title: Amide hydrogen/deuterium exchange & MALDI-TOF mass spectrometry analysis of Pak2 activation.
abstract: Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry(1,2,3). The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein(4). H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection(2), instead of a HPLC/ESI (electrospray ionization)-MS system(5,6). The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A(2;) (sPLA(2;)). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation(7,8,9).
authors.affiliation: Department of Chemistry, Tunghai University. howardhsu@thu.edu.tw
authors.forename: Yuan-Hao
authors.initials: YH
authors.lastname: Hsu
authors.forename: Jolinda A
authors.initials: JA
authors.lastname: Traugh
mesh_terms: D000577:Amides / Q000737:chemistry*; D053148:Caspase 3 / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D054462:p21-Activated Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000577:Amides; D054462:p21-Activated Kinases; D053148:Caspase 3