|
Journal of Proteins and Proteomics
Number of Followers: 2  Open Access journalISSN (Print) 0975-8151 - ISSN (Online) 2524-4663 Published by Springer-Verlag  [2468 journals]
|
- Rice bean acid phosphatase (Vigna umbellata Thunb.): substrate specificity
and inactivation studies
Abstract: Rice bean acid phosphatase exhibited a broad range of substrate specificity. The highest activity was observed with p-nitrophenylphosphate while, substrates like phenylphosphate, phosphoenolpyruvate, adenosine diphosphate, and 1-naphthyl phosphate showed % activities between 100 and 40%. Other phosphate esters displayed little activity (15–40%); no activity was observed with phytic acid. The specificity constant values for phenylphosphate and 1-naphthylphosphate are maximum and equal. Others follow the decreasing sequence: p-nitrophenylphosphate, phosphoenolpyruvate, and adenosine diphosphate, respectively. Thermal inactivation of acid phosphatase carried at 55, 60, and 65 °C with the t1/2 values 45.45, 9.708, and 1.54 min, respectively. The D-values were 151.5, 32.4, and 5.175 min; the Z-value was 6.82 °C. The thermal inactivation carried at 60 °C in the presence of additives brought an increase in the t1/2 values; citric acid (11.278 min), polyethylene glycol (11.278 min), and bovine serum albumin (16.48 min), respectively. The energy of activation in the enzyme inactivation was 309.7 kJmol−1. The value of ∆H and ∆G at 60 °C were 306.93 and 100.478 kJ mol−1. The ∆S was + 0.62 kJmol−1 K−1. There was a complete loss in activity when the enzyme incubated for an hour in the presence of 10 M urea or 5 M guanidine hydrochloride. The inactivation kinetics was carried out in the presence of 9 M urea and 4 M guanidine hydrochloride; the t1/2 values with both urea and guanidine hydrochloride were 55.55 min.
PubDate: 2023-08-21
- Tyrosine inhibits the Mycobacterium tuberculosis protein tyrosine
phosphatase MptpA
Abstract: The catalytic action of the low-molecular weight protein tyrosine phosphatase (MptpA), expressed by Mycobacterium tuberculosis, is known to be essential for the pathogenicity of the bacterium. Candidate MptpA inhibitors are, therefore, actively sought and evaluated, and important work has been dedicated to the identification of competitive inhibitors. Here, we show that the activity of MptpA at the expense of phosphotyrosine (pTyr) is promptly inhibited by tyrosine, whereas the p-nitrophenol generated from p-nitrophenyl phosphate antagonizes the enzyme to a limited extent. We also report here a Ki of 512 ± 26 µM of MptpA for tyrosine, a value approximately one order of magnitude lower than the corresponding Ki for orthophosphate. Remarkably, the inhibition of MptpA observed when pTyr was used as substrate was effectively relieved by tyrosinase, the action of which was responsible for maintaining zero-order kinetics up to the generation of 200–400 µM of reaction product. Overall, the different sensitivity of MptpA towards tyrosine and p-nitrophenol suggests a significant inhibitory role of the α-aminocarboxylic acid moiety of the aromatic amino acid. This may, therefore, suggest the design of MptpA competitive inhibitors targeting the portion of the enzyme active site which is distal from the phosphate-binding region.
