Protein pIShifts due to Posttranslational Modifications in the Separation and Characterization of Proteins

2005 ◽  
Vol 77 (9) ◽  
pp. 2745-2755 ◽  
Author(s):  
Kan Zhu ◽  
Jia Zhao ◽  
David M. Lubman ◽  
Fred R. Miller ◽  
Timothy J. Barder
Keyword(s):  
Posttranslational Modifications

scholarly journals Insights into the Functionality of the Putative Residues Involved in Enterocin AS-48 Maturation

2010 ◽  
Vol 76 (21) ◽  
pp. 7268-7276 ◽  
Author(s):  
Rubén Cebrián ◽  
Mercedes Maqueda ◽  
José Luis Neira ◽  
Eva Valdivia ◽  
Manuel Martínez-Bueno ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Efficiency ◽  
Specific Protein ◽  
Culture Conditions ◽  
Site Directed Mutagenesis ◽  
Circular Structure ◽  
Cleavage Enzyme ◽  
Site Recognition ◽  
Biosynthetic Machinery

ABSTRACT AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81 His-1Ile ) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

scholarly journals Biochemical and molecular characterization of N66 from the shell ofPinctada mazatlanica

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7212
Author(s):  
Crisalejandra Rivera-Perez ◽  
Catalina Magallanes-Dominguez ◽  
Rosa Virginia Dominguez-Beltran ◽  
Josafat Jehu Ojeda-Ramirez de Areyano ◽  
Norma Y. Hernandez-Saavedra
Keyword(s):  
Posttranslational Modifications ◽  
Protein A ◽  
Pearl Oyster ◽  
Peptide Sequence ◽  
Calcium Carbonate Precipitation ◽  
Base Pairs ◽  
Shell Matrix ◽  
Glycosylation Sites ◽  
Shell Matrix Proteins

Mollusk shell mineralization is a tightly controlled process made by shell matrix proteins (SMPs). However, the study of SMPs has been limited to a few model species. In this study, the N66 mRNA of the pearl oysterPinctada mazatlanicawas cloned and functionally characterized. The full sequence of the N66 mRNA comprises 1,766 base pairs, and encodes one N66 protein. A sequence analysis revealed that N66 contained two carbonic anhydrase (CA) domains, a NG domain and several glycosylation sites. The sequence showed similarity to the CA VII but also with its homolog protein nacrein. The native N66 protein was isolated from the shell and identified by mass spectrometry, the peptide sequence matched to the nucleotide sequence obtained. Native N66 is a glycoprotein with a molecular mass of 60–66 kDa which displays CA activity and calcium carbonate precipitation ability in presence of different salts. Also, a recombinant form of N66 was produced inEscherichia coli, and functionally characterized. The recombinant N66 displayed higher CA activity and crystallization capability than the native N66, suggesting that the lack of posttranslational modifications in the recombinant N66 might modulate its activity.

scholarly journals N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation

2021 ◽  
Author(s):  
Benjamin H. Meyer ◽  
Ben A. Wagstaff ◽  
Panagiotis S. Adam ◽  
Sonja-Verena Albers ◽  
Helge C. Dorfmueller
Keyword(s):  
Posttranslational Modifications ◽  
Biochemical Characterization ◽  
Similar Process ◽  
List Type ◽  
Histidine Residue ◽  
Functional Importance ◽  
Domains Of Life ◽  
Distant Relation ◽  
Identification And Characterization

AbstractProtein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis.HighlightsFirst identification and characterization of a thermostable β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea.A highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24.Agl24-like homologs are broadly distributed among Archaea.The eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis.

scholarly journals Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS

2020 ◽  
Vol 19 (6) ◽  
pp. 1005-1016 ◽  
Author(s):  
James A. Wilkins ◽  
Krista Kaasik ◽  
Robert J. Chalkley ◽  
Alma L. Burlingame
Keyword(s):  
Cell Culture ◽  
Posttranslational Modifications ◽  
High Performance ◽  
Direct Approach ◽  
Metabolic Labeling ◽  
Tissue Samples ◽  
Mixed Disulfide ◽  
Cell Extracts ◽  
Prenylated Proteins

