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1.
Anal Chem ; 96(15): 5860-5868, 2024 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-38567987

RESUMO

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.


Assuntos
Hidrogéis , Nanopartículas , Hidrogéis/química , Cromatografia de Fase Reversa/métodos , Polietilenoglicóis/química , Aerossóis
2.
J Chromatogr A ; 1710: 464414, 2023 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-37806043

RESUMO

In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.


Assuntos
Oligonucleotídeos , RNA Guia de Sistemas CRISPR-Cas , Espectrometria de Massas , Cromatografia Líquida/métodos , Oligonucleotídeos/análise , Interações Hidrofóbicas e Hidrofílicas
3.
bioRxiv ; 2023 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-37609276

RESUMO

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties following applied stresses, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel compositions is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, Bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e. proteins), and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with high resolution mass spectrometry and a Fourier-transform based deconvolution algorithm. To our knowledge, this is the first RPLC-CAD method for characterizing the critical quality attributes of supramolecular hydrogels. We envision this analytical strategy could be generalized to characterize other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

4.
Anal Chem ; 95(6): 3180-3186, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-36606446

RESUMO

In recent years, CRISPR-Cas9 genome editing has become an important technology in biomedical research and has demonstrated tremendous therapeutic potential. With Cas9 endonuclease, the use of single guide ribonucleic acids (sgRNAs) allows for sequence-specific cutting on target double-stranded deoxyribonucleic acids. Therefore, the design and quality of sgRNAs can greatly affect the efficiency and specificity of genome editing. Mass spectrometry (MS) has been a powerful tool to detect molecular features and sequence a variety of biomolecules; however, as the sizes of oligonucleotides get larger, it becomes more challenging to desalt samples and achieve high-quality intact spectra with effective fragmentation. Here, we develop a simple but effective online column-based clean-up method (reversed-phase column in a size exclusion mode) that removes formulation salts and metal adducts from larger oligonucleotides upon entering the mass spectrometer in a consistent manner. Using the top-down approach without any nuclease digestion, we characterized and sequenced 100-nucleotide-long sgRNAs by higher-energy collision dissociation (HCD), collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and activated electron photodetachment (a-EPD). In a single 10 min liquid chromatography-tandem MS (LC-MS/MS) run, CID yielded the best sequence coverage, of 67%. When adding complementary UVPD and a-EPD runs, we achieved 80% overall sequence coverage and 100% cleavages for the variable sequence, the first 20 nucleotides from the 5' end. This LC-MS/MS platform provides a facile top-down workflow to analyze and sequence larger chemically modified oligonucleotides with no sample treatment.


Assuntos
Espectrometria de Massas em Tandem , Raios Ultravioleta , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Elétrons
5.
J Pharm Biomed Anal ; 211: 114622, 2022 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-35131673

RESUMO

In pharmaceutical development, structural elucidation of small molecules from process related impurities and degradation products is an essential component. As one of the most important methods in the toolbox, high resolution mass spectrometry (HRMS) and specifically tandem mass spectrometry (MS/MS) often provide fast and informative structural insights. However, many small molecule drugs containing certain biological relevant pharmacophores result in limited numbers of fragments when using traditional collision based fragmentation techniques, such as higher energy collisional dissociation (HCD), due to its inherent preference of cleaving the weakest bond first. As an alternative, ultraviolet photodissociation (UVPD), which irradiates the precursor ion with high energy photons, can lead to more extensive fragmentation from the readily UV absorbing small molecules. Here, we showcase the advantage of UVPD over HCD on pyrrolidine and piperidine containing molecules derivatized from a model compound, telmisartan. While HCD only yielded a single, highly abundant ion resulting from the pyrrolidine and pipieridine ring cleavage, UVPD generated rich and structurally informative fragment ions. UVPD is an attractive and powerful alternative for traditional fragmentation techniques for small molecule structural elucidation.


Assuntos
Espectrometria de Massas em Tandem , Raios Ultravioleta , Íons , Piperidinas , Pirrolidinas , Espectrometria de Massas em Tandem/métodos
6.
J Chromatogr A ; 1665: 462839, 2022 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-35093620

RESUMO

Guide ribonucleic acid (gRNA) is a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. The single stranded guide RNA (sgRNA) is the most commonly used gRNA in application. Evaluation of the impurity profile of synthetic sgRNA is important for any CRISPR genome editing experiments. However, the large molecular size, complex impurity profile and unique secondary structure pose many challenges in the analysis of sgRNA by ion pairing reversed-phase liquid chromatography (IP-RPLC), the commonly used method. In this work, we developed a generic IP-RPLC method for guide RNA analysis. We found that large pore size of stationary phase was the most critical column parameter to achieve high resolution separation of sgRNA while particle structure, particle size and surface chemistry had less impact. Our results indicated that charge interaction was the most critical mechanism for retention and mass transfer had less impact on the performance of separation. An IP-RPLC/mass spectrometry (MS) method was also developed with a specific practice to reduce adducts and enable intact MS analysis of sgRNAs. The generic IP-RPLC method demonstrates its feasibility to serve as a release, stability, characterization and in-process control testing method for synthetic sgRNA products.