PubDate: 2023-08-17
- Differential proteomics reveals overexpression of ferroptosis-related
proteins in cervical cancer tissue
Abstract: Cervical cancer, the fourth most common female cancer, is one of the major global health challenges. Because of the high prevalence rate, women are frequently screened for cervical cancer using Pap smears; however, the low sensitivity of Pap smear results in a late-stage diagnosis of cervical cancer. This study was designed to identify candidate biomarkers of cervical cancer, which can be explored for their utility as diagnostic markers and/or therapeutic targets. Cervical cancer and healthy tissue proteins were identified by tandem mass spectrometry (LC–MS/MS) and quantified by a label-free method. Candidate biomarkers were selected by applying stringent statistical methods. A total of 201 differentially expressed proteins were identified, which displayed statistical power to classify cervical cancer and control groups. The top proteins with higher abundance in cervical cancer included integrin beta-2, catenin alpha-1, stathmin, and tax1-binding protein 3; while the top proteins with higher abundance in healthy tissue samples included alpha-2-HS-glycoprotein, serotransferrin, afamin, hemopexin, plasminogen, apolipoprotein A-II, and immunoglobulin lambda variable 1–51. Stathmin and afamin expressions were also validated by western blot analysis. Upregulated proteins in cervical cancer were mainly associated with cell adhesion, protein folding, inflammatory disorders, and tumorigenesis. The KEGG pathways over-represented in cervical cancer were antigen processing and presentation, cell adhesion, spliceosome, adherens junction, and ferroptosis. Applying stringent statistical selection criteria, 46 DEPs were selected as candidate biomarkers of cervical cancer, which can be explored for their utility as diagnostic markers and/or therapeutic targets of cervical cancer.
PubDate: 2023-08-12
- A review on-analytical tools in proteomics
Abstract: Background Proteomics is an analytical technique employed for the identification and quantitative analysis of total protein content in a cell, tissue or organism using various methods and tools. Proteomics succeeds in genomics (study of set of DNA) and transcriptomics (study of set of RNA) of biological systems. A proteome is an array of proteins generated by a particular organism, system, or biological condition. Though, an organism’s genome is more or less constant, a proteome is more complicated than a genome as cells generate a variety of sets of protein at different times or under different situations, such as cellular development and differentiation, cell cycle or carcinogenesis. Therefore, new approaches for capturing crucial biological information have been developed. Proteomics-based technologies are used for several research purposes including detection of various diagnostic markers, vaccine candidates as well as understanding pathogenicity mechanisms, changes in expression patterns in response to various signals, and interpretation of functional protein pathways in various diseases. Analytical methods like two-dimensional gel electrophoresis, mass spectrometry and various hyphenated techniques have been developed. Main body This review highlights various analytical tools used in qualitative and quantitative analysis of proteomics and novel approaches for the use of proteomics in disease diagnostics, as well as their other applications. Conclusion This review shall help academicians and researchers as a reference guide to update their knowledge on analytical tools for proteomics as well as the application of proteomics.
PubDate: 2023-07-24
- Calreticulin: a quintessential multifaceted protein with therapeutic
potential
Abstract: Calreticulin is primarily an endoplasmic reticulum (ER) resident protein which has also been identified at different cellular sites. Its multicompartment presence is in congruence with its multifaceted nature, performing diverse biological functions. Inside ER, it participates in proteostasis allowing protein folding and preventing misfolding, while interacting with other chaperones including calnexin, protein disulphide isomerse (PDI), etc. Calcium homeostasis in ER is among the initial and well-studied roles of calreticulin. Besides, it also plays vital roles in adipocyte differentiation, stress response, cardiogenesis and cardiovascular functioning. Calreticulin is considered to be a key modulator of integrin-dependent cell adhesion and cell signaling. The occasional presence of calreticulin on the cell surface during physiological stress makes it an important prognostic marker for various pathological conditions. Calreticulin can modulate immune response since its presence on the cell surface generates signals responsible for immunological cell death assisting in presentation of antigens derived from dying or damaged cells to T-cells. Calreticulin is also important in the assembly and loading of antigens to major histocompatibility complex class-I molecules. Calreticulin is a quintessential multifaceted protein performing diverse functions at multiple locations in the cell. Significance of its varied physiological roles and implications of its dysregulation along with its diagnostic and therapeutic potential has been presented in this review.
