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Andrea GarganoUniversity of Amsterdam | UVA · Analytical Chemistry
Andrea Gargano
PhD
About
70
Publications
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1,162
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Introduction
Additional affiliations
January 2019 - January 2025
January 2016 - present
University of Amsterdam and VU University Amsterdam
Position
- Researcher
September 2013 - January 2016
Education
January 2010 - April 2013
Publications
Publications (70)
Online comprehensive two-dimensional liquid chromatography (LC × LC) has become an attractive option for the analysis of complex non-volatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). LC × LC complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrome...
Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In p...
Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of...
In this study, we optimized a polymerization mixture to synthesize poly(acrylamide-co-N,N′-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 separati...
Understanding the relation between chemical characteristics and properties of synthetic polymers is one of the challenges faced by analytical chemists in industry. This is a complex task, as polymers are not synthesized as single molecule, but are populations of chemically similar compounds with distributions over several properties. The latter inc...
The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatograph...
Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the method's reduced sensitivity which can be linked to the high flow rates of the second separatio...
Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs), e.g., charge variants, oxidation, etc., during production and storage, necessitating evaluation of the resulting PTMs as critical quality attributes (CQA) for protein quality and safety. Charge variants PTMs can be separated using capillary zone electrophoresis (...
Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC×LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the methods' reduced sensitivity that can be linked to the high flow rates of the second separation d...
The end groups of poly(lactide-co-glycolide) (PLGA) play a crucial role in determining the properties of polymers used in drug delivery systems. Research has shown that the encapsulation efficiency in PLGA microspheres can vary significantly between ester-terminated and acid-terminated PLGA. To study the functionality-type distribution (FTD) in PLG...
The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatograph...
Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans including microheterogeneity at the same amino acid residue (glycosite). Top-down mass spectrometry (MS) allows precise identification and localization of glycans on intact proteins, and coupling t...
While the advent of modern analytical technology has allowed scientists to determine the complexity of mixtures, it also spurred the demand to understand these sophisticated mixtures better. Chemical transformation can...
The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The high heterogeneity that glycated proteins carry makes their detection and identification challenging. In this study, we investigated two novel...
Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC×LC-HRMS) has not yet been widely applied to the analysis of complex protein digests such as cell lysates. One reason is the reduced sensitivity of the methods, with second separation dimensions that r...
We constructed a high-performance biosensor for detecting uric acid by immobilizing an engineered urate oxidase on gold nanoparticles deposited on a carbon-glass electrode. This biosensor showed a low limit-of-detection (9.16 nM), a high sensitivity (14 μA/μM), a wide range of linearity (50 nM-1 mM), and more than 28 days lifetime.
SuFEx click chemistry has established itself as a formidable tool to rapidly and effectively link chemical structures. Despite tremendous advancements in the field in recent years, the installment of the crucial -SO2F handle still requires the use of purposely designed, expensive, and non-atom economical reagents. However, the use of the most obvio...
Polymers’ performance, biodegradability and recyclability can be tuned during synthesis by adopting monomers with differ-ent chemical characteristics. Recent research has shown the aptness of enzyme hydrolysis to depolymerize polyester under mild conditions with respect to chemical approaches. Herein, we engineered a cutinase from T. cellulosilytic...
Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs)...
In this research, we developed an online comprehensive two-dimensional liquid chromatographic (LC×LC) method hyphenated with high-resolution mass spectrometry (HRMS) for the non-targeted identification of poly- and perfluorinated compounds (PFASs) in fire-fighting aqueous-film forming foams (AFFFs). The method exploited the combination of mixed-mod...
Native Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a useful tool for the characterization of proteins in their native state. However, in many cases the conditions needed to realize the hyphenation of SEC with MS require relative high activation...
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, compr...
In the second part of this review article, the recent progress in supercritical fluid chromatography (SFC) for enantiomeric separations is evaluated. With the substantial developments carried out over the past years in instrumentation, columns, and detector hyphenation, the interest in chiral SFC has been steadily growing in various fields. In comb...
The quantification of the enantiomers of racemic substances is of great importance in the development and regulation of pharmaceutical compounds. Active ingredients are often chiral; typically, only one of the stereoisomers has the desired pharmacokinetic and/or pharmacodynamic properties. Therefore, the stereoisomer distribution of chiral drug pro...
Comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive separation technique that allows achieving high peak capacities and information on chemical correlations. Unfortunately, its application in industrial practice is still not widespread due to limiting factors such as complex method development, tedious method optimization...
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 compre...
Among all the –omics approaches, proteomics and metabolomics have received increased attention over the last decade. Both approaches have reached a certain level of maturity, showing their relevance in numerous clinical applications, including biomarkers discovery, improved diagnosis, staging, and prognosis of diseases, as well as a better knowledg...