Assuntos
Sistemas CRISPR-Cas , Cromatografia de Fase Reversa , Espectrometria de Massas , RNA , RNA Guia de Cinetoplastídeos
7.
Biochemistry ; 60(14): 1108-1119, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33755420

RESUMO

Methods for maintaining membrane proteins in their native state after removal from the lipid bilayer are essential for the study of this important class of biomacromolecules. Common solubilization strategies range from the use of detergents to more complex systems that involve a polypeptide working in concert with lipids or detergents, such as nanodiscs, picodiscs, and peptidiscs, in which an engineered protein or synthetic peptide surrounds the membrane protein along with a lipid sheath. Picodiscs employ the protein saposin A, which naturally functions to facilitate lipid degradation in the lysozome. Saposin A-amphiphile complexes therefore tend to be most stable at acidic pH, which is not optimal for most membrane protein applications. In search of new picodisc assemblies, we have explored pairings of saposin A or other saposin proteins with a range of detergents, and we have identified a number of combinations that spontaneously co-assemble at neutral pH. The resulting picodiscs are stable for weeks and have been characterized by size-exclusion chromatography, native mass spectrometry, and small angle X-ray scattering. The new assemblies are formed by double-tail detergents rather than more traditional single-tail detergents; the double-tail detergents can be seen as structurally intermediate between single-tail detergents and common lipids. In addition to saposin A, an engineered variant of saposin B (designated saposin BW) forms picodisc assemblies. These findings provide a framework for future efforts to solubilize membrane proteins with multiple picodisc systems that were previously unknown.


Assuntos
Detergentes/química , Saposinas/química , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Saposinas/genética
8.
Anal Chem ; 92(22): 15096-15103, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33108180

RESUMO

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.


Assuntos
Cromatografia Líquida/métodos , Ciclotrons , Análise de Fourier , Imunoconjugados/análise , Espectrometria de Massas em Tandem/instrumentação , Imunoconjugados/química , Isomerismo , Fatores de Tempo
9.
Nat Commun ; 11(1): 3903, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764543

RESUMO

Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>1010 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide high-resolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibody-free strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.


Assuntos
Análise Química do Sangue/métodos , Proteínas Sanguíneas/análise , Espectrometria de Massas/métodos , Proteômica/métodos , Troponina I/sangue , Biomarcadores/sangue , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Nanotecnologia , Processamento de Proteína Pós-Traducional , Proteoma/análise , Reprodutibilidade dos Testes , Albumina Sérica Humana/análise
10.
Anal Chem ; 92(12): 8584-8590, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32442374

RESUMO

A current trend in drug development involves the use of high molecular weight, branched, and functionalized polymers for protein conjugation and drug delivery. Accurately characterizing these polymers is critical to control the product quality, to monitor the stability, and ultimately to ensure the drug efficacy and patient safety. However, due to the heterogeneity in size, the multiplicity of functional groups, and the highly convoluted charge-distribution profile in mass spectra, the characterization of these polymers is highly challenging from both chromatography and mass spectrometry perspectives. To overcome these challenges, we developed a strategy utilizing charge-reduction mass spectrometry (CRMS) coupled with two-dimensional HPLC (2D-LC). We then applied the workflow to characterize a 40 kDa 8-arm polyethylene glycol (PEG) functionalized with a maleimide terminal group for protein conjugation. The development was carried out in stages, where first we focused on the development of a CRMS method to simplify the charge profile of the polymers and then coupled it to HPLC to obtain discernible mass spectra of key impurities and degradants. Second, the CRMS method was applied to an investigation of the size-variant impurity resolved by reversed-phase size-exclusion 2D-LC. Finally, a separate size-exclusion reversed-phase 2D-LC-CRMS method was developed to capture a wider range of process-related impurities and reaction intermediates from the PEG-maleimide polymers throughout the conjugation process. The combination of these experiments using the 2D-LC-CRMS strategy enables the sensitive characterization of the entire impurity profile of the high molecular weight multifunctionalized PEG-maleimide conjugation intermediate.


Assuntos
Maleimidas/química , Polietilenoglicóis/química , Proteínas/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Peso Molecular , Software
11.
J Am Soc Mass Spectrom ; 30(12): 2561-2570, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31792770

RESUMO

Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Domínio Catalítico , Modelos Moleculares , Fosforilação , Subunidades Proteicas/química , Proteínas Recombinantes/química
12.
Anal Chem ; 91(18): 11661-11669, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31442030

RESUMO

Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcomes. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of coexisting variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris; BV) and lysine (Kadcyla; T-DM1) conjugated ADCs at the subunit level (∼25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the microvariants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we accurately measured average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and microvariants, which potentially opens up a new avenue to characterize ADCs.


