The potential of polyaniline-coated stationary phase in hydrophilic interaction liquid chromatography-based solid-phase extraction for glycopeptide enrichment.

Glycopeptide enrichment is a crucial step in glycoproteomic analysis, often achieved through solid-phase extraction (SPE) on polar stationary phases in hydrophilic interaction liquid chromatography (HILIC). This study explores the potential of polyaniline (PANI)-coated silica gel for enriching human immunoglobulin G (IgG). Experimental conditions were varied to assess their impact on glycopeptide enrichment efficiency, comparing PANI-cotton wool SPE with conventional cotton wool as SPE sorbents. Two formic acid concentrations (0.1% and 1%) in elution solvent were tested, revealing that higher concentrations led to earlier elution of studied glycopeptides, especially for sialylated glycopeptides. Substituting formic acid with acetic acid increased the interaction of neutral glycopeptides with the PANI-modified sorbent, while sialylated glycopeptides showed no significant change in enrichment efficiency. Acetonitrile concentration in the elution solvent (5%, 10%, and 20%) affected the enrichment efficiency with most glycopeptides eluting at the lowest acetonitrile concentration. The acetonitrile concentration in conditioning and washing solutions (65%, 75%, and 85%) played a crucial role; at 65% acetonitrile, glycopeptides were least retained on the stationary phase, and neutral glycopeptides were even detected in the flow-through fraction. This study shows the potential of in-house-prepared PANI-modified sorbents for SPE-HILIC glycopeptide enrichment, highlighting the crucial role of tuning experimental conditions in sample preparation to enhance enrichment efficiency and selectivity.

IgG glycopeptide enrichment using hydrophilic interaction chromatography-based solid-phase extraction on an aminopropyl column.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion.

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, ß-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

Microplastics and nanoplastics toxicity assays: A revision towards to environmental-relevance in water environment.

Plastic pollution poses a serious risk to the oceans, freshwater ecosystems, and land-based agricultural production. Most plastic waste enters rivers and then reaches the oceans, where its fragmentation process begins and the forming of microplastics (MPs) and nanoplastics (NPs). These particles increase their toxicity by the exposition to external factors and binding environmental pollutants, including toxins, heavy metals, persistent organic pollutants (POPs), halogenated hydrocarbons (HHCs), and other chemicals, which further and cumulatively increase the toxicity of these particles. A major disadvantage of many MNPs in vitro studies is that they do not use environmentally relevant microorganisms, which play a vital role in geobiochemical cycles. In addition, factors such as the polymer type, shapes, and sizes of the MPs and NPs, their exposure times and concentrations must be taken into account in in vitro experiments. Last but not least, it is important to ask whether to use aged particles with bound pollutants. All these factors affect the predicted effects of these particles on living systems, which may not be realistic if they are insufficiently considered. In this article, we summarize the latest findings on MNPs in the environment and propose some recommendations for future in vitro experiments on bacteria, cyanobacteria, and microalgae in water ecosystems.

Not so dangerous? PET microplastics toxicity on freshwater microalgae and cyanobacteria.

