scholarly journals Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

2022 ◽  
Author(s):  
Lingjun Li ◽  
Yatao Shi ◽  
Zihui Li ◽  
Bin Wang ◽  
Xudong Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Global Analysis ◽  
Protein Structures ◽  
Isotopic Labeling ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Mouse Tissues ◽  
Global Mapping

Abstract Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 691 citrullination sites and 81 homocitrullination sites from 432 and 63 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation for further deciphering their physiological and pathological roles.

scholarly journals Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

2021 ◽  
Author(s):  
Lingjun Li ◽  
Yatao Shi ◽  
Zihui Li ◽  
Bin Wang ◽  
Xudong Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Global Analysis ◽  
Protein Structures ◽  
Isotopic Labeling ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Mouse Tissues ◽  
Global Mapping

Abstract Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 1,198 citrullination sites and 108 homocitrullination sites from 619 and 79 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation to further decipher their physiological and pathological roles.

The proteomic future: where mass spectrometry should be taking us

2012 ◽  
Vol 444 (2) ◽  
pp. 169-181 ◽  
Author(s):  
Jay J. Thelen ◽  
Ján A. Miernyk
Keyword(s):  
Mass Spectrometry ◽  
Gel Electrophoresis ◽  
Protein Structures ◽  
Research Area ◽  
Mass Accuracy ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Post Translational Modifications ◽  
Peptide Separation ◽  
Research Areas

A newcomer to the -omics era, proteomics, is a broad instrument-intensive research area that has advanced rapidly since its inception less than 20 years ago. Although the ‘wet-bench’ aspects of proteomics have undergone a renaissance with the improvement in protein and peptide separation techniques, including various improvements in two-dimensional gel electrophoresis and gel-free or off-gel protein focusing, it has been the seminal advances in MS that have led to the ascension of this field. Recent improvements in sensitivity, mass accuracy and fragmentation have led to achievements previously only dreamed of, including whole-proteome identification, and quantification and extensive mapping of specific PTMs (post-translational modifications). With such capabilities at present, one might conclude that proteomics has already reached its zenith; however, ‘capability’ indicates that the envisioned goals have not yet been achieved. In the present review we focus on what we perceive as the areas requiring more attention to achieve the improvements in workflow and instrumentation that will bridge the gap between capability and achievement for at least most proteomes and PTMs. Additionally, it is essential that we extend our ability to understand protein structures, interactions and localizations. Towards these ends, we briefly focus on selected methods and research areas where we anticipate the next wave of proteomic advances.

Identification of Multiple Oxidative Post-Translational Modifications of Plasma Albumin in Patients with Pulmonary Hypertension of Sickle Cell Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4814-4814
Author(s):  
Adam Odhiambo ◽  
David Perlman ◽  
Martin Steinberg ◽  
Catherine E. Costello ◽  
Mark E. McComb ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Pulmonary Hypertension ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Plasma Proteins ◽  
Maldi Mass Spectrometry ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Redox Biology ◽  
Extensive Evaluation

Abstract Background: Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in patients with SCA and PH, oxidative post-translational modifications (PTMs) occur on plasma proteins and are important in disease pathogenesis. We previously reported the identification of one PTM, a malondialdehyde adduct on peptide 146–159 of albumin and sought to do a more extensive evaluation of this protein to determine the presence of other abnormalities. Methods: Plasma was obtained from subjects with: SCA and PH (n=5); SCA steady-state without PH (n=4); Pulmonary Arterial Hypertension (PAH) (n=4); no evidence of cardiopulmonary disease (n=4). Platelet-poor plasma was separated into albumin-enriched and albumin-depleted fractions. The albumin-enriched fraction was subjected to proteolytic digestion by trypsin and studied by matrix-assisted-laser desorption/ionization (MALDI) mass spectrometry (MS) and liquid chromatography (LC)-MS/MS tandem mass spectrometry. Proteomic analyses were performed on all samples and post-translational modifications characterized by MS/MS. Results: We have characterized several additional peptides of albumin from patients with PH of SCA that bear lipid peroxidation and glycation adducts. Our comprehensive LC-MS/MS results have allowed us to identify 4-hydroxynonenal (HNE), hexose (HEX) and malonyl adduction at numerous, distinct cysteine, histidine, lysine, and threonine albumin residues. Conclusion: Increased oxidant burden and altered redox biology is characteristic of PH in SCA. Plasma proteins, such as albumin, are a target for oxidants and changes in their structure may play a role in disease pathogenesis.

