Targeting macrophages with phosphatidylserine-rich liposomes as a potential antigen-specific immunotherapy for type 1 diabetes.

Type 1 diabetes (T1D) results from a breakdown in immunological tolerance, with pivotal involvement of antigen-presenting cells. In this context, antigen-specific immunotherapies have been developed to arrest autoimmunity, such as phosphatidylserine (PS)-liposomes. However, the role of certain antigen-presenting cells in immunotherapy, particularly human macrophages (Mφ) in T1D remains elusive. The aim of this study was to determine the role of Mφ in antigen-specific immune tolerance and T1D. To that end, we evaluated Mφ ability to capture apoptotic-body mimicking PS-liposomes in mice and conducted a phenotypic and functional characterisation of four human monocyte-derived Mφ (MoMφ) subpopulations (M0, M1, M2a and M2c) after PS-liposomes uptake. Our findings in mice identified Mφ as the most phagocytic cell subset in the spleen and liver. In humans, while phagocytosis rates were comparable between T1D and control individuals, PS-liposome capture dynamics differed among Mφ subtypes, favouring inflammatory (M1) and deactivated (M2c) Mφ. Notably, high nanoparticle concentrations did not affect macrophage viability. PS-liposome uptake by Mφ induced alterations in membrane molecule expression related to immunoregulation, reduced secretion of IL-6 and IL-12, and diminished autologous T-cell proliferation in the context of autoantigen stimulation. These results underscore the tolerogenic effects of PS-liposomes and emphasize their potential to target human Mφ, providing valuable insights into the mechanism of action of this preclinical immunotherapy.

Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.

Determination of Sialic Acid Isomers from Released N-Glycans Using Ion Mobility Spectrometry.

Complex carbohydrates are ubiquitous in nature and represent one of the major classes of biopolymers. They can exhibit highly diverse structures with multiple branched sites as well as a complex regio- and stereochemistry. A common way to analytically address this complexity is liquid chromatography (LC) in combination with mass spectrometry (MS). However, MS-based detection often does not provide sufficient information to distinguish glycan isomers. Ion mobility-mass spectrometry (IM-MS)─a technique that separates ions based on their size, charge, and shape─has recently shown great potential to solve this problem by identifying characteristic isomeric glycan features such as the sialylation and fucosylation pattern. However, while both LC-MS and IM-MS have clearly proven their individual capabilities for glycan analysis, attempts to combine both methods into a consistent workflow are lacking. Here, we close this gap and combine hydrophilic interaction liquid chromatography (HILIC) with IM-MS to analyze the glycan structures released from human alpha-1-acid glycoprotein (hAGP). HILIC separates the crude mixture of highly sialylated multi-antennary glycans, MS provides information on glycan composition, and IMS is used to distinguish and quantify α2,6- and α2,3-linked sialic acid isomers based on characteristic fragments. Further, the technique can support the assignment of antenna fucosylation. This feature mapping can confidently assign glycan isomers with multiple sialic acids within one LC-IM-MS run and is fully compatible with existing workflows for N-glycan analysis.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis.

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.

Analysis of Intact Glycoproteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be regarded as a key tool to rapidly obtain molecular mass information of intact glycoproteins in glycoproteomic studies and quality control of recombinant biopharmaceuticals. However, MALDI-TOF MS of these glycosylated compounds is a tricky task due to its low ionization efficiency and fragmentation of labile groups such as sialic acids.Here, we offer the reader a practical overview of the available methodologies for the confident analysis of intact glycoproteins with different glycosylation degree by MALDI-TOF MS. The three proposed methods fulfil the requirements of reproducibility and low extent of glycan fragmentation required to successfully analyze intact glycoproteins.

Alterations in the Glycan Profile of Mouse Transferrin: New Insights in Collagen-Induced Arthritis.

Transferrin purification from mice serum samples by immunoaffinity chromatography (IAC) was optimized in order to study the possible modifications occurring in its glycans in collagen-induced arthritis (CIA) samples. SDS-PAGE and nanoLC-MS/MS were used to monitor the IAC purification performance. Afterward, a relative quantification of mouse transferrin (mTf) glycan isomers using [12C6]/[13C6]-aniline was used to unequivocally detect alterations in the glycan profile of CIA mice. In addition, multivariate data analysis was applied to identify the most meaningful glycan isomers for the discrimination between control and pathological samples. Partial least-squares discriminant analysis (PLS-DA) revealed that five out of fifteen mTf glycan isomers could be potential biomarkers of CIA, most of them corresponding to highly sialylated structures (H6N5S3_2, H6N5S3_3, and H5N4S3_2). Moreover, some of these glycan isomers also seemed to be related with the progression of CIA, especially H6N5S2 and H6N5S3_2, as their overexpression increased with the clinical score of the pathology. Hence, the established methodology not only provides valuable information to find glycan-based biomarkers of CIA, but also leaves the door open to evaluate, in the future, glycosylation changes of many other inflammatory diseases, in which transferrin has been described to be altered.

Analysis of glycopeptide biomarkers by on-line TiO2 solid-phase extraction capillary electrophoresis-mass spectrometry.