PubDate: 2023-06-26
DOI: 10.1007/s42485-023-00111-x
- An insight into the structural analysis of α-crystallin of
habitat-specific fish: a computational approach
Abstract: α-crystallin is a major eye lens protein, comprising up to 40% of total lens protein. It is composed of two subunits, αA and αB and even shares a common central domain of about 90 residues with variable N- and C-terminal extensions. For the establishment of an evolutionary inter-relationship, an elucidation of the structure and alignment of protein sequences is crucial. In the present study, a bioinformatics approach was adopted to explore the possible structure, sequence and phylogenetic relationship of α-crystallin (both subunits αA and αB) from ten habitat-specific fish species, (freshwater and saltwater) and compared with a standard sequence of Bos taurus species. The sequence of Bos taurus was predicted to be a close homologue of the fish species. Wet lab experiments such as NMR are not only expensive and time consuming but are suitable for small proteins having less than 150 amino acids, so a preliminary computer-aided approach has been selected for structural analysis of α-crystallin of fish species. Our analysis shows that the secondary structures of bovine α-crystallin revealed no considerable differences as compared to that of the crystallins of the habitat-specific fish and that the presence of β- sheets was predominant in all structures. Though no significant differences in the αA subunits were revealed yet some structural variations were observed for αB subunits which had been confirmed by MSA analysis. The 3D structure of the protein hasn’t been elucidated yet so a computational analysis estimated no major differences in structures of crystallin for either bovine or the fish species except that saltwater fish proteins possess more favourable states and higher reliabilities. At the same time, the RMSF values of α-crystallin computed by CABS Flex 2.0 showed lesser values in the case of freshwater fish species which state the possible favourable structures of freshwater fish species. The stabilities of αB- subunits were revealed from the physiochemical parameters computed as compared to αA subunits of the respective proteins for all species. The Kyle-Doolittle Plots revealed the predominance of hydrophilic amino acids in both subunits of α-crystallin for all species and it is a cytosolic protein that has been determined using the TMHMM server. Considerate differences were revealed in the case of the αB subunit but not for the αA subunit, for all species as deciphered from Clustal omega which may indicate that the differences in α-crystallin occur primarily due to the αB subunit. Homology modeling revealed that bovine α-crystallin showed a greater sequence homology with most fish species, especially zebrafish. From our study, no major differences in α- crystallin structure could be deciphered between fish species in terms of habitat. The structure of human crystallin is a complex model so fish species were chosen as models which would be beneficial for humans in terms of drug designing as well.
PubDate: 2023-06-17
DOI: 10.1007/s42485-023-00107-7
- A quantitative proteomic approach revealed variations of salivary proteome
during high altitude de-acclimatization
Abstract: De-acclimatization occurs when an individual returns to sea level after long term exposure to high altitude. Reports on high altitude de-acclimatization document symptoms, physiological changes, and modulations in cardiovascular and nervous systems when individuals descend to sea level. However, there is currently no available data regarding the molecular networks affected by de-acclimatization. The lack of omics-based studies holds necessity of studying molecular signatures and pathways involved in high-altitude de- acclimatization using proteomics-based approach by taking saliva as a diagnostic sample. In this study, we employed iTRAQ based LC-MS/MS approach for comparison between saliva samples obtained from humans ascended from sea level to high altitude and then descended back to sea level followed by pathway analysis using ingenuity pathway analysis (IPA) and validation using immunoassays. Saliva samples were initially taken in normoxic conditions (Baseline 216 m above sea level) with further sampling done at high-altitude exposure (4420 m above sea level) for 30 days (HAD 30) followed by sampling after de- acclimatization (Baseline 216 m above sea level) for 30 days (DI 30). Nearly 152 proteins were found to be differentially expressed in the de-acclimatized group as compared to acclimatized group and normoxia indicating modulated canonical pathways as glycolysis and gluconeogenesis. Carbohydrate metabolism was found to be significantly activated pathway in response to de-acclimatization to high altitude. Collectively, this study provided the initial advancement in understanding high-altitude de-acclimatization mediated events through salivary proteome.
