scholarly journals In Silico Proteome Cleavage Reveals Iterative Digestion Strategy for High Sequence Coverage

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jesse G. Meyer
Keyword(s):  
Posttranslational Modifications ◽  
Rna Splicing ◽  
Shotgun Proteomics ◽  
Peptide Sequence ◽  
Sequence Coverage ◽  
Proteome Coverage ◽  
Protein Inference ◽  
Peptide Length ◽  
Splicing Variants

In the postgenome era, biologists have sought to measure the complete complement of proteins, termed proteomics. Currently, the most effective method to measure the proteome is with shotgun, or bottom-up, proteomics, in which the proteome is digested into peptides that are identified followed by protein inference. Despite continuous improvements to all steps of the shotgun proteomics workflow, observed proteome coverage is often low; some proteins are identified by a single peptide sequence. Complete proteome sequence coverage would allow comprehensive characterization of RNA splicing variants and all posttranslational modifications, which would drastically improve the accuracy of biological models. There are many reasons for the sequence coverage deficit, but ultimately peptide length determines sequence observability. Peptides that are too short are lost because they match many protein sequences and their true origin is ambiguous. The maximum observable peptide length is determined by several analytical challenges. This paper explores computationally how peptide lengths produced from several common proteome digestion methods limit observable proteome coverage. Iterative proteome cleavage strategies are also explored. These simulations reveal that maximized proteome coverage can be achieved by use of an iterative digestion protocol involving multiple proteases and chemical cleavages that theoretically allow 92.9% proteome coverage.

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 Cloning and Characterization of Multiple RNA Splicing Variants of LDH-C Gene in Human and Rat

2013 ◽  
Vol 5 (4) ◽  
pp. 182-189
Author(s):  
Qinglian Zhang ◽  
Min Yang ◽  
Jiagui Jin ◽  
Qinghua He ◽  
Jinhu Ma
Keyword(s):  
Rna Splicing ◽  
Splicing Variants

scholarly journals Splicing variants impact in thyroid normal physiology and pathological conditions

2009 ◽  
Vol 53 (6) ◽  
pp. 709-715 ◽  
Author(s):  
Elizabete Rosária de Miranda ◽  
Luiz De Marco ◽  
Maria Marta Sarquis Soares
Keyword(s):  
Rna Splicing ◽  
Protein Localization ◽  
Mrna Translation ◽  
Peptide Sequence ◽  
Disease States ◽  
Pathological Conditions ◽  
Splicing Variants ◽  
Localization Errors ◽  
And Function ◽  
Splicing Patterns

RNA splicing is an essential, precisely regulated process that occurs after gene transcription and before mRNA translation, in which introns may be removed and exons, retained. Variability in splicing patterns is a major source of protein diversity from the genome and function to generate a tremendously diverse proteome from a relatively small number of genes. Changes in splice site choice can determine different effects on the encoded protein. Small changes in peptide sequence can alter ligand binding, enzymatic activity, allosteric regulation, or protein localization. Errors in splicing regulation have been implicated in a number of different disease states. This study reviewed the mechanisms of splicing and their repercussion in endocrinology, emphasizing its importance in some thyroid physiological and pathological conditions.

scholarly journals A protein standard that emulates homology for the characterization of protein inference algorithms

2017 ◽  
Author(s):  
Matthew The ◽  
Fredrik Edfors ◽  
Yasset Perez-Riverol ◽  
Samuel H. Payne ◽  
Michael R. Hoopmann ◽  
...  
Keyword(s):  
Shotgun Proteomics ◽  
Tryptic Peptides ◽  
Protein Fragments ◽  
E Coli ◽  
Protein Inference ◽  
Inference Algorithms ◽  
False Sense ◽  
Inference Methods ◽  
Natural Way

AbstractA natural way to benchmark the performance of an analytical experimental setup is to use samples of known content, and see to what degree one can correctly infer the content of such a sample from the data. For shotgun proteomics, one of the inherent problems of interpreting data is that the measured analytes are peptides and not the actual proteins themselves. As some proteins share proteolytic peptides, there might be more than one possible causative set of proteins resulting in a given set of peptides and there is a need for mechanisms that infer proteins from lists of detected peptides. A weakness of commercially available samples of known content is that they consist of proteins that are deliberately selected for producing tryptic peptides that are unique to a single protein. Unfortunately, such samples do not expose any complications in protein inference. For a realistic benchmark of protein inference procedures, there is, therefore, a need for samples of known content where the present proteins share peptides with known absent proteins. Here, we present such a standard, that is based on E. coli expressed human protein fragments. To illustrate the usage of this standard, we benchmark a set of different protein inference procedures on the data. We observe that inference procedures excluding shared peptides provide more accurate estimates of errors compared to methods that include information from shared peptides, while still giving a reasonable performance in terms of the number of identified proteins. We also demonstrate that using a sample of known protein content without proteins with shared tryptic peptides can give a false sense of accuracy for many protein inference methods.

scholarly journals Optimization of filtering criterion for SEQUEST database searching to improve proteome coverage in shotgun proteomics

2007 ◽  
Vol 8 (1) ◽  
pp. 323 ◽  
Author(s):  
Xinning Jiang ◽  
Xiaogang Jiang ◽  
Guanghui Han ◽  
Mingliang Ye ◽  
Hanfa Zou
Keyword(s):  
Shotgun Proteomics ◽  
Database Searching ◽  
Proteome Coverage

scholarly journals Fabrication and characterization of hydrogels formed from designer coiled-coil fibril-forming peptides

RSC Advances ◽  
2017 ◽  
Vol 7 (44) ◽  
pp. 27260-27271 ◽  
Author(s):  
A. F. Dexter ◽  
N. L. Fletcher ◽  
R. G. Creasey ◽  
F. Filardo ◽  
M. W. Boehm ◽  
...  
Keyword(s):  
Carbonic Acid ◽  
Coiled Coil ◽  
Peptide Sequence ◽  
Fabrication And Characterization

A peptide sequence was designed to form α-helical fibrils and hydrogels at physiological pH, utilising transient buffering by carbonic acid.

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.

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
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