Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

RecA levels modulate biofilm development in Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii, a Gram-negative opportunistic pathogen, are difficult to eradicate due to the bacterium's propensity to quickly gain antibiotic resistances and form biofilms, a protective bacterial multicellular community. The A. baumannii DNA damage response (DDR) mediates the antibiotic resistance acquisition and regulates RecA in an atypical fashion; both RecALow and RecAHigh cell types are formed in response to DNA damage. The findings of this study demonstrate that the levels of RecA can influence formation and dispersal of biofilms. RecA loss results in surface attachment and prominent biofilms, while elevated RecA leads to diminished attachment and dispersal. These findings suggest that the challenge to treat A. baumannii infections may be explained by the induction of the DDR, common during infection, as well as the delicate balance between maintaining biofilms in low RecA cells and promoting mutagenesis and dispersal in high RecA cells. This study underscores the importance of understanding the fundamental biology of bacteria to develop more effective treatments for infections.

Progressive mechanical and structural changes in anterior cerebral arteries with Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease and the main cause for dementia. The irreversible neurodegeneration leads to a gradual loss of brain function characterized predominantly by memory loss. Cerebrovascular changes are common neuropathologic findings in aged subjects with dementia. Cerebrovascular integrity is critical for proper metabolism and perfusion of the brain, as cerebrovascular remodeling may render the brain more susceptible to pulse pressure and may be associated with poorer cognitive performance and greater risk of cerebrovascular events. The objective of this study is to provide understanding of cerebrovascular remodeling with AD progression. Anterior cerebral arteries (ACAs) from a total of 19 brain donor participants from controls and pathologically diagnosed AD groups (early-Braak stages I-II; intermediate-Braak stages III-IV; and advanced-Braak stages V-VI) were included in this study. Mechanical testing, histology, advanced optical imaging, and mass spectrometry were performed to study the progressive structural and functional changes of ACAs with AD progression. Biaxial extension-inflation tests showed that ACAs became progressively less compliant, and the longitudinal stress in the intermediate and advanced AD groups was significantly higher than that from the control group. With pathological AD development, the inner and outer diameters of the ACAs remained almost unchanged; however, histology study revealed progressive smooth muscle cell atrophy and loss of elastic fibers which led to compromised structural integrity of the arterial wall. Multiphoton imaging demonstrated elastin degradation at the media-adventitia interface, which led to the formation of an empty band of 21.0 ± 15.4 µm and 32.8 ± 9.24 µm in width for the intermediate and advanced AD groups, respectively. Furthermore, quantitative birefringence microscopy showed disorganized adventitial collagen with AD development. Mass spectrometry analysis provided further evidence of altered collagen content and other extracellular matrix (ECM) molecule and smooth muscle cell changes that were consistent with the mechanical and structural alterations. Collectively, our study provides understanding of the mechanical and structural cerebrovascular deterioration in cerebral arteries with AD, which may be related to neurodegenration and pathology in the brain.

Analysis of complex proteoglycans using serial proteolysis and EThcD provides deep N- and O-glycoproteomic coverage.

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-energy collisional dissociation (EThcD) to optimize protein sequence coverage and glycopeptide identification from proteoglycans. In many cases, the use of HCD alone allows the identification of more glycopeptides. However, the localization of glycoforms on multiply glycosylated peptides has remained elusive. We demonstrate the use of EThcD for the confident assignment of glycan compositions on multiply glycosylated peptides. Dense glycosylation on proteoglycans is key to their biological function; thus, developing tools to identify and quantify doubly glycosylated peptides is of interest. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples.

Progressive Mechanical and Structural Changes in Anterior Cerebral Arteries with Alzheimer's Disease.