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO2-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O126-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O126 glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg L-1 and 10-50 mg L-1 for O126 glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N83-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature.

Multivariate data analysis for the detection of human alpha-acid glycoprotein aberrant glycosylation in pancreatic ductal adenocarcinoma.

Relative quantification of human alpha-acid glycoprotein (hAGP) glycan isomers using [12C6]/[13C6]-aniline in combination with multivariate data analysis is proposed as an efficient method for the identification of pancreatic ductal adenocarcinoma (PDAC) glycan biomarkers in serum samples. Intact and desialylated glycans from hAGP, purified from serum samples of patients with PDAC and chronic pancreatitis (ChrP), were labeled with aniline and analyzed by µZIC-HILIC-MS. Afterwards, partial least squares discriminant analysis (PLS-DA) was applied to the relative areas obtained for all glycan isomers in the different samples: pathological (ChrP or PDAC) versus healthy samples. Seven intact glycan isomers with α2-6 linked sialic acids, five of them also fucosylated, were the most meaningful to distinguish between PDAC and ChrP patients. The desialylated glycan isomers also identified by PLS-DA as potential biomarker candidates confirmed that antenna but also core fucosylation could be involved in PDAC. The analysis of intact and desialylated glycan isomers in combination with the multivariate data analysis revealed that the triantennary glycan with two fucoses of hAGP could have in the future a relevant role in the differentiation of patients with PDAC from those with ChrP. SIGNIFICANCE: Multivariate data analysis is currently being used in many omics fields for biomarker discovery. However, to date, no glycomics studies have applied chemometric tools combined with mass spectrometry in a preclinical research. In this work, this methodology has been used to identify altered glycosylation of human alpha-acid glycoprotein in pancreatic ductal adenocarcinoma (PDAC). The obtained results reveal that the triantennary glycan with two fucoses could have a great biomarker potential as it was relevant to differentiate PDAC and chronic pancreatitis (ChrP) patients.

Zwitterionic-hydrophilic interaction capillary liquid chromatography coupled to tandem mass spectrometry for the characterization of human alpha-acid-glycoprotein N-glycan isomers.

In this work, a µZIC-HILIC-MS/MS methodology was established in negative ion mode for the characterization of glycan isomers. The possibility to separate the glycan isomers by the µZIC-HILIC strategy coupled to a high resolution tandem mass spectrometry detection permitted us to obtain valuable information about each glycan structure. The most important diagnostic ion fragments previously described to characterize structural features of glycans, were evaluated in this study using hAGP as model glycoprotein. The assignation of hAGP glycan isomers performed in our previous work using the GRIL strategy in combination with exoglycosidase digestion [1] was used in this paper to confirm or discard some ion fragments reported in the literature and delve into the structural characterization of glycan isomers. Sialic acid as well as fucose linkage-type glycan isomers were assigned using this approach and daughter ions with higher diagnostic value were determined. The location of α2-3/α2-6 sialic acids on antennas and a deeper characterization of several highly sialylated tri- and tetraantennary glycans was also possible using the established MS/MS method. Moreover, relying on the characterization performed in Ref. [1], core and antenna fucosylation were differentiated in this work using specific ion fragments obtained in the tandem mass spectra. This methodology was also applied to hAGP purified from control and pathological serum samples, which corroborated its robustness and its potential for finding novel glycan-based biomarkers in patho-glycomic studies.

Analysis of O-Glycopeptides by Acetone Enrichment and Capillary Electrophoresis-Mass Spectrometry.

Acetone precipitation was evaluated as a rapid, simple, low-cost, and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovine α1-acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3), and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides, and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behavior of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterward, partial least-squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O126-glycopeptide glycoforms of rhEPO because of their applicability in biopharmaceutical quality control and doping analysis.

Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion.

In this study, a ZIC-HILIC-MS methodology for the analysis of N-glycan isomers was optimized to obtain greater detection sensitivity and thus identify more glycan structures in hAGP. In a second step, this method was combined with glycan reductive isotope labelling (GRIL) through [(12)C6]/[(13)C6]-aniline and exoglycosidase digestion to characterize the different glycan isomers. The GRIL method allows the peak areas resulting from two different labelled samples to be compared, since neither retention time shifts nor variations in the ionization of glycans between these samples are obtained. First, sialic acid linkage assignations were performed for most hAGP glycan isomers with α2-3 sialidase digestion. Bi-, tri- and tetraantennary glycan isomers with different terminal sialic acid linkages to galactose (α2-3 or α2-6) were assigned, and the potential of this technique for the structural characterization of isobaric isomers was therefore demonstrated. Furthermore, fucose linkage isomers of hAGP glycans were also characterized using this isotope-labelling approach in combination with α1-3,4 fucosidase and ß1-4 galactosidase digestion. α1-3 antennary fucoses and α1-6 core fucosylation were detected in hAGP fucosylated glycans. These established methodologies can be extremely useful for patho-glycomic studies to characterize glycoproteins of biomedical interest and find novel glycan isomers that could be used as biomarkers in cancer research.