PubDate: 2023-06-16
DOI: 10.1007/s42485-023-00109-5
- Will “CLINICAL PROTEOMICS” lead to the discovery of new biomarkers for
dilated cardiomyopathy (DCM)'
PubDate: 2023-06-10
DOI: 10.1007/s42485-023-00110-y
- Comprehensive review on some food-derived bioactive peptides with
anti-hypertension therapeutic potential for angiotensin-converting enzyme
(ACE) inhibition
Abstract: Angiotensin-converting enzyme (ACE) inhibitory peptides have lately attracted interest since functional foods that help maintain homeostatic regulations have been developed. Rarely discussed are the intrinsic ACE-peptide interactions and their positioning, both of which help illustrate the ACE inhibitory functionalities in food-derived peptides. In this study, 173 ACE inhibitory peptides were collated using the UWM-BIOPEP database. The sequences were grouped into short, medium, and long peptides. The hydrophobicity/hydrophilicity property of peptides was analyzed using Peptide2 and the peptide binding site on ACE was predicted using PepSite2. Peptide residues interacting with ACE were denoted as reactive amino acids. Molecular docking analysis was conducted to simulate ACE-peptide binding and delineate the roles of reactive amino acids at the ultimate, penultimate, and antepenultimate positions of N—(N1, N2, and N3) and C—(C1, C2, and C3) terminals. Peptide2 analysis suggested that hydrophobic property was prominent in the peptides. The C-terminals were prominent in ACE binding for long-chained peptides through interaction with ACE hotspots. Moreover, branched-chain amino acids (BCAA) such as leucine and isoleucine were crucial at the N-terminals. The bulky side chain of BCAA forms a hydrophobic shield that protects the Zn-peptide chelate complex from water attacks. The hydrophobic fence in turn stabilizes the disrupted tetrahedral Zn-coordinate complex of ACE. This finding provided a thorough exploration of how peptide structures are related and what function they play in ACE inhibitory action. The database analysis, therefore, gave a clearer insight and comprehensive understanding into the protein-peptide interactions and provided a mechanistic explanation.
PubDate: 2023-05-17
DOI: 10.1007/s42485-023-00106-8
- Evolutionary aspects of mutation in functional motif and
post-translational modifications in SARS-CoV-2 3CLpro (Mpro): an in-silico
study
Abstract: SARS CoV-2 is the virus that caused the COVID-19 pandemic. The main protease is one of the most prominent pharmacological targets for developing anti-COVID-19 therapeutic drugs (Mpro); SARS-CoV-2 replication is dependent on this component. SARS CoV-2’s Mpro/cysteine protease is quite identical to SARS CoV-1’s Mpro/cysteine protease. However, there is limited information on its structural and conformational properties. The present study aims to perform a complete in silico evaluation of Mpro protein’s physicochemical properties. The motif prediction, post-translational modifications, effect of point mutation, and phylogenetic links were studied with other homologs to understand the molecular and evolutionary mechanisms of these proteins. The Mpro protein sequence was obtained in FASTA format from the RCSB Protein Data Bank. The structure of this protein was further characterized and analyzed using standard bioinformatics methods. According to Mpro’s in-silico characterization, the protein is a basic, non-polar, and thermally stable globular protein. The outcomes of the phylogenetic and synteny study showed that the protein’s functional domain amino acid sequence is substantially conserved. Furthermore, it has undergone many changes at the motif level over time from porcine epidemic diarrhoea virus to SARS-CoV 2, possibly to achieve various functions. Several post-translational modifications (PTMs) were also observed, and the possibilities of changes in Mpro protein exhibit additional orders of peptidase function regulation. During heatmap development, the effect of a point mutation on the Mpro protein was seen. This protein’s structural characterization will aid in a better understanding of its function and mechanism of action.