Microalgae and cyanobacteria are among the most important primary producers and are responsible for the production of 50-80% of the oxygen on Earth. They can be significantly affected by plastic pollution, as the vast majority of plastic waste ends up in rivers and then the oceans. This research focuses on green microalgae Chlorella vulgaris (C. vulgaris), Chlamydomonas reinhardtii (C. reinhardtii), filamentous cyanobacterium Limnospira (Arthrospira) maxima (L.(A.) maxima) and how they are affected by environmentally relevant PET-MPs (polyethylene-terephtalate microplastics). Manufactured PET-MPs have asymmetric shape, size between 3 and 7 µm and were used in concentrations ranging from 5 mg/L to 80 mg/L. The highest inhibitory rate of growth was found in C. reinhardtii (-24%). Concentration-dependent changes in chlorophyll a composition were found in C. vulgaris and C. reinhardtii, not in L. (A.) maxima. Furthermore, cell damage was detected in all three organisms by CRYO-SEM (shriveling, cell wall disruption), but the cyanobacterium was the least damaged. A PET-fingerprint was detected on the surface of all tested organisms using FTIR, indicating the adherence of PET-MPs. The highest rate of PET-MPs adsorption was detected in L. (A.) maxima. Specifically, characteristic spectra were observed at ∼721, 850, 1100, 1275, 1342, and 1715 cm-1 which are specific for functional groups of PET-MPs. Nitrogen and carbon content significantly increased in L. (A.) maxima under exposure to 80 mg/L due to the PET-MPs adherence and mechanical stress. In all three tested organisms, weak exposure-related ROS generation was detected. In general, cyanobacteria seem to be more resistant to the effects of MPs. However, organisms in the aquatic environment are exposed to MPs over a longer time scale, so it is important to use the present findings for further longer-term experiments on environmentally relevant organisms.

Liquid chromatography and capillary electrophoresis in glycomic and glycoproteomic analysis.

Glycosylation is one of the most significant and abundant post-translational modifications in cells. Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycomic and glycoproteomic analysis is highly challenging because of the large diversity of structures, low abundance, site-specific heterogeneity, and poor ionization efficiency of glycans and glycopeptides in mass spectrometry (MS). MS is a key tool for characterization of glycans and glycopeptides. However, MS alone does not always provide full structural and quantitative information for many reasons, and thus MS is combined with some separation technique. This review focuses on the role of separation techniques used in glycomic and glycoproteomic analyses, liquid chromatography and capillary electrophoresis. The most important separation conditions and results are presented and discussed.

Prediction of Intact N-Glycopeptide Retention Time Windows in Hydrophilic Interaction Liquid Chromatography.

Analysis of protein glycosylation is challenging due to micro- and macro-heterogeneity of the attached glycans. Hydrophilic interaction liquid chromatography (HILIC) is a mode of choice for separation of intact glycopeptides, which are inadequately resolved by reversed phase chromatography. In this work, we propose an easy-to-use model to predict retention time windows of glycopeptides in HILIC. We constructed this model based on the parameters derived from chromatographic separation of six differently glycosylated peptides obtained from tryptic digests of three plasma proteins: haptoglobin, hemopexin, and sex hormone-binding globulin. We calculated relative retention times of different glycoforms attached to the same peptide to the bi-antennary form and showed that the character of the peptide moiety did not significantly change the relative retention time differences between the glycoforms. To challenge the model, we assessed chromatographic behavior of fetuin glycopeptides experimentally, and their retention times all fell within the calculated retention time windows, which suggests that the retention time window prediction model in HILIC is sufficiently accurate. Relative retention time windows provide complementary information to mass spectrometric data, and we consider them useful for reliable determination of protein glycosylation in a site-specific manner.

Comparison of human IgG glycopeptides separation using mixed-mode hydrophilic interaction/ion-exchange liquid chromatography and reversed-phase mode.

Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.

Comparison of Different HILIC Stationary Phases in the Separation of Hemopexin and Immunoglobulin G Glycopeptides and Their Isomers.

Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.

Rifampin-Releasing Triple-Layer Cross-Linked Fresh Water Fish Collagen Sponges as Wound Dressings.