Assuntos
Ado-Trastuzumab Emtansina/química , Brentuximab Vedotin/química , Imunoconjugados/análise , Imunoconjugados/química , Ado-Trastuzumab Emtansina/análise , Brentuximab Vedotin/análise , Cromatografia de Fase Reversa , Cisteína/química , Transporte de Elétrons , Lisina/química , Espectrometria de Massas em Tandem/métodos
13.
Nano Res ; 12(6): 1473-1481, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31341559

RESUMO

A reproducible synthetic strategy was developed for facile large-scale (200 mg) synthesis of surface silanized magnetite (Fe3O4) nanoparticles (NPs) for biological applications. After further coupling a phosphate-specific affinity ligand, these functionalized magnetic NPs were used for the highly specific enrichment of phosphoproteins from a complex biological mixture. Moreover, correlating the surface silane density of the silanized magnetite NPs to their resultant enrichment performance established a simple and reliable quality assurance control to ensure reproducible synthesis of these NPs routinely in large scale and optimal phosphoprotein enrichment performance from batch-to-batch. Furthermore, by successful exploitation of a top-down phosphoproteomics strategy that integrates this high throughput nanoproteomics platform with online liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we were able to specifically enrich, identify, and characterize endogenous phosphoproteins from highly complex human cardiac tissue homogenate. This nanoproteomics platform possesses a unique combination of scalability, specificity, reproducibility, and efficiency for the capture and enrichment of low abundance proteins in general, thereby enabling downstream proteomics applications.

14.
Nat Methods ; 16(7): 587-594, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31249407

RESUMO

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.


Assuntos
Benchmarking , Espectrometria de Massas/métodos , Proteínas/química , Desnaturação Proteica , Processamento de Proteína Pós-Traducional , Proteômica
15.
Nat Methods ; 16(5): 417-420, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988469

RESUMO

We report the identification of a photocleavable anionic surfactant, 4-hexylphenylazosulfonate (Azo), which can be rapidly degraded by ultraviolet irradiation, for top-down proteomics. Azo can effectively solubilize proteins with performance comparable to that of sodium dodecyl sulfate (SDS) and is compatible with mass spectrometry. Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of post-translational modifications. Moreover, Azo is simple to synthesize and can be used as a general SDS replacement in SDS-polyacrylamide gel electrophoresis.


Assuntos
Compostos Azo/química , Eletroforese em Gel de Poliacrilamida/métodos , Proteômica/métodos , Dodecilsulfato de Sódio/química , Tensoativos/química , Compostos Azo/efeitos da radiação , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Proteínas de Membrana/análise , Fotólise , Dodecilsulfato de Sódio/efeitos da radiação , Solubilidade , Tensoativos/efeitos da radiação , Raios Ultravioleta
16.
Anal Chem ; 91(6): 3835-3844, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30758949

RESUMO

Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.


Assuntos
Cromatografia em Gel/instrumentação , Ciclotrons , Análise de Fourier , Espectrometria de Massas/instrumentação , Proteômica/instrumentação , Humanos , Miocárdio/metabolismo
17.
Anal Chem ; 90(24): 14643, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30522269
18.
Anal Chem ; 90(12): 7135-7138, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29846060

RESUMO

Therapeutic monoclonal antibodies (mAbs) are an important class of drugs for a wide spectrum of human diseases. Liquid chromatography (LC) coupled to mass spectrometry (MS) is one of the techniques in the forefront for comprehensive characterization of analytical attributes of mAbs. Among various protein chromatography modes, hydrophobic interaction chromatography (HIC) is a popular offline nondenaturing separation technique utilized to purify and analyze mAbs, typically with the use of non-MS-compatible mobile phases. Herein we demonstrate for the first time, the application of direct HIC-MS and HIC-tandem MS (MS/MS) with electron capture dissociation (ECD) for analyzing intact mAbs on quadrupole-time-of-flight (Q-TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, respectively. Our method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles of mAbs as well as sequence and structural characterization of the complementarity-determining regions in an online configuration.


Assuntos
Anticorpos Monoclonais/análise , Interações Hidrofóbicas e Hidrofílicas , Cromatografia Líquida , Humanos , Espectrometria de Massas
19.
Anal Chem ; 90(8): 4935-4939, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29565561

RESUMO

Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray ionization (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus, the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation, multiphosphorylation noticeably affected ESI-MS quantification of phosphoproteins likely due to differential ionization/detection efficiency between unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated ß-casein (24 kDa).


Assuntos
Fosfoproteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caseínas/análise , Caseínas/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/metabolismo , Fosfopeptídeos/análise , Fosfoproteínas/metabolismo , Fosforilação , Proteômica
20.
Anal Chem ; 90(1): 110-127, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29161012
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