Disarm The Bacteria: What Temperate Phages Can Do.

In the field of phage applications and clinical treatment, virulent phages have been in the spotlight whereas temperate phages received, relatively speaking, less attention. The fact that temperate phages often carry virulent or drug-resistant genes is a constant concern and drawback in temperate phage applications. However, temperate phages also play a role in bacterial regulation. This review elucidates the biological properties of temperate phages based on their life cycle and introduces the latest work on temperate phage applications, such as on host virulence reduction, biofilm degradation, genetic engineering and phage display. The versatile use of temperate phages coupled with their inherent properties, such as economy, ready accessibility, wide variety and host specificity, make temperate phages a solid candidate in tackling bacterial infections.

Effects of Anthraquinones on Immune Responses and Inflammatory Diseases.

The anthraquinones (AQs) and derivatives are widely distributed in nature, including plants, fungi, and insects, with effects of anti-inflammation and anti-oxidation, antibacterial and antiviral, anti-osteoporosis, anti-tumor, etc. Inflammation, including acute and chronic, is a comprehensive response to foreign pathogens under a variety of physiological and pathological processes. AQs could attenuate symptoms and tissue damages through anti-inflammatory or immuno-modulatory effects. The review aims to provide a scientific summary of AQs on immune responses under different pathological conditions, such as digestive diseases, respiratory diseases, central nervous system diseases, etc. It is hoped that the present paper will provide ideas for future studies of the immuno-regulatory effect of AQs and the therapeutic potential for drug development and clinical use of AQs and derivatives.

High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry.

Antibody-drug conjugates (ADCs) are one of the fastest growing classes of anticancer therapies. Combining the high targeting specificity of monoclonal antibodies (mAbs) with cytotoxic small molecule drugs, ADCs are complex molecular entities that are intrinsically heterogeneous. Primary sequence variants, varied drug-to-antibody ratio (DAR) species, and conformational changes in the starting mAb structure upon drug conjugation must be monitored to ensure the safety and efficacy of ADCs. Herein, we have developed a high-throughput method for the analysis of cysteine-linked ADCs using trapped ion mobility spectrometry (TIMS) combined with top-down mass spectrometry (MS) on a Bruker timsTOF Pro. This method can analyze ADCs (∼150 kDa) by TIMS followed by a three-tiered top-down MS characterization strategy for multi-attribute analysis. First, the charge state distribution and DAR value of the ADC are monitored (MS1). Second, the intact mass of subunits dissociated from the ADC by low-energy collision-induced dissociation (CID) is determined (MS2). Third, the primary sequence for the dissociated subunits is characterized by CID fragmentation using elevated collisional energies (MS3). We further automate this workflow by directly injecting the ADC and using MS segmentation to obtain all three tiers of MS information in a single 3-min run. Overall, this work highlights a multi-attribute top-down MS characterization method that possesses unparalleled speed for high-throughput characterization of ADCs.

Tumor microenvironment: a prospective target of natural alkaloids for cancer treatment.

Malignant tumor has become one of the major diseases that seriously endangers human health. Numerous studies have demonstrated that tumor microenvironment (TME) is closely associated with patient prognosis. Tumor growth and progression are strongly dependent on its surrounding tumor microenvironment, because the optimal conditions originated from stromal elements are required for cancer cell proliferation, invasion, metastasis and drug resistance. The tumor microenvironment is an environment rich in immune/inflammatory cells and accompanied by a continuous, gradient of hypoxia and pH. Overcoming immunosuppressive environment and boosting anti-tumor immunity may be the key to the prevention and treatment of cancer. Most traditional Chinese medicine have been proved to have good anti-tumor activity, and they have the advantages of better therapeutic effect and few side effects in the treatment of malignant tumors. An increasing number of studies are giving evidence that alkaloids extracted from traditional Chinese medicine possess a significant anticancer efficiency via regulating a variety of tumor-related genes, pathways and other mechanisms. This paper reviews the anti-tumor effect of alkaloids targeting tumor microenvironment, and further reveals its anti-tumor mechanism through the effects of alkaloids on different components in tumor microenvironment.

Comparative Membrane N-Glycomics of Different Breast Cancer Cell Lines To Understand Breast Cancer Brain Metastasis.

The mechanism of brain metastatic breast cancer has gained attention because of its increased incidence rate and its low survival rate. Aberrant protein glycosylation is thought to be a contributing factor in this metastatic mechanism, in which metastatic cancer cells can pass through the blood-brain barrier (BBB). The cell membrane is the outermost layer of a cell and in direct contact with the environment and with other cells, making membrane glycans especially important in many biological processes that include mediating cell-cell adhesion, cell signaling, and interactions. Thus, membrane glycomics has attracted more interest for a variety of disease studies in recent years. To reveal the role that membrane N-glycans play in breast cancer brain metastasis, in this study, membrane enrichment was achieved by ultracentrifugation. Liquid chromatography-tandem mass spectrometry was employed to analyze enriched membrane N-glycomes from five breast cancer cell lines and one brain cancer cell line. Relative quantitative glycomic data from each cell line were compared to MDA-MB-231BR, which is the brain-seeking cell line. The higher sialylation level observed in MDA-MB-231BR suggested the importance of sialylation as it might assist with cell invasion and the penetration of the BBB. Some highly sialylated N-glycans, such as HexNAc5Hex6DeoxyHex1NeuAc3 and HexNAc6Hex7DeoxyHex1NeuAc3, exhibited higher abundances in 231BR, indicating their possible contributions to breast cancer brain metastasis as well as their potential to be indicators for the breast cancer brain metastasis.

Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.

8-plex LC-MS/MS Analysis of Permethylated N-Glycans Achieved by Using Stable Isotopic Iodomethane.

Glycosylation is an important post-translational modification of proteins. Many diseases, such as cancer, have proved to be related to aberrant glycosylation. High throughput quantitative methods have gained attention recently in the study of glycomics. With the development of high-resolution mass spectrometry, the sensitivity of detection in glycomics has largely improved; however, most of the commonly used MS-based techniques are focused on relative quantitative analysis, which can hardly provide direct comparative glycomic quantitation results. In this study, we developed a novel multiplex glycomic analysis method on an LC-ESI-MS platform. Reduced glycans were stable isotopic labeled during the permethylation procedure, with the use of iodomethane reagents CH2DI, CHD2I, CD3I, 13CH3I, 13CH2DI, 13CHD2I, 13CD3I, and CH3I. Up to 8-plex glycomic profiling was possible in a single analysis by LC-MS, and a 100 k mass resolution was sufficient to allow a baseline resolution of the mass differences among the 8-plex labeled glycans. The major advantages of this method are that it overcomes quantitative fluctuations caused by nanoESI, it facilitates a level of comparative quantitative glycomic analysis that accurately reflects the quantitative information in samples, and it dramatically shortens analysis time. Quantitation validation was tested on glycans released from bovine fetuin and model glycoprotein mixtures (RNase B, bovine fetuin, and IgG) with good linearity (R2 = 0.9884) and a dynamic range from 0.1 to 10. The 8-plex strategy was successfully applied to a comparative glycomic study of cancer cell lines. The results demonstrate that different distributions of sialylated glycans are related to the metastatic properties of cell lines and provide important clues for a better understanding of breast cancer brain metastasis.

Enhanced Quantitative LC-MS/MS Analysis of N-linked Glycans Derived from Glycoproteins Using Sodium Deoxycholate Detergent.

Protein glycosylation is a common protein post-translational modification (PTM) in living organisms and has been shown to associate with multiple diseases, and thus may potentially be a biomarker of such diseases. Efficient protein/glycoprotein extraction is a crucial step in the preparation of N-glycans derived from glycoproteins prior to LC-MS analysis. Convenient, efficient and unbiased sample preparation protocols are needed. Herein, we evaluated the use of sodium deoxycholate (SDC) acidic labile detergent to release N-glycans of glycoproteins derived from biological samples such as cancer cell lines. Compared to the filter-aided sample preparation approach, the sodium deoxycholate (SDC) assisted approach was determined to be more efficient and unbiased. SDC removal was determined to be more efficient when using acidic precipitation rather than ethyl acetate phase transfer. Efficient extraction of proteins/glycoproteins from biological samples was achieved by combining SDC lysis buffer and beads beating cell disruption. This was suggested by a significant overall increase in the intensities of N-glycans released from cancer cell lines. Additionally, the use of SDC approach was also shown to be more reproducible than those methods that do not use SDC.

Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures.

Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well-known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75 °C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 nonisomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (P < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood-brain barrier during brain metastasis, were observed.

LC-MS/MS isomeric profiling of permethylated N-glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients.

Early stage detection and cancer treatment demand the identification of reliable biomarkers. Over the past decades, efforts have been devoted to assess the variation of glycosylation level as well as the glycan structures of proteins in blood or serum, associated with the development and/or progression of several cancers, including liver. Herein, an LC-MS/MS-based analysis was conducted to define the glycosylation patterns of haptoglobin glycoprotein derived from sera collected from cirrhotic and hepatocellular carcinoma (HCC) patients. The haptoglobin samples were extracted from serum using an antibody-immobilized column prior to the release of N-glycans. A comparison of non-isomeric and isomeric permethylated glycan forms was achieved using C18 and porous graphitic carbon (PGC) columns, respectively. In the case of C18-LC-MS/MS analysis, 25 glycan structures were identified of which 10 sialylated structures were found to be statistically significant between the two cohorts. Also, 8 out of 34 glycan structures identified by PGC-LC-MS/MS were found to be statistically significant, suggesting that isomeric distributions of a particular glycan composition were different in abundances between the two cohorts. The glycan isoform patterns distinguished early stage HCC from cirrhotic patients. Both retention times and tandem mass spectra were utilized to determine the specific isomeric glycan structures. All of the glycan isomers, which were statistically significant, were either branch fucosylated or composed of α-2,6 linked sialic acid moieties. The result of this study demonstrates the potential importance of isomeric separation for defining disease prompted aberrant glycan changes. The levels of several glycan isoforms effectively distinguished early stage HCC from cirrhosis.

