Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips.

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

[A case of botulism in the Czech Republic and current possibilities for detecting the neurotoxin produced by Clostridium botulinum].

In the Czech Republic, botulism is a rare life-threatening disease. A total of 155 cases have been reported since 1960; according to the ISIN (formerly EPIDAT) database, there have been only three isolated cases since 2013, with the exception of a single occurrence of familial botulism in 2013. In our work, we present the occurrence of botulism after ingestion of pâté of untraceable origin by a couple who were hospitalized for botulotoxin food poisoning in July 2022. Their neurological symptoms were dominated by dysarthria. After administration of antibotulinum serum, their condition improved significantly. Patient samples were analyzed using affinity carriers and MALDI mass spectrometry, a modern highly sensitive technique for detecting the presence of botulinum neurotoxins. Unlike traditional detection by a difficult and costly biological experiment on mice, the above analysis does not require the killing of laboratory animals.

Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

Three-Dimensional Printed Target Plates for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Three-dimensional printing (3D printing) is a fast-growing technology with high impact in industry, medicine, and the life sciences. Fused deposition modeling (FDM), which uses plastic filaments extruded through a heated nozzle, is the most common 3D printing technology for creation of objects. In this work, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plates printed by FDM technology using conductive plastic material were evaluated for their detection capability of proteins and peptides. The 3D printed MALDI targets were validated by analysis of different types of bacteria and compared with commercially available MBT BioTargets. The results indicate that 3D printed MALDI targets are comparable to standard MBT BioTargets and stainless-steel targets and may be used for different MALDI-TOF MS applications. The 3D printing allows easy manufacturing of MALDI targets with different dimensions and spot geometry. Moreover, the 3D printed MALDI targets are disposable, cheap, and easy to produce. These features make them a suitable cost-effective alternative to conventional targets for any MALDI MS analysis.

Dynamic secretome of Trichomonas vaginalis: Case study of ß-amylases.

The secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host-parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and ß-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used ß-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two ß-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two ß-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite.

Identification of Human Enzymes Oxidizing the Anti-Thyroid-Cancer Drug Vandetanib and Explanation of the High Efficiency of Cytochrome P450 3A4 in its Oxidation.

The metabolism of vandetanib, a tyrosine kinase inhibitor used for treatment of symptomatic/progressive medullary thyroid cancer, was studied using human hepatic microsomes, recombinant cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs). The role of CYPs and FMOs in the microsomal metabolism of vandetanib to N-desmethylvandetanib and vandetanib-N-oxide was investigated by examining the effects of CYP/FMO inhibitors and by correlating CYP-/FMO-catalytic activities in each microsomal sample with the amounts of N-desmethylvandetanib/vandetanib-N-oxide formed by these samples. CYP3A4/FMO-activities significantly correlated with the formation of N-desmethylvandetanib/ vandetanib-N-oxide. Based on these studies, most of the vandetanib metabolism was attributed to N-desmethylvandetanib/vandetanib-N-oxide to CYP3A4/FMO3. Recombinant CYP3A4 was most efficient to form N-desmethylvandetanib, while FMO1/FMO3 generated N-oxide. Cytochrome b5 stimulated the CYP3A4-catalyzed formation of N-desmethylvandetanib, which is of great importance because CYP3A4 is not only most efficient in generating N-desmethylvandetanib, but also most significant due to its high expression in human liver. Molecular modeling indicated that binding of more than one molecule of vandetanib into the CYP3A4-active center can be responsible for the high efficiency of CYP3A4 N-demethylating vandetanib. Indeed, the CYP3A4-mediated reaction exhibits kinetics of positive cooperativity and this corresponded to the in silico model, where two vandetanib molecules were found in CYP3A4-active center.

Detection and Quantification of Carbohydrate-Deficient Transferrin by MALDI-Compatible Protein Chips Prepared by Ambient Ion Soft Landing.

