Hydrophilic Interaction Chromatography Coupled to Ultraviolet Photodissociation Affords Identification, Localization, and Relative Quantitation of Glycans on Intact Glycoproteins.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans.

Consolidating two-dimensional liquid chromatography-high-resolution tandem mass spectrometry (LC×LC-HRMS/MS) technique for the non-targeted analysis of poly- and perfluorinated substances: A trial on aqueous film-forming foams.

To date, poly- and perfluoroalkyl substances (PFAS) represent a real threat for their environmental persistence, wide physicochemical variability, and their potential toxicity. Thus far a large portion of these chemicals remain structurally unknown. These chemicals, therefore, require the implementation of complex non-targeted analysis workflows using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for their comprehensive detection and monitoring.

Functionality-type and chemical-composition separation of poly(lactide-co-glycolide) using gradient elution normal-phase liquid chromatography with basic and acidic additives.

End groups of poly(Lactide-co-glycolide) (PLGA) play an important role in determining the properties of polymers for use in drug delivery systems. For instance, it has been reported that the encapsulation efficiency in PLGA microspheres varies significantly between ester-terminated and acid-terminated PLGA. More importantly, the in-vivo degradation time of such polymer excipients is influenced by the functional end-group of the copolymer used.

Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.

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 chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins.

Comprehensive Two-Dimensional Liquid Chromatography-High-Resolution Mass Spectrometry for Complex Protein Digest Analysis Using Parallel Gradients.

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 separation dimension (D). This results in higher dilution factors and the need for flow splitters to couple to ESI-MS.

Identification of heavily glycated proteoforms by hydrophilic-interaction liquid chromatography and native size-exclusion chromatography - High-resolution mass spectrometry.

Background: 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 characterization of heavily glycated proteins at the intact level is of high interest as it allows to describe co-occurring protein modifications. However, the high heterogeneity of glycated protein makes this process challenging, and novel methods are required to accomplish this.

Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from a single sample injection - Intact protein workflow.

Background: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone.

Sample transformation in online separations: how chemical conversion advances analytical technology.

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 be used to provide insights into properties of complex samples such as degradation pathways or molecular heterogeneity that are otherwise unaccessible. In this article, we explore how sample transformation is exploited across different application fields to empower analytical methods.

Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes.

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 labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry.

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), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins.

Development of a comprehensive two-dimensional liquid chromatographic mass spectrometric method for the non-targeted identification of poly- and perfluoroalkyl substances in aqueous film-forming foams.

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-mode weak anion exchange-reversed phase with a octadecyl stationary phase, separating PFASs according to ionic classes and chain length. To develop and optimize the LC × LC method we used a reference training set of twenty-four anionic PFASs, representing the main classes of compounds occurring in AFFFs and covering a wide range of physicochemical properties.

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

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.

Investigation of the effects of solvent-mismatch and immiscibility in normal-phase × aqueous reversed-phase liquid chromatography.

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 and solvent-incompatibility (such as solvent-strength mismatch or immiscibility experienced during fraction transfer). A severe case of solvent-incompatibility is encountered in the comprehensive coupling of normal-phase LC and reversed-phase LC (NPLC × RPLC).

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

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).

Fast determination of functionality-type × molecular-weight distribution of propoxylates with varying numbers of hydroxyl end-groups using gradient-normal-phase liquid chromatography × ultra-high pressure size-exclusion chromatography.

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 include, for example, molecular weight, nature of end-groups (functionality), and chemical composition.

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins.

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 reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC.

Development of comprehensive two-dimensional low-flow liquid-chromatography setup coupled to high-resolution mass spectrometry for shotgun proteomics.

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 for HRMS analysis, but they tend to be lengthy.

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc -Glycosylation.

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). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging.

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

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.

Accurate modelling of the retention behaviour of peptides in gradient-elution hydrophilic interaction liquid chromatography.

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.

Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms.

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 glycoforms of glycoproteins.