OBJECTIVES: Surgical wounds resulting from biofilm-producing microorganisms represent a major healthcare problem that requires new and innovative treatment methods. Rifampin is one of a small number of antibiotics that is able to penetrate such biofilms, and its local administration has the potential to serve as an ideal surgical site infection protection and/or treatment agent. This paper presents two types (homogeneous and sandwich structured) of rifampin-releasing carbodiimide-cross-linked fresh water fish collagen wound dressings. METHODS: The dressings were prepared by means of the double-lyophilization method and sterilized via gamma irradiation so as to allow for testing in a form that is able to serve for direct clinical use. The mechanical properties were studied via the uniaxial tensile testing method. The in vivo rifampin-release properties were tested by means of a series of incubations in phosphate-buffered saline. The microbiological activity was tested against methicillin-resistant staphylococcus aureus (MRSA) employing disc diffusion tests, and the in vivo pharmacokinetics was tested using a rat model. A histological examination was conducted for the study of the biocompatibility of the dressings. RESULTS: The sandwich-structured dressing demonstrated better mechanical properties due to its exhibiting ability to bear a higher load than the homogeneous sponges, a property that was further improved via the addition of rifampin. The sponges retarded the release of rifampin in vitro, which translated into at least 22 hours of rifampin release in the rat model. This was significantly longer than was achieved via the administration of a subcutaneous rifampin solution. Microbiological activity was proven by the results of the disc diffusion tests. Both sponges exhibited excellent biocompatibility as the cells penetrated into the scaffold, and virtually no signs of local irritation were observed. CONCLUSIONS: We present a novel rifampin-releasing sandwich-structured fresh water fish collagen wound dressing that has the potential to serve as an ideal surgical site infection protection and/or treatment agent.

Glycan-specific precipitation of glycopeptides in high organic content sample solvents used in HILIC.

The composition of a sample solvent has a crucial impact on separations in hydrophilic interaction liquid chromatography (HILIC). In this short communication, we studied the effect of an organic modifier in the sample solvent on the solubility of different tryptic glycopeptides of hemopexin and haptoglobin proteins. The results showed that the solubility of glycopeptides in solvents with a high acetonitrile content depends on the type of attached N-glycan. We observed lower solubility in larger glycans attached to the same peptide backbone, and we demonstrated that glycopeptides containing sialic acids precipitate more readily than those without sialic acid. Therefore, the sample solvent composition in HILIC must be carefully optimized for accurate quantitative data collection and for adequate separation.

Interaction of heparin and tetraarginine in capillary electrophoresis: Implication for analytical applications.

Interactions between heparin and tetraarginine in an acidic background electrolyte were investigated in capillary electrophoresis. The results showed that tetraarginine and heparin form a stable complex that migrates toward the anode immediately after coming into contact. When a zone of tetraarginine at a mg/mL concentration level passes through a zone of heparin at a µg/mL concentration level, tetraarginine is gradually removed by the formation of the complex that migrates in the opposite direction, thereby decreasing the tetraarginine peak area. The variation of the tetraarginine peak area as a function of the unfractionated heparin concentration was linear within the range 2-20 µg/mL, which enables us to detect and determine heparin concentrations undetectable with a UV detector. The same behavior was confirmed for low molecular weight heparin.

Numbers of non-severe as well as cerebral malaria cases are decreasing with higher altitude in Burundi Highlands, but still remains high.

OBJECTIVE: We aimed to assess the occurrence of malaria-positive cases in 4 rural Burundian hospitals during December 2011 placed at different altitudes above sea level. METHODS: Diagnosis of malaria was made upon considering clinical symptoms, microscopic evaluation and rapid diagnostic test results. We performed analysis of patient's clinical data collected in 4 hospitals in Burundi to compare the occurrence of malaria at different altitudes. RESULTS: The lowest incidence of malaria during December 2011 was detected at Murago Hospital (606 cases per month, 47.6%), which is located in the highest altitude, and the highest occurrence was in Gasura (1,559 cases, 91.3%), then in Rutovu (732 cases, 81.2%) and Buraniro (4,436 cases, 78.6%). Compared with other types of consultations (gynecological, HIV/AIDS, other tropical diseases), malaria was the most frequent reason for medical consultation. CONCLUSION: We have observed the lowest occurrence of malaria at hospitals located in the highest altitude. Despite the lower number of malaria cases in higher altitudes, its impact on public health should not be underestimated.