Posttranslational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction, and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labeling strategies in cell culture have been reported. However, their applicability is limited to cell culture systems and does not allow for analysis of tissue samples. The chemical characteristics (hydrophobicity, low abundance, highly basic charge) of many of the c-terminal regions of prenylated proteins have impaired the use of standard proteomic workflows. In this context, we sought a direct approach to the problem in order to examine these proteins in tissue without the use of labeling. Here we demonstrate that prenylated proteins can be captured on chromatographic resins functionalized with mixed disulfide functions. Protease treatment of resin-bound proteins using chymotryptic digestion revealed peptides from many known prenylated proteins. Exposure of the protease-treated resin to reducing agents and hydro organic mixtures released c-terminal peptides with intact prenyl groups along with other enzymatic modifications expected in this protein family. Database and search parameters were selected to allow for c-terminal modifications unique to these molecules such as CAAX box processing and c-terminal methylation. In summary, we present a direct approach to enrich and obtain information at a molecular level of detail about prenylation of proteins from tissue and cell extracts using high-performance LC-MS without the need for metabolic labeling and derivatization.

scholarly journals Identification of α1-Antitrypsin Variants in Plasma with the Use of Proteomic Technology

2001 ◽  
Vol 47 (11) ◽  
pp. 2012-2022 ◽  
Author(s):  
Kevin Mills ◽  
Philippa B Mills ◽  
Peter T Clayton ◽  
Andrew W Johnson ◽  
David B Whitehouse ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Metabolic Diseases ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complete Characterization ◽  
Proteomic Technology ◽  
Flight Mass Spectrometry ◽  
Type Ia ◽  
Specific Proteins ◽  
Polypeptide Structure

Abstract Background: Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins. Methods: We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. Results: Complete characterization of the polypeptide sequences and glycosylation of α1-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known α1-antitrypsin deficiencies and congenital disorder of glycosylation type Ia. Conclusions: This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.

scholarly journals Middle‐Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms

PROTEOMICS ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 1700442 ◽  
Author(s):  
Tingting Jiang ◽  
Michael E. Hoover ◽  
Matthew V. Holt ◽  
Michael A. Freitas ◽  
Alan G. Marshall ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Histone H4 ◽  
Cell Cycle Dependence ◽  
Cycle Dependence

scholarly journals Characterization of cellular oxidative stress response by stoichiometric redox proteomics

2020 ◽  
Author(s):  
Tong Zhang ◽  
Matthew J. Gaffrey ◽  
Xiaolu Li ◽  
Wei-Jun Qian
Keyword(s):  
Oxidative Stress ◽  
Posttranslational Modifications ◽  
Protein Function ◽  
Regulatory Networks ◽  
Total Oxidation ◽  
Redox Proteomics ◽  
Thiol Redox ◽  
Cysteine Thiols ◽  
In Cells

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including S-glutathionylation (SSG), S-nitrosylation (SNO), S-sulfenylation (SOH), and S-sulfhydration (SSH). These modifications can impact various aspects of protein function such as activity, binding, conformation, localization, and interactions with other molecules. To identify novel redox proteins in signaling and regulation, it is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs. Mass spectrometry (MS)-based redox proteomics has emerged as the primary platform for broad characterization of thiol PTMs in cells and tissues. Herein we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs at physiological or oxidative stress conditions and highlight some recent applications. Considering the relative maturity of available methods, emphasis will be on two types of modifications: 1) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and 2) S-glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives towards a better understanding of cellular redox regulatory networks in cells and tissues

scholarly journals Characterization of posttranslational modifications in neuron-specific class III beta-tubulin by mass spectrometry.

1991 ◽  
Vol 88 (11) ◽  
pp. 4685-4689 ◽  
Author(s):  
J. E. Alexander ◽  
D. F. Hunt ◽  
M. K. Lee ◽  
J. Shabanowitz ◽  
H. Michel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Specific Class ◽  
Class Iii ◽  
Beta Tubulin
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