. Example experiments, which leverage the speed of the IR-MALDESI-MS system to monitor NISTmAb reduction, protein autophosphorylation, and compound binding kinetics in near real time, are demonstrated.
Assuntos
Produtos Biológicos , Espectrometria de Massas por Ionização por Electrospray , Descoberta de Drogas , Lasers , Proteínas/química , Sais , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Electron transfer dissociation (ETD) is an analytically useful tool for primary structure interrogation of intact proteins, but its utility is limited by higher-order reactions with the products. To inhibit these higher-order reactions, first-generation fragment ions are kinetically excited by applying an experimentally tailored parallel ion parking waveform during ETD (ETD-PIP). In combination with subsequent ion/ion proton transfer reactions, precursor-to-product conversion was maximized as evidenced by the consumption of more than 90% of the 21 kDa Protein G precursor to form ETD product ions. The employment of ETD-PIP increased sequence coverage to 90% from 80% with standard ETD. Additionally, the inhibition of sequential electron transfers was reflected in the high number of complementary ion pairs from ETD-PIP (90%) compared to standard ETD (39%).
Assuntos
Elétrons , Proteínas , Transporte de Elétrons , Íons , Análise de SequênciaRESUMO
Methodology for sequence analysis of â¼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8murea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3-9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms ofN-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion-ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.
Assuntos
Anticorpos Monoclonais/metabolismo , Proteólise , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional , Fatores de TempoRESUMO
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
Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of â¼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.
Assuntos
Espectrometria de Massas , Ligantes , Espectrometria de Massas/métodos , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Anidrases Carbônicas/análise , Ligação Proteica , Tirosina Quinase da Agamaglobulinemia/metabolismo , Tirosina Quinase da Agamaglobulinemia/química , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Humanos , Proteínas/química , Proteínas/metabolismo , Proteínas/análiseRESUMO
Deconvolution from intact protein mass-to-charge spectra to mass spectra is essential to generate interpretable data for mass spectrometry (MS) platforms coupled to ionization sources that produce multiply charged species. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) can be used to analyze intact proteins in multiwell microtiter plates with speed matching small molecule analyses (at least 1 Hz). However, the lack of compatible deconvolution software has limited its use in high-throughput screening applications. Most existing automated deconvolution software packages work best for data generated from LC-MS, and to the best of our knowledge, there is no software capable of performing fast plate-based mass spectral deconvolution. Herein we present the use of a new workflow in ProSight Native for the deconvolution of protein spectra from entire well plates that can be completed within 3 s. First, we successfully demonstrated the potential increased throughput benefits produced by the combined IR-MALDESI-MS - ProSight Native platform using protein standards. We then conducted a screen for Bruton's tyrosine kinase (BTK) covalent binders against a well-annotated compound collection consisting of 2232 compounds and applied ProSight Native to deconvolute the protein spectra. Seventeen hits including five known BTK covalent inhibitors in the compound set were identified. By alleviating the data processing bottleneck using ProSight Native, it may be feasible to analyze and report covalent screening results for >200,000 samples in a single day.
Assuntos
Espectrometria de Massas , Proteínas , Proteínas/química , SoftwareRESUMO
We have enabled parallel ion parking on a modified Orbitrap Elite™ as a way to control ion-ion proton transfer reactions via selective activation of a range of ions. The result is the concentration of the majority of ion current from multiple charge states of each precursor proteoform into a single charge state, maximizing signal intensity and increasing effective sensitivity compared to conventional MS1 spectra. These techniques were applied in an on-line HPLC, data-dependent MS/MS analysis of intact E. coli ribosomal proteins with HCD fragmentation. With one injection, all but two ribosomal proteins were selected for fragmentation and subsequently identified. The techniques described facilitate rapid identification of intact proteins in complex mixtures and an enhanced ability to observe proteins of low abundance.