Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

Lam, Hoang-Trang; Josserand, Jacques; Lion, Niels; Girault, Hubert H.

doi:10.1021/pr0606023

Cited by 38 publications

(36 citation statements)

References 49 publications

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“…A threshold of 5% probability (p Ͻ 0.05) of a false positive was used for all Mascot searches, and a decoy database was used in the peptide OGE searches to estimate a false positive rate. Searches were conducted on a custom-built database consisting of 138,151 A. caninum DNA sequences (effective database size, 828,906 sequences) deposited in the NCBI databases as of November 19,2007. Single peptide identifications were verified manually, and acceptable spectra were manually annotated.…”

Section: Methodsmentioning

confidence: 99%

“…Although a very useful approach, such studies are labor-intensive and relatively insensitive because of the extensive processing of gel fragments. The relatively new technique of OGE (17)(18)(19) focuses peptides or proteins according to their isoelectric point. Focusing in this way allows for relatively large scale fractionations and reduces the processing required before mass spectrometry.…”

mentioning

confidence: 99%

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Proteomics Analysis of the Excretory/Secretory Component of the Blood-feeding Stage of the Hookworm, Ancylostoma caninum

Mulvenna

Hamilton

Nagaraj

et al. 2009

Molecular & Cellular Proteomics

163

166

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Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting ϳ600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three ␤-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/ PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Proteomics Analysis of the Excretory/Secretory Component of the Blood-feeding Stage of the Hookworm, Ancylostoma caninum

Mulvenna

Hamilton

Nagaraj

et al. 2009

Molecular & Cellular Proteomics

163

166

View full text Add to dashboard Cite

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“…34 Modeling of two proteins with ten charged states under an electric field of 30,000 V/m within a pH range of 6-9 was done by Shim et al 33 IEF of three peptides: leucine enkephalin, angiotensin II, and a peptide from the human proteome digestion were modeled in an IPG gel with pH gradient. 24 The peptide charge slope at the isoelectric point was determined to be an important parameter in focusing dynamics to determine the optimal separation-well width for best focusing.…”

Section: Introductionmentioning

confidence: 99%

Modeling the Electromobility of Type-I Collagen Molecules in the Electrochemical Fabrication of Dense and Aligned Tissue Constructs

Uquillas

Akkuş

2012

Ann Biomed Eng

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Isoelectric focusing (IEF) of type-I collagen molecules is a technology with proven efficacy to produce dense and aligned collagen-based biomaterials. The forces and mechanisms during IEF of collagen molecules in carrier ampholyte-free environments remain unknown. This study presents theoretical framework describing the congregation of collagen molecules along the isoelectric point (pI). A single molecule was modeled as a rod-like particle, distributed homogeneously between parallel electrodes. Upon application of electrical current, molecules migrated to the pI. The results showed that self-aggregation of collagen molecules along the pI occurred due to formation of a non-linear pH gradient that rendered the anodic side acidic, and the cathodic side basic. This pH profile and the amphoteric nature of collagen resulted in positively charged molecules at the anode and negatively charged molecules at the cathode. Therefore, repulsive electrostatic forces aided self-aggregation of molecules along the pI. The model could effectively validate the pI of collagen, the pI location, and predict that the instantaneous velocity acting on a molecule at the anode was higher than those velocities at the cathode. This fundamental information represents the baseline theory upon which we can expand our knowledge to the production of biomaterials to engineer soft tissues.

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“…Isoelectric Focusing (IEF) Using the OFFGEL System-IEF of cross-linked A␤ 1-40 was carried out as described before (26,27). Briefly, peptide samples were desalted and concentrated by protein phase separation and precipitation as described previously (28).…”

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confidence: 99%

Tissue Transglutaminase-mediated Glutamine Deamidation of β-Amyloid Peptide Increases Peptide Solubility, Whereas Enzymatic Cross-linking and Peptide Fragmentation May Serve as Molecular Triggers for Rapid Peptide Aggregation

Schmid

Condemi

Tuchscherer

et al. 2011

Journal of Biological Chemistry

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Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGasemediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of ␤-amyloid peptide (A␤), resulting in structural changes of monomeric A␤. Using high resolution mass spectrometry (MS) of cross-linked A␤ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH 3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of A␤ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGasemediated A␤ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of A␤ 1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of A␤ peptide may serve as an important seed for aggregation.

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Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

Cited by 38 publications

References 49 publications

Proteomics Analysis of the Excretory/Secretory Component of the Blood-feeding Stage of the Hookworm, Ancylostoma caninum

Proteomics Analysis of the Excretory/Secretory Component of the Blood-feeding Stage of the Hookworm, Ancylostoma caninum

Modeling the Electromobility of Type-I Collagen Molecules in the Electrochemical Fabrication of Dense and Aligned Tissue Constructs

Tissue Transglutaminase-mediated Glutamine Deamidation of β-Amyloid Peptide Increases Peptide Solubility, Whereas Enzymatic Cross-linking and Peptide Fragmentation May Serve as Molecular Triggers for Rapid Peptide Aggregation

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