Publications by authors named "Marta Passamonti"

4 Publications

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Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain.

Anal Chem 2022 04 5;94(15):5909-5917. Epub 2022 Apr 5.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Boulevard, Richland, Washington 99354, United States.

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.
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http://dx.doi.org/10.1021/acs.analchem.2c00139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9003935PMC
April 2022

Poly(acrylamide--,'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content.

Anal Chem 2021 12 22;93(48):16000-16007. Epub 2021 Nov 22.

Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, Amsterdam 1098 XH, The Netherlands.

In this study, we optimized a polymerization mixture to synthesize poly(acrylamide--,'-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable ( between 2.5 × 10 and 1.40 × 10 m) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.
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http://dx.doi.org/10.1021/acs.analchem.1c03473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8655738PMC
December 2021

Fabrication of polymer monoliths within the confines of non-transparent 3D-printed polymer housings.

J Chromatogr A 2020 Jul 12;1623:461159. Epub 2020 May 12.

Van't Hoff Institute for Molecular Sciences, Science Park, University of Amsterdam 1098 HX Amsterdam, Netherlands; The Centre for Analytical Sciences Amsterdam (CASA), University of Amsterdam 1098 HX Amsterdam, Netherlands.

In the last decade, 3D-printing has emerged as a promising enabling technology in the field of analytical chemistry. Fused-deposition modelling (FDM) is a popular, low-cost and widely accessible technique. In this study, RPLC separations are achieved by in-situ fabrication of porous polymer monoliths, directly within the 3D-printed channels. Thermal polymerization was employed for the fabrication of monolithic columns in optically non-transparent column housings, 3D-printed using two different polypropylene materials. Both acrylate-based and polystyrene-based monoliths were created. Two approaches were used for monolith fabrication, viz. (i) in standard polypropylene (PP) a two-step process was developed, with a radical initiated wall-modification step 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, followed by a polymerization step to generate the monolith; (ii) for glass-reinforced PP (GPP) a silanization step or wall modification preceded the polymerization reaction. The success of wall attachment and the morphology of the monoliths were studied using scanning electron microscopy (SEM), and the permeability of the columns was studied in flow experiments. In both types of housings polystyrene-divinylbenzene (PS-DVB) monoliths were successfully fabricated with good wall attachment. Within the glass-reinforced polypropylene (GPP) printed housing, SEM pictures showed a radially homogenous monolithic structure. The feasibility of performing liquid-chromatographic separations in 3D-printed channels was demonstrated.
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http://dx.doi.org/10.1016/j.chroma.2020.461159DOI Listing
July 2020

Confinement of Monolithic Stationary Phases in Targeted Regions of 3D-Printed Titanium Devices Using Thermal Polymerization.

Anal Chem 2020 02 13;92(3):2589-2596. Epub 2020 Jan 13.

Van't Hoff Institute for Molecular Sciences , University of Amsterdam , 1090GD Amsterdam , The Netherlands.

In this study, we have prepared thermally initiated polymeric monolithic stationary phases within discrete regions of 3D-printed titanium devices. The devices were created with controllable hot and cold regions. The monolithic stationary phases were first locally created in capillaries inserted into the channels of the titanium devices. The homogeneity of the monolith structure and the interface length were studied by scanning a capacitively coupled conductivity contactless detector (CD) along the length of the capillary. Homogeneous monolithic structures could be obtained within a titanium device equipped with a hot and cold jacket connected to two water baths. The confinement method was optimized in capillaries. The sharpest interfaces (between monolith and empty channel) were obtained with the hot region maintained at 70 °C and the cold region at 4 or 10 °C, with the latter temperature yielding better repeatability. The optimized conditions were used to create monoliths bound directly to the walls of the titanium channels. The fabricated monoliths were successfully used to separate a mixture of four intact proteins using reversed-phase liquid chromatography. Further chromatographic characterization showed a permeability () of ∼4 × 10 m and a total porosity of 60%.
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http://dx.doi.org/10.1021/acs.analchem.9b04298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003155PMC
February 2020
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