Alzheimer disease (AD) is a neurodegenerative disease and the main cause for dementia. The irreversible neurodegeneration leads to a gradual loss of brain function characterized predominantly by memory loss. Cerebrovascular changes are common neuropathologic findings in aged subjects with dementia. Cerebrovascular integrity is critical for proper metabolism and perfusion of the brain, as cerebrovascular remodeling may render the brain more susceptible to pulse pressure and may be associated with poorer cognitive performance and greater risk of cerebrovascular events. The objective of this study is to provide understanding of cerebrovascular remodeling with AD progression. A total of 28 brain donor participants with human anterior cerebral artery (ACA) from controls and pathologically diagnosed AD groups (early - Braak stages I-II; intermediate - Braak stages III-IV; and advanced - Braak stages V-VI) were included in this study. Mechanical testing, histology, advanced optical imaging, and mass spectrometry were performed to study the progressive structural and functional changes of ACAs with AD progression. Biaxial extension-inflation tests showed that ACAs became progressively less compliant, and the longitudinal stress in the intermediate& advanced AD groups was significantly higher than that from the control group. With pathological AD development, the inner and outer diameter of ACA remained almost unchanged; however, histology study revealed progressive smooth muscle cell atrophy and loss of elastic fibers which led to compromised structural integrity of the arterial wall. Multiphoton imaging demonstrated elastin degradation at the media-adventitia interface, which led to the formation of an empty band of 21.0 ± 15.4 µm and 32.8 ± 9.24 µm in width for the intermediate& advanced AD groups, respectively. Furthermore, quantitative birefringence microscopy showed disorganized adventitial collagen with AD development. Mass spectrometry analysis provided further evidence of altered collagen content and other extracellular matrix (ECM) molecule and smooth muscle cell changes that were consistent with the mechanical and structural alterations. Collectively, our study provides understanding of the mechanical and structural cerebrovascular deterioration in cerebral arteries with AD, which may be related to neurodegenration and pathology in the brain.

Bibliometric Analysis of International Medical Graduates and Professorship Promotion in Neurosurgery.

BACKGROUND: International medical graduates (IMGs) comprise ∼25% of physicians in the United States. Differences in promotion rates from assistant to associate to full professorship based on medical school location have been understudied. We aim to stratify odds of professional advancement by 3 categories: IMG with U.S. residency, IMG with international residency, and U.S. medical with U.S. residency training. METHODS: We created and queried a database after exclusions of 1334 neurosurgeons including multiple demographic factors: academic productivity and promotion rates. Stratified logistic regression modeled odds of promotion including the variables: decades out of training, Scopus h-index, gender, and training location. Odds ratios (ORs) and 95% confidence intervals (CIs) for each variable were calculated. RESULTS: Significant predictors of increased associate versus assistant professorship included decades out of training (OR = 2.519 [95% CI: 2.07-3.093], P < 0.0001) and Scopus h-index (OR = 1.085 [95% CI: 1.064-1.108], P < 0.0001) while international medical school with U.S. residency (OR = 0.471 [95% CI: 0.231-0.914], P = 0.0352) was associated with decreased promotion. Significant predictors of associate versus full professorship were decades out of training (OR = 2.781 [95% CI: 2.268-3.444], P < 0.0001) and Scopus h-index (OR = 1.064 [95% CI: 1.049-1.080], P < 0.0001). Attending medical school or residency internationally was not associated with odds of full professorship. CONCLUSIONS: Time out of residency and Scopus h-index were associated with higher academic rank regardless of career level. Attending medical school internationally with U.S. residency was associated with lower odds of associate professorship promotion over 10 years. There was no relationship between IMG and full professorship promotion. IMGs who attended residency internationally did not have lower promotion rates. These findings suggest it may be harder for IMGs to earn promotion from assistant to associate professor in neurosurgery.

Mass spectrometry methods for analysis of extracellular matrix components in neurological diseases.

The brain extracellular matrix (ECM) is a highly glycosylated environment and plays important roles in many processes including cell communication, growth factor binding, and scaffolding. The formation of structures such as perineuronal nets (PNNs) is critical in neuroprotection and neural plasticity, and the formation of molecular networks is dependent in part on glycans. The ECM is also implicated in the neuropathophysiology of disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Schizophrenia (SZ). As such, it is of interest to understand both the proteomic and glycomic makeup of healthy and diseased brain ECM. Further, there is a growing need for site-specific glycoproteomic information. Over the past decade, sample preparation, mass spectrometry, and bioinformatic methods have been developed and refined to provide comprehensive information about the glycoproteome. Core ECM molecules including versican, hyaluronan and proteoglycan link proteins, and tenascin are dysregulated in AD, PD, and SZ. Glycomic changes such as differential sialylation, sulfation, and branching are also associated with neurodegeneration. A more thorough understanding of the ECM and its proteomic, glycomic, and glycoproteomic changes in brain diseases may provide pathways to new therapeutic options.

In-Depth Matrisome and Glycoproteomic Analysis of Human Brain Glioblastoma Versus Control Tissue.

