PXD016024-1

PXD016024 is an original dataset announced via ProteomeXchange.

Title	Intact Transition Epitope Mapping- Thermodynamic Weak-force Order (ITEM-TWO)
Description	Most important characteristics of antibodies are that they typically strongly bind to specific epitopes, thereby expressing low dissociation constants (KDs) and high Gibbs free binding energies (delta Gs). In this study, we describe an off-line nano-electrospray mass spectrometry method, termed ITEM-TWO, which enables one to simultaneously identify epitopes and obtain characteristic gas phase dissociation constants of antibody - epitope interactions in a single experiment. To validate the method, we characterize the interaction between an antiHistag antibody and its epitope peptide obtained from a tryptic digest of a His-tag containing beta actin. In a mixture of solution 1 (tryptic digest of a His-tag containing recombinant human beta-actin protein in 30 mM ammonium bicarbonate) and solution 2 (antiHis-tag antibody in 200 mM ammonium acetate) the specific immune complexes form (molar ratio 2.2 : 1). Without any purification steps this mixture (solution 3) is then electrosprayed. With the aid of a quadrupole ion filter, the immune complex ions are separated from unbound peptide ions. Increasing the voltage difference (DeltaCV) in the subsequent collision cell results in collision induced dissociation (CID) by which the epitope peptide(s) is(are) released from the immune complex. The mass(es) of the complex-released peptide(s) is(are) then measured in a ToF analyzer by which the epitope is identified. A step-wise increase in DeltaCV allows the simultaneous determination of the intensities of (i) the surviving ionized immune complexes together with (ii) released epitope peptide ions plus (iii) the left behind antibody ions. From the ions´ normalized intensity ratios are deduced the apparent gas phase dissociation constants (KD#m0g) and the apparent dissociation energies over temperature values (delta G#m0g / T) of the gas phase dissociation processes.
HostingRepository	PRIDE
AnnounceDate	2019-11-07
AnnouncementXML	Submission_2019-11-07_00:31:28.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Michael Kreutzer
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q-Tof Ultima; Synapt MS

Revision	Datetime	Status	ChangeLog Entry
0	2019-10-25 04:33:09	ID requested
⏵ 1	2019-11-07 00:46:11	announced

Danquah BD, Yefremova Y, Opuni KFM, R, ö, wer C, Koy C, Glocker MO, Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO). J Proteomics, 212():103572(2020) [pubmed]

submitter keyword: antibody specificity determination, antibody affinity determination

Prof. Dr. Michael O. Glocker
contact affiliation	Proteome Center Rostock Department for Proteome Research Institute of Immunology Medical Faculty and Natural Science Faculty University of Rostock Schillingallee 69 18057 Rostock Germany
contact email	michael.glocker@med.uni-rostock.de
lab head
Michael Kreutzer
contact affiliation	University Medical Center Rostock
contact email	michael.kreutzer@med.uni-rostock.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/11/PXD016024

PRIDE project URI

• PRIDE
  1. PXD016024
     1. Label: PRIDE project
     2. Name: Intact Transition Epitope Mapping- Thermodynamic Weak-force Order (ITEM-TWO)