Peptide Mass Fingerprinting after Less Specific In-Gel Proteolysis Using MALDI-LTQ-Orbitrap and 4-Chloro-α-cyanocinnamic Acid

2010 ◽  
Vol 9 (5) ◽  
pp. 2619-2629 ◽  
Author(s):  
Dimitrios G. Papasotiriou ◽  
Thorsten W. Jaskolla ◽  
Stavroula Markoutsa ◽  
Dominic Baeumlisberger ◽  
Michael Karas ◽  
...  
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Peptide Mass ◽  
Cyanocinnamic Acid

Proteomic Analysis of Medicinal Plant Calotropis Gigantea by insilico Peptide Mass Fingerprinting

2020 ◽  
Vol 16 ◽  
Author(s):  
Saad Ur Rehman ◽  
Muhammad Rizwan ◽  
Sajid Khan ◽  
Azhar Mehmood ◽  
Anum Munir
Keyword(s):  
Medicinal Plant ◽  
Structure Prediction ◽  
3D Structure ◽  
Peptide Mass Fingerprinting ◽  
Protein Docking ◽  
Receptor Protein ◽  
Human Leukemia ◽  
Protein Database ◽  
Calotropis Gigantea ◽  
Peptide Mass

: Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. Background: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in a database and identified. Method: The calculation of insilico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from MASCOT online server. Anticancer probability is calculated from the iACP server, docking of active peptides is done by CABS-dock the server. Objective: The purpose of the study is to identify the peptides having anti-cancerous properties by in-silico peptide mass fingerprinting. Results : The anti-cancerous peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the Active chain, the human leukemia protein docking with peptides shows the accurate binding. Conclusion : These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

scholarly journals The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin

2019 ◽  
Vol 7 (9) ◽  
pp. 313 ◽  
Author(s):  
Pamela Ruiz ◽  
Miguel Balado ◽  
Juan Carlos Fuentes-Monteverde ◽  
Alicia E. Toranzo ◽  
Jaime Rodríguez ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Iron Uptake ◽  
Peptide Mass Fingerprinting ◽  
Iron Limitation ◽  
Fish Pathogen ◽  
Fish Pathogens ◽  
Iron Deprivation ◽  
Peptide Mass ◽  
Photobacterium Damselae ◽  
Vibrio Ordalii

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

scholarly journals Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2625
Author(s):  
Muzammeer Mansor ◽  
Jameel R. Al-Obaidi ◽  
Nurain Nadiah Jaafar ◽  
Intan Hakimah Ismail ◽  
Atiqah Farah Zakaria ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Chloroform Solution ◽  
Milk Proteins ◽  
Peptide Mass Fingerprinting ◽  
Extraction Methods ◽  
Dairy Goats ◽  
Goat’S Milk ◽  
Peptide Mass ◽  
Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

scholarly journals Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears

2002 ◽  
Vol 366 (2) ◽  
pp. 511-520 ◽  
Author(s):  
Benjamin L. SCHULZ ◽  
David OXLEY ◽  
Nicolle H. PACKER ◽  
Niclas G. KARLSSON
Keyword(s):  
Molecular Mass ◽  
Peptide Mass Fingerprinting ◽  
High Molecular Mass ◽  
Tear Fluid ◽  
Protein Variant ◽  
Peptide Mass ◽  
Composite Gel ◽  
Molecular Masses ◽  
Protein Components ◽  
Sialyl Lea

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

scholarly journals Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting

PROTEOMICS ◽  
2002 ◽  
Vol 2 (2) ◽  
pp. 157-163 ◽  
Author(s):  
Julie M. Pratt ◽  
Duncan H. L. Robertson ◽  
Simon J. Gaskell ◽  
Isabel Riba-Garcia ◽  
Simon J. Hubbard ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Protein Identification ◽  
Peptide Mass Fingerprinting ◽  
Stable Isotope Labelling ◽  
Isotope Labelling ◽  
Peptide Mass

scholarly journals The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 923-935 ◽  
Author(s):  
Nicole Hansmeier ◽  
Andreas Albersmeier ◽  
Andreas Tauch ◽  
Thomas Damberg ◽  
Robert Ros ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
Direct Repeat ◽  
Peptide Mass Fingerprinting ◽  
Gene Encoding ◽  
Force Microscopy ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Rapid Amplification

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

scholarly journals A Novel Bipartite Double-Stranded RNA Mycovirus from the White Root Rot Fungus Rosellinia necatrix: Molecular and Biological Characterization, Taxonomic Considerations, and Potential for Biological Control

2009 ◽  
Vol 83 (24) ◽  
pp. 12801-12812 ◽  
Author(s):  
Sotaro Chiba ◽  
Lakha Salaipeth ◽  
Yu-Hsin Lin ◽  
Atsuko Sasaki ◽  
Satoko Kanematsu ◽  
...  
Keyword(s):  
Biological Control ◽  
Root Rot ◽  
Sequence Similarity ◽  
Peptide Mass Fingerprinting ◽  
Dsrna Virus ◽  
Rosellinia Necatrix ◽  
Virus Family ◽  
White Root Rot ◽  
Double Stranded Rna ◽  
Peptide Mass

ABSTRACT White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5′ (24 nt) and 3′ (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5′ untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3′-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ∼50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

OLAV-PMF:  A Novel Scoring Scheme for High-Throughput Peptide Mass Fingerprinting

2004 ◽  
Vol 3 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Jérôme Magnin ◽  
Alexandre Masselot ◽  
Christoph Menzel ◽  
Jacques Colinge
Keyword(s):  
High Throughput ◽  
Peptide Mass Fingerprinting ◽  
Peptide Mass
Sign in / Sign up

Export Citation Format

Share Document