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001-es BibID:	BIBFORM090030
Első szerző:	Révész Ágnes
Cím:	Tailoring to Search Engines : bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence / Ágnes Révész, Márton Gyula Milley, Kinga Nagy, Dániel Szabó, Gergő Kalló, Éva Csősz, Károly Vékey, László Drahos
Dátum:	2021
ISSN:	1535-3893
Megjegyzések:	Bottom-up proteomics relies on identification of peptides from tandem mass spectra, usually via matching against sequence databases. Confidence in a peptide-spectrum match can be characterized by a score value given by the database search engines, and it depends on the information content and the quality of the spectrum. The latter are influenced by experimental parameters, of which the collision energy is the most important one in the case of collision-induced dissociation. We examined how the identification score of the Byonic and Andromeda (MaxQuant) engines varies with collision energy for more than a thousand individual peptides from a HeLa tryptic digest on a QTof instrument. We thereby extended our earlier study on Mascot scores and corroborated its findings on the potential bimodal nature of this energy dependence. Optimal energies as a function of m/z show comparable linear trends for the three engines. On the basis of peptide-level results, we designed methods with one or two liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs and various collision energy settings and assessed their practical performance in peptide and protein identification from the HeLa standard sample. A 10-40% gain in various measures, such as the number of identified proteins or sequence coverage, was obtained over the factory default settings. Best performing methods differ for the three engines, suggesting that the experimental parameters should be fine-tuned to the choice of the engine. We also recommend a simple approach and provide reference data to ease the transfer of the optimized methods to other mass spectrometers relevant for proteomics. We demonstrate the utility of this approach on an Orbitrap instrument. Data sets can be accessed via the MassIVE repository (MSV000086379).
Tárgyszavak:	Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk bottom-up proteomics collision energy optimization database search
Megjelenés:	Journal Of Proteome Research. - 20 : 1 (2021), p. 474-484. -
További szerzők:	Milley Márton Gyula Nagy Kinga (1988-) (agrár) Szabó Dániel (1986-) (gépészmérnök) Kalló Gergő (1989-) (molekuláris biológus) Csősz Éva (1977-) (biokémikus, molekuláris biológus) Vékey Károly Drahos László
Pályázati támogatás:	GINOP-2.3.3-15-2016-00020 GINOP NKFIH PD-132135 Egyéb NKFIH K 131762 Egyéb
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001-es BibID:	BIBFORM069265
Első szerző:	Wang, Dongdong
Cím:	Antigen Identification and Characterization of Lung Cancer Specific Monoclonal Antibodies Produced by mAb Proteomics / Wang Dongdong, Hincapie Marina, Guergova-Kuras Mariana, Kadas Janos, Takacs Laszlo, Karger Barry L.
Dátum:	2010
ISSN:	1535-3893
Megjegyzések:	A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globallyproduced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of nativeglycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), togenerate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma wassubjected to 3-sequential steps of affinity fractionation, including high abundant plasma proteindepletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization.In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs ofhigh differentiating power against a matched, pooled normal plasma sample. After production of largequantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification,SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to becomplement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the globalstrategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a KD ofroughly 10-9 M and to be 2 orders of magnitude lower than the reduced form, demonstrating thepower of the mAb proteomics technology in generating mAbs to the natural form of the proteinsin blood. The binding of this mAb to the -chain of haptoglobin was also dependent on glycosylationon this chain. The characterization of mAbs in this work reveals that the global mAb proteomicsprocess can generate high-quality lung cancer specific mAbs capable of recognizing proteins intheir native state.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban monoclonal antibody mAb proteomics antibody characterization glycoprotein haptoglobin conformational epitope lung cancer
Megjelenés:	Journal Of Proteome Research. - 9 : 4 (2010), p. 1834-1842. -
További szerzők:	Hincapie, Marina Guergova-Kuras, Mariana Kádas János (1976-) (molekuláris biológus, biokémikus, kertészmérnök) Takács László (1955-) Karger, Barry
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