Adeno-associated viruses (AAVs) are common vectors for emerging gene therapies due to their lack of pathogenicity in humans. Here, we present our investigation of the viral proteins (i.e., VP1, VP2, and VP3) of the capsid of AAVs via top-down mass spectrometry (MS). These proteins, ranging from 59 to 81 kDa, were chromatographically separated using hydrophilic interaction liquid chromatography and characterized in the gas-phase by high-resolution Orbitrap Fourier transform MS. Complementary ion dissociation methods were utilized to improve the overall sequence coverage. By reducing the overlap of product ion signals via proton transfer charge reduction on the Orbitrap Ascend BioPharma Tribrid mass spectrometer, the sequence coverage of each VP was significantly increased, reaching up to ∼40% in the case of VP3. These results showcase the improvements in the sequencing of proteins >30 kDa that can be achieved by manipulating product ions via gas-phase reactions to obtain easy-to-interpret fragmentation mass spectra.

The structure of zwitterion has great impact on the separation properties of zwitterionic hydrophilic stationary phases. To better understand the role of anionic groups of zwitterions, a novel carboxybetaine-based zwitterionic monolithic column was first prepared through thermo-initiated copolymerization of functional monomer (3-acrylamidopropyl)-dimethyl-(2-carboxymethyl) ammonium (CBAA) and crosslinker ethylene dimethacrylate (EDMA) within 100 μm ID capillary. The optimal poly(CBAA-co-EDMA) monolithic column exhibited satisfactory mechanical and chemical stability, good repeatability, high column efficiency (96,000 plates/m), and excellent separation performance for different classes of polar compounds (i.e., phenols, monophosphate nucleotides, urea and allantoin). A comparative study was then performed among three zwitterionic hydrophilic stationary phases containing different anionic groups, i.e. poly(CBAA-co-EDMA) (carboxybetaine), poly(2-{2-(methacryloyloxy) ethyldimethylammonium}ethyl n-butyl phosphate-co-EDMA) (phosphocholine), and poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine-co-EDMA) (sulfobetaine) using benzoic acid derivatives, amine compounds, nucleobases and nucleosides as model analytes. The carboxybetaine-based monolithic column exhibited much higher positive zeta-potential and hydrophilicity, which endows it with a stronger retention capacity for acidic and neutral compounds, but sulfobetaine-based monolithic column exhibited much higher selectivity and retention capacity for the amines. Moreover, their enrichment efficiencies for N-glycopeptides were also evaluated based on their different hydrophilicity, and it was observed that the poly(CBAA-co-EDMA) monolithic material captured 4-8 times more N-glycopeptides compared to the other two materials.

Hydrophilic interaction liquid chromatography (HILIC) based on polar stationary phases has vital research significance in the separation of polar compounds. Numerous HILIC stationary phases with different structures have been developed, which do not have universal properties and broad selectivity, making it a challenge to select the suitable column based on the properties of the samples. Consequently, it is particularly important to develop a bonded phase capable of separating a wide variety of samples, while having enhanced retention, improved selectivity, symmetric peak shape and good stability. Herein, a novel nitrogen-containing heterocyclic bonded phase with multiple functionalities, such as thioether, amino and hydroxyl groups (named AMTA) was employed as HILIC stationary phase. Detailed chromatographic evaluations were carried out, and the results showed that it was superior to other hydrophilic chromatographic columns in terms of selectivity, peak shapes and practical sample separation. Lastly, it has been verified that AMTA exhibited high orthogonality with the XBridge C18 column of reversed-phase liquid chromatography (RPLC) mode. In summary, we anticipate our assay to be instructive to other researchers in developing the HILIC stationary phase.

Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.

In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.

BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required.
RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates.
UNASSIGNED: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Etimicin is a typical aminoglycoside antibiotic (AG). High performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD) method is a commonly used method for determining impurities in Etimicin. However, due to the poor reproducibility, low sensitivity and narrow linear range of the ELSD, high-throughput quantitative analysis of impurities in Etimicin currently poses a challenge. In this study, a sensitive method using hydrophilic interaction liquid chromatography coupled with charged aerosol detector (HILIC-CAD) was developed for the analysis of the impurities in Etimicin. The liquid phase conditions for determination impurities in Etimicin were optimized using Box Behnken design (BBD) and response surface methodology (RSM), resulting in satisfactory separation and optimal CAD output signal. We also studied the influence of CAD parameters on the signal-to-noise ratio and linearity of Etimicin and its impurities. This method has also been proven to be effective in separating impurities from two other typical AGs, Isepamicin and Amikacin. In the method validation, the coefficient of determination (R2) of Etimicin, Isepamicin and Amikacin and their impurities were all greater than 0.999, within the range of 0.5-50 μg/mL. The average recoveries of the impurities of three typical AGs were 99.03 %-101.22 %, RSDs all were less than 2.5 % for intra-day and inter-day precision, with good precision and accuracy. The developed HILIC-CAD quantification method was sensitive, accurate and highly selective for quantitative analysis of impurities in the AGs without need ion-pairing reagents, which is ensure the public medication safety. The method is first reported application of HILIC-CAD method for quantitative analysis of the impurities in AGs.

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol μL-1 for glycopeptides, 0.005 fmol μL-1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, β-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer.

Sampling and chromatography-mass spectrometry methods were investigated to measure atmospheric amines and aminoamides. Amines and their amide derivatives play significant roles in new particle formation (NPF) in the atmosphere, especially diamines and aminoamides have higher NPF potentials compared to monoamines. For amine sampling, silica gel tube collection and formic acid extraction gave good overall recoveries (>93 ± 8%) for mono-, di-, tri-, tetramines, and aminoamides. Two chromatography methods were subjected to analyze the extracted amines. One involved direct analysis using hydrophilic interaction liquid chromatography with carboxyl or diol group functioned separation column (carboxyl-HILIC or diol-HILIC), and the other utilized derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and subsequent reversed-phase chromatography (HPLC). Separated amines were detected by electrospray ionization and tandem mass spectrometry in both cases. DBD-F-HPLC method provided good sensitivity for mono- and all polyamines (limit of detection (LOD) < 4.6 nM, relative standard deviation (RSD) for 100 nM < 9.2%). However, aminoamides could not be detected by DBD-F-HPLC. Carboxyl-HILIC provided good sensitivities for mono- and diamines and aminoamides (LOD < 1.6 nM, RSD < 4.8%). Forest air measurement was performed and data obtained by carboxyl-HILIC and DBD-F-HPLC showed good agreement for 1,3-diaminopropane, 1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine) (R2 = 0.9215-0.9739, n = 7-14). Carboxyl-HILIC method was the best for the amine analysis, and combination with silica gel tube sampling provides atmospheric monitoring available. The developed method can be used not only to study atmospheric chemistry of diamines and aminoamides but also to analyze flavor/odor of foods, flowers and wastes.

The chromatographic behavior of the selected compounds was studied under conditions of hydrophilic interaction liquid chromatography (HILIC). The effect of mobile phase composition on the retention in different chromatographic systems was systematically examined using high-performance thin-layer chromatography. The sorbents of different polarity and adsorption characteristics were selected and mixtures of water and organic solvents of various compositions, from pure water to pure organic solvent were used as mobile phases. Increasing the amount of water in the mobile phase leads to a conversion of the separation mechanism, and the retention curves have a characteristic \"U\" shape. The conversion between the adsorption and partition mechanisms is most likely continuous and depends on the chemical nature of separated substances, the stationary phase as well as on organic component of the mobile phase. Silica gel can be considered the most suitable stationary phase for the systematic investigation of the chromatographic behavior of the test compounds, whereas acetonitrile was the most suitable solvent. The obtained results contribute to the understanding of the dominant separation mechanism, the type, and the intensity of the interactions between separated substances with both stationary and mobile phases. Besides, the lipophilicity parameters obtained under HILIC conditions were evaluated and correlated with the calculated values.