PubDate: 2023-04-13
DOI: 10.1007/s42485-023-00105-9
- Bungarus fasciatus venom from eastern and north-east India: venom
variation and immune cross-reactivity with Indian polyvalent antivenoms
Abstract: Bungarus fasciatus is one of the medically important elapid snakes of South and South-eastern Asia and is responsible for several snakebite incidents some of which were fatal. In this study, the venom compositional variation of Bungarus fasciatus from three different geographical locations in eastern and north-east India: two adjacent villages of Hooghly (West Bengal), Aizawl (Mizoram) and Guwahati (Assam) are reported. In vitro biochemical assays exhibit variation in phospholipase A2 activity, fibrinogenolytic activity, caseinolytic activity and anti-coagulation activity. The immunoreactivity of three Indian polyvalent antivenoms against the venoms revealed incomplete recognition. Bharat Serums antivenom demonstrated that at a venom–antivenom ratio of 1:16, the antivenom exhibited different immunocapturing abilities for all the venom samples. The percentage of non-retained fractions was highest for Guwahati (60.00%) and lowest for Hooghly 1 (18.91%). The study demonstrates intra-population (or inter-individual) variation of B. fasciatus venom from two nearby locations of Hooghly (West Bengal), intra-specific variation of B. fasciatus from three geographical locations and also inter-specific venom variation with B. caeruleus from Tamil Nadu. Thus, the venom variation leads to partial immune cross-reactivity by Indian polyvalent antivenoms. Inclusion of non-recognized venom proteins in the immunization mixture during antivenom production would help to improve the efficacy of the antivenom. Further study of the neutralizing ability of Indian polyvalent antivenoms against medically important snakes from different geographical regions would help to understand the effectiveness of the antivenoms and would invariably assist in the designing and development of safe and effective antivenoms.
PubDate: 2023-01-16
DOI: 10.1007/s42485-022-00104-2
- Correction: Digging deeper into the immunopeptidome: characterization of
post‑translationally modified peptides presented by MHC I
PubDate: 2023-01-02
DOI: 10.1007/s42485-022-00103-3
- Back2Basics: animal lectins: an insight into a highly versatile
recognition protein
Abstract: The rapid advancement of molecular research has contributed to the discovery of ‘Lectin’, a carbohydrate-binding protein which specifically interacts with receptors on surface glycan moieties that regulate various critical cellular activities. The first animal lectin reported was ‘the asialoglycoprotein receptor’ in mammalian cells which helped analyze how animal lectins differ in glycoconjugate binding. Animal lectins are classified into several families, depending on their diverse cellular localization, and the binding specificities of their Carbohydrate-Recognition Domain (CRD) modules. Earlier characterization of animal lectins classified them into two structural families, the C-type (Ca2+-dependent binding) and S-type galectins (sulfhydryl-dependent binding) lectins. The C-type lectin includes the most significant animal lectins, such as endocytic receptors, mannose receptors, selectins, and collectins. The recent developments in research based on the complexity of the carbohydrate ligands, the metabolic processes they perform, their expression levels, and their reliance on divalent cations have identified more than 100 animal lectins and classified them into around 13 different families, such as Calnexin, F-lectin, Intelectin, Chitinase-like lectin, F-box lectin, etc. Understanding their structure and expression patterns have aided in defining their significant functions including cell adhesion, antimicrobial activity, innate immunity, disease diagnostic biomarkers, and drug delivery through specific carbohydrate–protein interactions. Such extensive potential roles of animal lectins made it equally important to plant lectins among researchers. Hence, the review focuses on providing an overview of animal lectins, their taxonomy, structural characteristics, and functions in diverse aspects interconnected to their specific carbohydrate and glycoconjugate binding. Graphical abstract
PubDate: 2022-12-29
DOI: 10.1007/s42485-022-00102-4
- Structure–function relationship of α-crystallin in the context of
vertebrate lens evolution and its role in eye disorders
Abstract: α- Crystallin, an oligomeric major constituent protein of the eye lens of vertebrates, was originally identified for its role in the lens’ transparency. In addition to having its micelle-like architecture, this protein has molecular chaperoning activity. Lens α-crystallin consists of two subunits, αA and αB, whose aggregate formation is necessary for molecular chaperoning activity. Isolation and characterization of α-crystallin from a wide range of vertebrates will help in understanding a better structure–function relationship in a broader sense, which is yet to be achieved. The stability, structure, aggregation and molecular chaperone activity of α-crystallin differ significantly from species to species. These differences clearly reflect specific structural changes of the protein, which, in turn, may contribute to the transparency and refractive power of the eye lens. Several eye diseases, such as diabetic retinopathy involve oxidative stress, followed by a decrease in total soluble lens proteins and a decreased amount of βB1 crystallins. Intraperitoneal injection of edaravone drugs, a member of the substituted 2-pyrazolin-5-one class, and its analogs are now being tried to revert back crystallin activity and inhibit hyperglycemia and oxidative stress-mediated eye cell damage. In the upcoming future, edaravone-like drugs or their analogs can be synthesized and targeted for better efficacy.