Glioblastoma (GBM) is the most common and malignant primary brain tumor. The extracellular matrix, also known as the matrisome, helps determine glioma invasion, adhesion, and growth. Little attention, however, has been paid to glycosylation of the extracellular matrix components that constitute the majority of glycosylated protein mass and presumed biological properties. To acquire a comprehensive understanding of the biological functions of the matrisome and its components, including proteoglycans (PGs) and glycosaminoglycans (GAGs), in GBM tumorigenesis, and to identify potential biomarker candidates, we studied the alterations of GAGs, including heparan sulfate (HS) and chondroitin sulfate (CS), the core proteins of PGs, and other glycosylated matrisomal proteins in GBM subtypes versus control human brain tissue samples. We scrutinized the proteomics data to acquire in-depth site-specific glycoproteomic profiles of the GBM subtypes that will assist in identifying specific glycosylation changes in GBM. We observed an increase in CS 6-O sulfation and a decrease in HS 6-O sulfation, accompanied by an increase in unsulfated CS and HS disaccharides in GBM versus control samples. Several core matrisome proteins, including PGs (decorin, biglycan, agrin, prolargin, glypican-1, and chondroitin sulfate proteoglycan 4), tenascin, fibronectin, hyaluronan link protein 1 and 2, laminins, and collagens, were differentially regulated in GBM versus controls. Interestingly, a higher degree of collagen hydroxyprolination was also observed for GBM versus controls. Further, two PGs, chondroitin sulfate proteoglycan 4 and agrin, were significantly lower, about 6-fold for isocitrate dehydrogenase-mutant, compared to the WT GBM samples. Differential regulation of O-glycopeptides for PGs, including brevican, neurocan, and versican, was observed for GBM subtypes versus controls. Moreover, an increase in levels of glycosyltransferase and glycosidase enzymes was observed for GBM when compared to control samples. We also report distinct protein, peptide, and glycopeptide features for GBM subtypes comparisons. Taken together, our study informs understanding of the alterations to key matrisomal molecules that occur during GBM development. (Data are available via ProteomeXchange with identifier PXD028931, and the peaks project file is available at Zenodo with DOI 10.5281/zenodo.5911810).

Matrisome changes in Parkinson's disease.

Extracellular matrix (ECM) proteins, collectively known as the matrisome, include collagens, glycoproteins, and proteoglycans. Alterations in the matrisome have been implicated in the neurodegenerative pathologies including Parkinson's disease (PD). In this work, we utilized our previously published PD and control proteomics data from human prefrontal cortex and focused our analysis on the matrisome. Among matrisome proteins, we observed a significant enrichment in the expression of type I collagen in PD vs. control samples. We then performed histological analysis on the same samples used for proteomics study, and examined collagen expression using picrosirius red staining. Interestingly, we observed similar trends in collagen abundance in PD vs. control as in our matrisome analysis; thus, this and other histological analyses will be useful as a complementary technique in the future to study the matrisome in PD with a larger cohort, and it may aid in choosing regions of interest for proteomic analysis. Additionally, collagen hydroxyprolination was less variable in PD compared to controls. Glycoproteomic changes in matrisome molecules were also observed in PD relative to aged individuals, especially related to type VI collagen and versican. We further examined the list of differentially expressed matrisome molecules using network topology-based analysis and found that angiogenesis indicated by alterations in decorin and several members of the collagen family was affected in PD. These findings collectively identified matrisome changes associated with PD; further studies with a larger cohort are required to validate the current results.

Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis.

Advancement in mass spectrometry has revolutionized the field of proteomics. However, there remains a gap in the analysis of protein post-translational modifications (PTMs), particularly for glycosylation. Glycosylation, the most common form of PTM, is involved in most biological processes; thus, analysis of glycans along with proteins is crucial to answering important biologically relevant questions. Of particular interest is the brain extracellular matrix (ECM), which has been called the "final Frontier" in neuroscience, which consists of highly glycosylated proteins. Among these, proteoglycans (PGs) contain large glycan structures called glycosaminoglycans (GAGs) that form crucial ECM components, including perineuronal nets (PNNs), shown to be altered in neuropsychiatric diseases. Thus, there is a growing need for high-throughput methods that combine GAG (glycomics) and PGs (proteomics) analysis to unravel the complete biological picture. The protocol presented here integrates glycomics and proteomics to analyze multiple classes of biomolecules. We use a filter-aided sample preparation (FASP) type serial in-solution digestion of GAG classes, including hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS), followed by peptides. The GAGs and peptides are then cleaned and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is an efficient and economical way of processing tissue or cell lysates to isolate various GAG classes and peptides from the same sample. The method is more efficient (single-pot) than available parallel (multi-pot) release methods, and removal of GAGs facilitates the identification of the proteins with higher peptide-coverage than using conventional-proteomics. Overall, we demonstrate a high-throughput & efficient protocol for mass spectrometry-based glycomic and proteomic analysis (data are available via ProteomeXchange with identifier PXD017513).