Direct comparison of derivatization strategies for LC-MS/MS analysis of N-glycans.

Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-Glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization.

The biosynthesis of glycans is a template-free process; hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS)-based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated; thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions, achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages. Graphical Abstract LC-MS and tandem MS of terminal galactose isomers.

Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.

LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers.

High-temperature LC-MS/MS of permethylated glycans derived from glycoproteins.

Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS-based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed-phase liquid chromatography (RPLC). Recently, RPLC-MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely-applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high-molecular-weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high-temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high-temperature LC method for both manual and automatic glycan identification.

LC-MS/MS of permethylated N-glycans derived from model and human blood serum glycoproteins.

LC-MS/MS is one of the most powerful tools for N-glycan structure elucidation; however, it is still challenging to identify some glycan structures with low abundance. In this study, we investigated the chromatographic behavior of permethylated N-glycans. The relationship between retention times versus molecular weight of dextran, dextrin, and model glycans was investigated. Also, the nonpolar surface area of glycans was calculated and compared to their experimental retention times. Both retention time and nonpolar surface area trends are similar when the intermolecular interaction is included in the calculation. Moreover, retention time corresponds to glycan types and branch types. The N-glycans analysis model, which combines high mass accuracy and retention time, was applied to confirm serum N-glycans. In total, there were 78 N-glycan compositions identified. A linear relationship between retention times and molecular weights were observed for each subgroup of glycan structures, for example, R(2) value for complex N-glycans was determined to be > 0.98. Moreover, the retention time could be further applied to distinguish between structural isomers as well as linkage isomers. MS/MS data were used to confirm the structural isomers.

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments.

A procedure for the analysis of site-specific and structure-specific fucosylation in alpha-1-antitrypsin.

A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.

Glycosylation and other PTMs alterations in neurodegenerative diseases: Current status and future role in neurotrauma.

Traumatic brain injuries (TBIs) present a chief public health threat affecting nations worldwide. As numbers of patients afflicted by TBI are expected to rise, the necessity to increase our understanding of the pathophysiological mechanism(s) as a result of TBI mounts. TBI is known to augment the risk of developing a number of neurodegenerative diseases (NDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD). Hence, it is rational to assume that a common mechanistic ground links the pathophysiology of NDs to that of TBIs. Through this review, we aim to identify the protein-protein interactions, differential proteins expression, and PTMs, mainly glycosylation, that are involved in the pathogenesis of both ND and TBI. OVID and PubMed have been rigorously searched to identify studies that utilized advanced proteomic platforms (MS based) and systems biology tools to unfold the mechanism(s) behind ND in an attempt to unveil the mysterious biological processes that occur postinjury. Various PTMs have been found to be common between TBI and AD, whereas no similarities have been found between TBI and PD. Phosphorylated tau protein, glycosylated amyloid precursor protein, and many other modifications appear to be common in both TBI and AD. PTMs, differential protein profiles, and altered biological pathways appear to have critical roles in ND processes by interfering with their pathological condition in a manner similar to TBI. Advancement in glycoproteomic studies pertaining to ND and TBI is urgently needed in order to develop better diagnostic tools, therapies, and more favorable prognoses.

Deciphering glycomics and neuroproteomic alterations in experimental traumatic brain injury: Comparative analysis of aspirin and clopidogrel treatment.

As populations age, the number of patients sustaining traumatic brain injury (TBI) and concomitantly receiving preinjury antiplatelet therapy such as aspirin (ASA) and clopidogrel (CLOP) is rising. These drugs have been linked with unfavorable clinical outcomes following TBI, where the exact mechanism(s) involved are still unknown. In this novel work, we aimed to identify and compare the altered proteome profile imposed by ASA and CLOP when administered alone or in combination, prior to experimental TBI. Furthermore, we assessed differential glycosylation PTM patterns following experimental controlled cortical impact model of TBI, ASA, CLOP, and ASA + CLOP. Ipsilateral cortical brain tissues were harvested 48 h postinjury and were analyzed using an advanced neuroproteomics LC-MS/MS platform to assess proteomic and glycoproteins alterations. Of interest, differential proteins pertaining to each group (22 in TBI, 41 in TBI + ASA, 44 in TBI + CLOP, and 34 in TBI + ASA + CLOP) were revealed. Advanced bioinformatics/systems biology and clustering analyses were performed to evaluate biological networks and protein interaction maps illustrating molecular pathways involved in the experimental conditions. Results have indicated that proteins involved in neuroprotective cellular pathways were upregulated in the ASA and CLOP groups when given separately. However, ASA + CLOP administration revealed enrichment in biological pathways relevant to inflammation and proinjury mechanisms. Moreover, results showed differential upregulation of glycoproteins levels in the sialylated N-glycans PTMs that can be implicated in pathological changes. Omics data obtained have provided molecular insights of the underlying mechanisms that can be translated into clinical bedside settings.