PubDate: 2022-12-07
DOI: 10.1007/s42485-022-00101-5
- Oncoproteomics: insight into current proteomic technologies in cancer
biomarker discovery and treatment
Abstract: Cancer is a disease with second largest fatalities in humans and the sheer complexity of the disease is an enigma to the scientific community. It continues to be a highly significant research area for the development of diagnostics and therapeutics. While a great deal of focus has been on genomics study of cancer, the fact that cancer tissues have altered protein expression and unique protein–protein interactions demands more attention. The role of proteins and peptides which are unique and aberrant in cancer tissues has generated a great deal of interest in cancer proteome and serve as important diagnostic and therapeutic targets in the form of biomarkers. Oncoproteomics along with computational and genomic advances is utilized for the discovery of biomarkers and modern therapeutics indispensable in cancer management and care; the focus areas in cancer research involving proteomic studies, technologies used and their advances, identification and quantification of proteins with greater sensitivity and resolution, and detection of post-translational modifications. In this review, we have discussed an overview of various technological advances in oncoproteomics and the therapeutic milestones achieved using the oncoproteome data leading to antibody engineering, peptidomimetics, bispecific antibodies, and other techniques.
PubDate: 2022-12-02
DOI: 10.1007/s42485-022-00100-6
- Identification of potential inhibitors of brain-specific CYP46A1 from
phytoconstituents in Indian traditional medicinal plants
Abstract: Cytochrome P450 46A1 (CYP46A1) is a crucial enzyme in brain that converts cholesterol to 24 (S) hydroxy cholesterol thereby increasing its polarity to facilitate removal of excess cholesterol from the CNS. The inhibition of CYP46A1 with several synthetic molecules has been investigated extensively for treatment of Alzheimer’s disease, Huntington’s disease, glaucoma, and in hippocampal neurons from aged mice. However, phytochemicals have received far little attention in studies involving development of potential CYP46A1 inhibitors. Thus, in the present study phytoconstituents from Indian traditional medicinal plants; Bacopa monnieri, Piper longum, and Withania somnifera, were virtually screened for interaction with CYP46A1 using computational tools. Out of three plants, six molecules from P. longum and three molecules from W. somnifera were shortlisted to study interactions with CYP46A1 based on the physio-chemical parameters. Fargesin, piperolactam A and coumaperine from P. longum showed the higher binding affinity and the values were − 10.3, − 9.5, − 9.0 kcal/moles respectively, whereas, withaferin A from W. somnifera had a binding affinity of − 12.9 kcal/mol. These were selected as potential modulators as they exhibited suitable interactions with active site residues; Tyr109, Leu112, Trp368, Gly369, and Ala474. The selected molecules were further subjected to molecular dynamics simulation. Further, the pharmacological properties of molecules were also predicted using ADMET calculator and the data revealed that all the selected compounds had good absorption as well as solubility characteristics. In addition, sesamin, fargesin, piperolactam A, and coumaperine had minimal or no toxic effects. Thus, the study successfully identified compounds from Indian medicinal plants that may serve as potential inhibitors of CYP46A1 or base structures to design novel CYP46A1 inhibitors, which may be effective in treating neurological conditions involving perturbed cholesterol homeostasis.