Global analysis of protein expression in muscle tissues of dermatomyositis/polymyosisits patients demonstrated an association between dysferlin and human leucocyte antigen A

Rheumatology ◽  
2019 ◽  
Vol 58 (8) ◽  
pp. 1474-1484 ◽  
Author(s):  
Yizhi Xiao ◽  
Honglin Zhu ◽  
Liya Li ◽  
Siming Gao ◽  
Di Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Muscle Tissue ◽  
Inflammatory Cell ◽  
Global Analysis ◽  
Inflammatory Cell Infiltration ◽  
Cell Infiltration ◽  
Biological Processes ◽  
Monocyte Chemoattractant Protein 1 ◽  
Transmembrane Glycoprotein ◽  
Muscle Tissues

Abstract Objectives DM and PM are characterized by myofibre damage with inflammatory cell infiltration due to the strong expressions of MHC class I HLA-A and monocyte chemoattractant protein-1 (MCP-1). Dysferlin (DYSF) is a transmembrane glycoprotein that anchors in the sarcolemma of myofibres. DYSF mutation is closely associated with inherited myopathies. This study aimed to determine the role of DYSF in the development of DM/PM. Methods Mass spectrometry was performed in muscle tissues from DM/PM patients and controls. The DYSF levels in muscle tissue, peripheral blood cells and serum were detected by Western blotting, IF, flow cytometry or ELISA. Double IF and co-immunoprecipitation were used to investigate the relationship between DYSF and HLA-A. Results Mass spectrometry and bioinformatics analysis findings suggested the dysregulated proteins in DM/PM patients participated in common biological processes and pathways, such as the generation of precursor metabolites and energy. DYSF was upregulated in the muscle tissue and serum of DM/PM patients. DYSF was mainly expressed in myofibres and co-localized with HLA-A and MCP-1. DYSF and HLA-A expressions were elevated in myocytes and endothelial cells after being stimulated by patient serum and IFN-β. However, no direct interactions were found between DYSF and HLA-A by co-immunoprecipitation. Conclusion Our study revealed the dysregulated proteins involved in common and specific biological processes in DM/PM patient samples. DYSF is upregulated and exhibits a potential role along with that of HLA-A and MCP-1 in inflammatory cell infiltration and muscle damage during the development of DM/PM.

Identification of Post-Translational Modifications by Mass Spectrometry

2013 ◽  
Vol 66 (7) ◽  
pp. 734 ◽  
Author(s):  
Armand G. Ngounou Wetie ◽  
Izabela Sokolowska ◽  
Alisa G. Woods ◽  
Costel C. Darie
Keyword(s):  
Mass Spectrometry ◽  
Structure Stability ◽  
Biological Processes ◽  
Protein Activity ◽  
Post Translational Modifications ◽  
Protein Modifications ◽  
Effector Molecules ◽  
Pathological Conditions ◽  
Activity Structure

Proteins are the effector molecules of many cellular and biological processes and are thus very dynamic and flexible. Regulation of protein activity, structure, stability, and turnover is in part controlled by their post-translational modifications (PTMs). Common PTMs of proteins include phosphorylation, glycosylation, methylation, ubiquitination, acetylation, and oxidation. Understanding the biology of protein PTMs can help elucidate the mechanisms of many pathological conditions and provide opportunities for prevention, diagnostics, and treatment of these disorders. Prior to the era of proteomics, it was standard to use chemistry methods for the identification of protein modifications. With advancements in proteomic technologies, mass spectrometry has become the method of choice for the analysis of protein PTMs. In this brief review, we will highlight the biochemistry of PTMs with an emphasis on mass spectrometry.

scholarly journals Global Analysis of Protein Sumoylation inSaccharomyces cerevisiae

2004 ◽  
Vol 279 (44) ◽  
pp. 45662-45668 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Erica S. Johnson ◽  
Steven I. Reed ◽  
John R. Yates
Keyword(s):  
Mass Spectrometry ◽  
Chromatin Remodeling ◽  
Global Analysis ◽  
Eukaryotic Cells ◽  
Transcriptional Elongation ◽  
Biological Processes ◽  
Pol Ii ◽  
Macromolecular Complexes ◽  
Cellular Factors ◽  
Biological Patterns