BACKGROUND: Transferrin is synthetized in the liver and is the most important iron-transport carrier in the human body. Severe alcohol consumption leads to alterations in glycosylation of transferrin. Mass spectrometry can provide fast detection and quantification of transferrin isoforms because they have different molecular masses. In this study, we used antibody chips in combination with MALDI-TOF MS for the detection and quantification of transferrin isoforms. METHODS: Protein chips were prepared by functionalization of indium tin oxide glass using ambient ion soft landing of electrosprayed antitransferrin antibody. Two microliters of patient serum was applied on the antibody-modified spots, and after incubation, washing, and matrix deposition, transferrin isoforms were detected by MALDI-TOF MS. Peak intensities of each transferrin form were used to calculate total carbohydrate-deficient transferrin (CDT). The CDT values obtained by the MALDI chip method were compared with the results obtained by a standard capillary electrophoresis (CE). RESULTS: The chip-based MALDI-TOF MS method was used for enrichment and detection of CDT from human serum. A sample cohort from 186 patients was analyzed. Of these samples, 44 were positively identified as belonging to alcoholic patients, whereas 142 were negative by the MALDI chip approach. The correlation of the data obtained by the CE and the chip-based MALDI was r = 0.986, 95% CI. CONCLUSIONS: Functionalized MALDI chips modified by antitransferrin antibody prepared by ambient ion soft landing were successfully used for detection and quantification of CDT from human sera.

Protein Chips Compatible with MALDI Mass Spectrometry Prepared by Ambient Ion Landing.

We present a technology that allows the preparation of matrix-assisted laser desorption/ionization (MALDI)-compatible protein chips by ambient ion landing of proteins and successive utilization of the resulting protein chips for the development of bioanalytical assays. These assays are based on the interaction between the immobilized protein and the sampled analyte directly on the protein chip and subsequent in situ analysis by MALDI mass spectrometry. The electrosprayed proteins are immobilized on dry metal and metal oxide surfaces, which are nonreactive under normal conditions. The ion landing of electrosprayed protein molecules is performed under atmospheric pressure by an automated ion landing apparatus that can manufacture protein chips with a predefined array of sample positions or any other geometry of choice. The protein chips prepared by this technique are fully compatible with MALDI ionization because the metal-based substrates are conductive and durable enough to be used directly as MALDI plates. Compared to other materials, the nonreactive surfaces show minimal nonspecific interactions with chemical species in the investigated sample and are thus an ideal substrate for selective protein chips. Three types of protein chips were used in this report to demonstrate the bioanalytical applications of ambient ion landing. The protein chips with immobilized proteolytic enzymes showed the usefulness for fast in situ peptide MALDI sequencing; the lectin-based protein chips showed the ability to enrich glycopeptides from complex mixtures with subsequent MALDI analysis, and the protein chips with immobilized antibodies were used for a novel immunoMALDI workflow that allowed the enrichment of antigens from the serum followed by highly specific MALDI detection.

Planar Functionalized Surfaces for Direct Immunoaffinity Desorption/Ionization Mass Spectrometry.

BACKGROUND: Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. METHODS: MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. RESULTS: The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. CONCLUSIONS: MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.

Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease.

Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2-3,6,8)-neuraminidase and ß(1-4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior.

Site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma.

Haptoglobin is a liver-secreted glycoprotein with four N-glycosylation sites. Its glycosylation was reported to change in several cancer diseases, which prompted us to examine site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma. To this end, we have used two-dimensional separation composed of hydrophilic interaction and nano-reverse phase chromatography coupled to QTOF mass spectrometry of the enriched glycopeptides. Our results show increased fucosylation of haptoglobin in liver disease with up to six fucoses associated with specific glycoforms of one glycopeptide. Structural analysis using exoglycosidase treatment and MALDI-MS/MS of detached permethylated glycans led to the identification of Lewis Y-type structures observed particularly in the pooled hepatocellular carcinoma sample. To confirm the increase of the Lewis Y structures observed by LC-MS, we have used immunoaffinity detection with Lewis Y-specific antibodies. The presence of multiply fucosylated Lewis Y glycoforms of haptoglobin in the disease context could have important functional implications.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Protein and site specificity of fucosylation in liver-secreted glycoproteins.

Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.

Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease.

Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

Exploring site-specific N-glycosylation microheterogeneity of haptoglobin using glycopeptide CID tandem mass spectra and glycan database search.

Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS .

LC-MS3 quantification of O-glycopeptides in human serum.

Quantitative analysis of site-specific glycosylation of proteins is a challenging part of glycoproteomic research. Multiple enrichment steps are typically required in the analytical workflows to achieve adequate characterization of the site-specific glycoforms. In spite of recent advances, quantitative workflows need further development. Here, we report a selective and sensitive MS2 followed by further fragmentation in the linear IT-MS analyzer (MS3) multiple reaction monitoring workflow mass spectrometric method for direct analysis of O-glycopeptides in difficult matrix such as serum. Method optimization was performed using two serum glycoproteins, hemopexin (HPX) and sex hormone binding globulin. With the optimized MS3 workflow, we were able to analyze major glycoforms of HPX directly in human serum. Quantification of the minor glycoforms of HPX and glycoforms of sex hormone binding globulin required enrichment of the protein because these analytes were below the sensitivity of the 4000 quadrupole ion trap hybrid mass spectrometer in the complex serum background. In conclusion, we present a quantitative method for site-specific analysis of O-glycosylation with general applicability to mucin-type glycoproteins. Our results document reliable application of the optimized MS3 multiple reaction monitoring workflow to the relative quantification of O-glycosylation microheterogeneity of HPX in human serum. Introduction of isotopically labeled standards would be desirable to achieve absolute quantification of the analytes. The possibility to analyze serum samples directly represents a significant improvement of the quantitative glycopeptide workflows with the potential for use in clinical applications.

Characterization of the interaction between the tumour suppressor p53 and heme and its role in the protein conformational dynamics studied by various spectroscopic techniques and hydrogen/deuterium exchange coupled with mass spectrometry.

The tumour suppressor p53 regulates the expression of a myriad of proteins that are important for numerous cellular processes, including apoptosis, cell cycle arrest, DNA repair, metabolism, and even autophagy and ferroptosis. Aside from DNA, p53 can interact with many types of partners including proteins and small organic molecules. The ability of p53 to interact with heme has been reported so far. In this study, we used various spectroscopic studies to conduct a thorough biophysical characterization of the interaction between p53 and heme concerning the oxidation, spin, coordination, and ligand state of heme iron. We found that the p53 oligomeric state and zinc biding ability are preserved upon the interaction with heme. Moreover, we described the effect of heme binding on the conformational dynamics of p53 by hydrogen/deuterium exchange coupled with mass spectrometry. Specifically, the conformational flexibility of p53 is significantly increased upon interaction with heme, while its affinity to a specific DNA sequence is reduced by heme. The inhibitory effect of DNA binding by heme is partially reversible. We discuss the potential heme binding sites in p53 with respect to the observed conformational dynamics changes and perturbed DNA-binding ability of p53 upon interaction with heme.

Insulin receptor Arg717 and IGF-1 receptor Arg704 play a key role in ligand binding and in receptor activation.

The insulin receptor (IR, with its isoforms IR-A and IR-B) and the insulin-like growth factor 1 receptor (IGF-1R) are related tyrosine kinase receptors. Recently, the portfolio of solved hormone-receptor structures has grown extensively thanks to advancements in cryo-electron microscopy. However, the dynamics of how these receptors transition between their inactive and active state are yet to be fully understood. The C-terminal part of the alpha subunit (αCT) of the receptors is indispensable for the formation of the hormone-binding site. We mutated the αCT residues Arg717 and His710 of IR-A and Arg704 and His697 of IGF-1R. We then measured the saturation binding curves of ligands on the mutated receptors and their ability to become activated. Mutations of Arg704 and His697 to Ala in IGF-1R decreased the binding of IGF-1. Moreover, the number of binding sites for IGF-1 on the His697 IGF-1R mutant was reduced to one-half, demonstrating the presence of two binding sites. Both mutations of Arg717 and His710 to Ala in IR-A inactivated the receptor. We have proved that Arg717 is important for the binding of insulin to its receptor, which suggests that Arg717 is a key residue for the transition to the active conformation.