PubDate: 2022-11-16
DOI: 10.1007/s42485-022-00098-x
- Evaluation of steric entanglement in coiled-coil and domain-swapped
protein interfaces using 3D printed models
Abstract: Oligomeric protein interfaces involve non-covalent attractive forces plus potential steric entanglement. 70 years ago, Crick proposed a “Knobs in Holes” model for coiled-coil protein interfaces. Subsequently, modifications to this model have been proposed, describing either a “leucine zipper”, “jigsaw puzzle”, or a “peptide Velcro” interface. These principally describe forms of steric entanglement that may enhance oligomer stability. However, such entanglement has not been rigorously evaluated since it is not possible to experimentally eliminate intrinsic non-covalent attractive forces. 3D printing provides a novel means to evaluate steric entanglement of protein interfaces in the absence of attractive forces. Surprisingly, quantitation of the energy required to dissociate various coiled-coil protein interfaces of 3D printed protein models suggests minimal steric entanglement. Conversely, evaluation of domain-swapped interfaces of symmetric protein oligomers, differing by circular permutation, identifies extensive potential steric entanglement. Combined with available experimental data, the results suggest that steric entanglement of a protein interface can contribute to kinetic trapping of both folding and unfolding pathways. Steric entanglement of protein interfaces is therefore postulated to be an undesirable property for naturally evolved and designed protein oligomers.
PubDate: 2022-10-12
DOI: 10.1007/s42485-022-00099-w
- Mass spectrometric analysis of chondroitin sulfate-linked peptides
Abstract: Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3–6 monosaccharides attached to specific serine residues can be extended by 20–200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.
PubDate: 2022-10-02
DOI: 10.1007/s42485-022-00092-3
- Pangenome and subtractive genomic analysis of Clostridioides difficile
reveals putative drug targets
Abstract: Clostridioides difficile is the major cause of antibiotic-associated diarrhea in hospitalized patients. The low susceptibility of this pathogen to first-line antibiotics coupled with the recurrence of its infection (CDI) has become a global concern that necessitates the need to explore novel drug targets against this pathogen. In this study, in-silico approaches through pangenome and subtractive genomic analysis were used to predict putative drug targets. A total of 2556 core genes were identified after pangenome analysis of which 173 were predicted to be essential and non-homologous to human host. Further analysis such as virulence effector function, subcellular localization, involvement in metabolic pathways, gene-enrichment analysis, physicochemical properties and druggability of the proteins were done. A total of 5 cytoplasmic proteins were finally predicted as novel putative drug targets. This study contributes immensely to the search of novel drug targets against C. difficile though further experimental validation is highly imperative.
PubDate: 2022-10-01
DOI: 10.1007/s42485-022-00097-y
- Rapid and deep plasma proteomics workflows for robust identification and
quantification of biomarkers of sickle cell anaemia
Abstract: Plasma serves as a rich source of protein biomarkers but in-depth proteomic analysis is challenging due to vast dynamic range of protein abundance. Pre-fractionation of plasma proteins is commonly practiced to enhance the proteome coverage but the protocols are time-expensive, suffer from flowchart complexity, and often less reproducible. Here, we explore multiple strategies of shotgun proteomics to optimize biomarker discovery workflows for Sickle Cell Anaemia (SCA) patients from Maharashtra, India. A deep proteomics workflow via off-line reverse phase ultra-high-pressure liquid chromatography-based fractionation of tryptic digested plasma peptides followed by optimized pooling of peptides based on charge and hydrophobicity yielded the best depth of plasma proteome with a trade-off of significantly long experimental time. Alternatively, a rapid analysis of tryptic digested plasma peptides via a shorter gradient mass spectrometry run saves time but quantifies only ~ 50% of the proteins than the deep workflow. Intriguingly, despite the difference in proteome coverage, more than 80% of known FDA and SCA biomarkers quantified in the deep workflow are also quantified in the rapid workflow. Given the practical difficulties of sample collection and plasma preservation in rural India, we propose the deep proteomics workflow for biomarker discovery in smaller cohorts and the rapid workflow for biomarker validations in larger cohorts. Targeted-proteomics-based strategies may be designed for the validation of missing biomarkers in the rapid workflow.
PubDate: 2022-09-19
DOI: 10.1007/s42485-022-00096-z