Although the modification of cellular factors by SUMO is an essential process inSaccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes.

scholarly journals Mass spectrometry-based analysis of glycoproteins and its clinical applications in cancer biomarker discovery

2017 ◽  
Vol 4 (7) ◽  
pp. 203-215
Author(s):  
Farid Abu Shammala
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Biomarker Discovery ◽  
Global Analysis ◽  
Structural Identification ◽  
Clinical Applications ◽  
Biological Processes ◽  
Complex Sample ◽  
Systematic Identification ◽  
Glycoprotein Glycosylation

Most proteins are glycosylated, glycosylation is one of the most important posttranslational modifications of proteins and plays essential roles in various biological processes. Aberration in the glycan moieties of glycoproteins is associated with many diseases. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. With recent advances in proteomics, analytical and computational technologies, glycoproteomics, the global analysis of glycoproteins, is rapidly emerging as a subfield of proteomics with high biological and clinical relevance. Glycoproteomics integrates glycoprotein enrichment and proteomics technologies to support the systematic identification and quantification of glycoproteins in a complex sample. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. Mass spectrometry (MS) has become a powerful tool for mapping glycoprotein glycosylation and detailed glycan structural determination. Especially, tandem mass spectrometry can provide highly informative fragments for structural identification of glycoproteins. This review provides an overview of the development of MS technologies and their applications in identification of abnormal glycoproteins and glycans in human serum to screen cancer biomarkers in recent years.

scholarly journals Intracellular proteome compartmentalization: a biotin ligase-based proximity labeling approach

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Olanrewaju Ayodeji Durojaye
Keyword(s):  
Mass Spectrometry ◽  
Underlying Mechanism ◽  
Cellular Structures ◽  
Biological Processes ◽  
Interacting Proteins ◽  
Cellular Processes ◽  
Biotin Ligase ◽  
Labeling Approach ◽  
Cellular Compartments

AbstractSpecialized biological processes occur in different regions and organelles of the cell. Additionally, the function of proteins correlate greatly with their interactions and subcellular localization. Understanding the mechanism underlying the specialized functions of cellular structures therefore requires a detailed identification of proteins within spatially defined domains of the cell. Furthermore, the identification of interacting proteins is also crucial for the elucidation of the underlying mechanism of complex cellular processes. Mass spectrometry methods have been utilized systematically for the characterization of the proteome of isolated organelles and protein interactors purified through affinity pull-down or following crosslinking. However, the available methods of purification have limited these approaches, as it is difficult to derive intact organelles of high purity in many circumstances. Furthermore, contamination that leads to the identification of false positive is widespread even when purification is possible. Here, we present a highlight of the BioID proximity labeling approach which has been used to effectively characterize the proteomic composition of several cellular compartments. In addition, an observed limitation of this method based on proteomic spatiotemporal dynamics, was also discussed.

IMRPS: Inserted and Modified Residues in Protein Structures. A database

2020 ◽  
Vol 53 (2) ◽  
pp. 569-573
Author(s):  
R. Santhosh ◽  
Namrata Bankoti ◽  
M. Gurudarshan ◽  
J. Jeyakanthan ◽  
K. Sekar
Keyword(s):  
User Interface ◽  
Protein Structure ◽  
Protein Data Bank ◽  
Graphical User Interface ◽  
Protein Structures ◽  
Quality Criteria ◽  
Data Bank ◽  
Biological Processes ◽  
Online Database ◽  
Post Translational Modifications

Modified residues present in proteins are the result of post-translational modifications (PTMs). These PTMs increase the functional diversity of the proteome and influence various biological processes and diseased conditions. Therefore, identification and understanding of PTMs in various protein structures is of great significance. In view of this, an online database, Inserted and Modified Residues in Protein Structures (IMRPS), has been developed. IMRPS is a derived database that furnishes information on the residues modified and inserted in the protein structures available in the Protein Data Bank (PDB). The database is equipped with a graphical user interface and has an option to view the data for non-redundant protein structures (25 and 90%) as well. A quality criteria cutoff has been incorporated to assist in displaying the specific set of PDB codes. The entire protein structure along with the inserted or modified residues can be visualized in JSmol. This database will be updated regularly (presently, every three months) and can be accessed through the URL http://cluster.physics.iisc.ac.in/imrps/.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

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