In this study, we optimized a polymerization mixture to synthesize polyacrylamide-co-N,N’-methylene bisacrylamide monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of crosslinker and the porogen composition on the separation...
Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to...
Bottom-up proteomics provides often small amounts of highly complex samples that cannot be analysed by direct mass spectrometry (MS). To gain a better insight in the sample composition, liquid chromatography (LC) and (comprehensive) two-dimensional liquid chromatography (2D-LC or LC×LC) can be coupled to the MS. Low-flow separations are attractive...
This chapter presents a methodical overview of recent research literature dealing with the characterization of glycoproteins by hydrophilic interaction chromatography separations coupled to mass spectrometry (HILIC-MS). This includes the analysis of intact glycosylated proteins as well as released glycans, glycopeptides, and protein subunits obtain...
Electrospray ionization (ESI) and other ambient ionization techniques have allowed a successful interface between liquid chromatography (LC) and mass spectrometry (MS). The coupling of these two high-resolution techniques has fostered the use of analytical science in various fields, including, but not limited to, the clinical, pharmaceutical, and f...
Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortuna...
In this study, we evaluate the use of various two-dimensional liquid chromatographic methods to characterize water-soluble, synthetically grafted bio-polymers, consisting of long poly(acrylic acid) chains and short maltodextrin grafts. The confirmation of the presence of grafting and the estimation of its extent is challenging. It is complicated by...
Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a cri...
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 homogen...
The applicability of models to describe peptide retention in hydrophilic interaction liquid chromatography (HILIC) was investigated. A tryptic digest of bovine-serum-albumin (BSA) was used as a test sample. Several different models were considered, including adsorption, mixed-mode, exponential, quadratic and Neue-Kuss models. Gradient separations w...
This review article discusses the novel separation and detection strategies that are considered promising in clinical metabolomics to enhance the metabolome coverage. It includes hydrophilic interaction chromatography (HILIC), supercritical fluid chromatography (SFC), multidimensional LC approaches, as well as ion-mobility mass spectrometry (IM-MS)...
Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glyco...
Polyether polyols are often used in formulated systems, but their complete characterization is challenging, because of simultaneous heterogeneities in chemical composition, molecular weight and functionality. One-dimensional liquid chromatography-mass spectrometry is commonly used to characterize polyether polyols. However, the separation power of...
Recent progress in top-down proteomics has driven the demand for chromatographic methods compatible with mass spectrometry (MS) that can separate intact proteins. Hydrophilic interaction liquid chromatography (HILIC) on amide stationary phases has recently shown good potential for the characterization of glycoforms of intact proteins. In the presen...
In this study we describe an approach to enhance the sensitivity of an online comprehensive two-dimensional liquid chromatography (LC × LC) high-resolution mass spectrometry method for the separation and detection of trace levels of anabolic-steroid residues in complex urine matrices. .
Compared to one-dimensional liquid chromatography (1D-LC), LC...
Recent advancements in separation science have resulted in the commercialization of multidimensional separation systems that provide higher peak capacities and, hence, enable a more-detailed characterization of complex mixtures. In particular, two powerful analytical tools are increasingly used by analytical scientists, namely online comprehensive...
On-line comprehensive two-dimensional liquid chromatography (LC×LC) offers ways to achieve high-performance separations in terms of peak capacity (exceeding 1000) and additional selectivity to realize applications that cannot be addressed with one-dimensional chromatography (1D-LC). However, the greater resolving power of LC×LC comes at the price o...
Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column which is typically narrow and long, whereas theand a second- dimension column that is wide and short. Shallow gradients at low flow rates a...
This article describes the design and optimization of an effective microwave-assisted multicomponent reaction to produce a novel class of phosphopeptidomimetic compounds. When using aminophosphonic acids (α, β, γ), aldehydes and isocyanides as reactants and alcohols as solvents, these building blocks are merged to functionalized amido-aminophosphon...
Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 μm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix. These polymers were then photografted in a single step with 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylic acid to...
We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid ch...
Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked") and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism...
Quinine- and quinidine-derived anion-exchanger chiral stationary phases are versatile tools for enantiomer separation of acidic compounds in high performance liquid chromatography (HPLC). This article demonstrates their recognition ability in specific HPLC applications, involving enantiomer resolution and plasmid topoisomer separation. The extensio...
The state-of-the-art in CEC enantiomer separations with monolithic capillary columns is comprehensively reviewed. The various types of monolithic columns comprising in situ organic polymer monoliths, molecularly imprinted polymer (MIP) monoliths, silica monoliths and monoliths made from particles are discussed with a focus on materials' synthesis,...
Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid...