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1.
Anal Chem ; 94(12): 5140-5148, 2022 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-35285615

RESUMEN

Intact-mass measurements are becoming increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS is an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein but also the mass range populated by the entire complement of proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation (with the glycan load of phenotype 2-1 being notably lighter) despite a significant overlap of their ionic signals. More detailed characterization of their glycosylation patterns is enabled by the recently introduced technique of cross-path reactive chromatography (XP-RC) with online MS detection, which combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains and provides a wealth of information on glycosylation patterns of the heavy chains. N-Glycosylation patterns of both haptoglobin phenotypes were found to be consistent with bi- and triantennary structures of complex type that exhibit significant level of fucosylation and sialylation. However, multivariate analysis of haptoglobin 1-1 reveals higher number of the triantennary structures, in comparison to haptoglobin 2-1, as well as a higher extent of fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of the intact-mass analysis supplemented by limited charge reduction, suggesting that the latter technique can be employed in situations when fast assessment of protein heterogeneity is needed (e.g., process analytical technology applications).


Asunto(s)
Haptoglobinas , Espectrometría de Masa por Ionización de Electrospray , Glicosilación , Haptoglobinas/química , Haptoglobinas/metabolismo , Análisis Multivariante , Proteínas/metabolismo
2.
Methods ; 144: 14-26, 2018 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-29702225

RESUMEN

Interfacing liquid chromatography (LC) with electrospray ionization (ESI) to enable on-line MS detection had been initially implemented using reversed phase LC, which in the past three decades remained the default type of chromatography used for LC/MS and LC/MS/MS studies of protein structure. In contrast, the advantages of other types of LC as front-ends for ESI MS, particularly those that allow biopolymer higher order structure to be preserved throughout the separation process, enjoyed relatively little appreciation until recently. However, the past few years witnessed a dramatic surge of interest in the so-called "native" (with "non-denaturing" being perhaps a more appropriate adjective) LC/MS and LC/MS/MS analyses within the bioanalytical and biophysical communities. This review focuses on recent advances in this field, with an emphasis on size exclusion and ion exchange chromatography as front-end platforms for protein characterization by LC/MS. Also discussed are the benefits provided by the integration of chemical reactions in the native LC/MS analyses, including both ion chemistry in the gas phase (e.g., limited charge reduction for characterization of highly heterogeneous biopolymers) and solution-phase reactions (using the recently introduced technique cross-path reactive chromatography).


Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Espectrometría de Masas/métodos , Conformación Proteica , Proteínas/metabolismo , Ligandos , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteínas/química
3.
Anal Chem ; 90(2): 1348-1355, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29240412

RESUMEN

Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g,, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide-reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide-linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using ß2-microglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as demonstrated in this work by oxidatively labeling mAb on the column, followed by reduction of its disulfide bonds and MS analysis of the extent of oxidation within each chain of the molecule).

4.
Analyst ; 141(3): 853-61, 2016 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-26646585

RESUMEN

Serum transferrin is a key player in iron homeostasis, and its ability to deliver iron to cells via the endosomal pathway critically depends on the presence of carbonate that binds this protein synergistically with ferric ion. Oxalate is another ubiquitous anionic species that can act as a synergistic anion, and in fact its interaction with transferrin is notably stronger compared to carbonate, preventing the protein from releasing the metal in the endosomal environment. While this raises concerns that high oxalate levels in plasma may interfere with iron delivery to tissues, concentration of free oxalate in blood appears to be a poor predictor of impeded availability of iron, as previous studies showed that it cannot displace carbonate from ferro-transferrin on a physiologically relevant time scale under the conditions mimicing plasma. In this work we present a new method that allows different forms of ferro-transferrin (carbonate- vs. oxalate-bound) to be distinguished from each other by removing this protein from plasma without altering the composition of the protein/metal/synergistic anion complexes, and determining their accurate masses using native electrospray ionization mass spectrometry (ESI MS). The new method has been validated using a mixture of recombinant proteins, followed by its application to the analysis of clinical samples of human plasma, demonstrating that native ESI MS can be used in clinical analysis.


Asunto(s)
Hierro/sangre , Transferrina/metabolismo , Trastorno Autístico/sangre , Carbonatos/sangre , Cromatografía en Gel , Humanos , Oxalatos/sangre , Albúmina Sérica , Espectrometría de Masa por Ionización de Electrospray
5.
J Pharm Biomed Anal ; 151: 133-144, 2018 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-29324282

RESUMEN

Monoclonal antibodies (mAbs) are the fastest growing class of biopharmaceuticals. The specific therapeutic tasks vary among different mAbs, which may include neutralization of soluble targets, activation of cytotoxic pathways, targeted drug delivery, and diagnostic imaging. The specific therapeutic goal defines which interactions of the antibody with its multiple physiological partners are most critical for function, and which ones are irrelevant or indeed detrimental. In this work, we explored the ability of the glycan chains to affect IgG1 interaction with two key receptor families, FcRn and γ-type Fc receptors, as well as the influence of glycan composition on the conformation and stability of the antibody molecule. Three different glycan-modified forms of IgG1 (fully deglycosylated, hypergalactosylated and hypersialylated) were produced and characterized alongside the unmodified mAb molecule. Biophysical measurements did not reveal any changes that would be indicative of alterations in the higher order structure or increased aggregation propensity for any of the three glycoforms compared to the unmodified mAb, although the CH2 domain was shown to have reduced thermal stability in the fully deglycosylated form. No significant changes were observed for the hypergalactosylated and hypersialylated forms of IgG1 with regards to binding to FcRn, FcγRIIA and FcγRIIIA, suggesting that neither half-life in circulation nor their ability to induce an immune response are likely to be affected by these modifications of the glycan chains. In contrast, no measurable binding was observed for the deglycosylated form of IgG1 with either FcγRIIA or FcγRIIIA, although this form of the antibody retained the ability to associate with FcRn. These highly specific patterns of attenuation of Fc receptor recognition can be exploited in the future for therapeutic purposes.


Asunto(s)
Inmunoglobulina G/análisis , Inmunoglobulina G/química , Polisacáridos/análisis , Polisacáridos/química , Animales , Fenómenos Bioquímicos , Fenómenos Biofísicos , Células CHO , Cricetinae , Cricetulus , Glicosilación , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
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