Characterization of Tertiary Structure States and Specific Noncovalent Complexes of Proteins by UV-Matrix-Assisted Laser-Desorption / Ionization Mass Spectrometry

摘要: Hydrogen/deuterium exchange reactions (H/D exchange) in solution have been used for probing protein structures and dynamics monitored using a broad spectrum of analytical methods including mass spectrometry. In order to study the effects Matrix-Assisted Laser-Desorption/Ionization Mass Spectrometry (MALDIMS) preparation techniques on structures, we applied on-target H/D reactions. Samples were prepared MALDI-MS by mixing aqueous peptide solutions with acidic matrix (pH 2) that contained organic cosolvents. labile protons peptides proteins this manner was found be > 90%, indicating denatured. By subsequent laser irradiation one single spot MALDI-target, depth-profiles deposited matrix/sample mixtures recorded revealed provide only top crystal layers. deeper layers molecular ions unexchanged hydrogens observed. No intermediate detected. contrast, results from H/D-exchange in-solution, which electrospray/ionization spectrometry (ESI-MS), consistent folded globular structures. Subsequently, conditions sample preparations altered substantially. 6aza-2-thiothymine (ATT) as water soluble dissolved 5mM ammonium citrate 5.5). Proteins buffers such bicarbonate mixed solution. Under these non-covalent complexes could analyzed specificity complex formation ascertained admixture non-specific did not yield detectable aggregate ions. Also, trypsin maintained its active structure presence matrix, shown digests after UV-MALDI-MS weight analyses.