After 8 h of incubation, LAESI mass spectra were recorded for each sample. ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Obtaining other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylinCcopper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the -hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the CHSSNNC residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of -hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of -sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylinCcopper(II) interactions demonstrates the power of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors Macbecin I of amyloid protein aggregation. Noncovalent supramolecular self-assembly of proteins and other biological macromolecules is an essential event in many important biochemical and cellular processes, such as cell signaling, cell growth and differentiation, cell-to-cell adhesion, and inflammation.1 The characterization of proteinCligand interactions is needed for understanding certain biochemical processes and the structural properties Ptgfr of protein complexes.1C3 Finding inhibitors of protein complex formation or aggregation, a common task in the screening of potential pharmaceuticals, requires information around the affinity and binding selectivity between numerous drug candidates and their protein targets. Islet amyloid polypeptide (IAPP) or amylin, a 37-residue polypeptide, cosecreted with insulin from pancreatic -cells, functions as a partner to insulin in regulating glucose homeostasis.4C7 Under normal physiological conditions, amylin exists as a soluble monomer in a random coil state. In type 2 diabetes mellitus (T2DM), however, this peptide is usually a major component of amyloid plaques in the pancreatic tissue of patients.8C10 High resolution electron and atomic force microscopy revealed that amylin can aggregate into insoluble -pleated IAPP amyloid fibrils in a time and concentration dependent manner.11C13 CD spectroscopy studies demonstrated that amylin self-association into oligomers and fibrils is strongly conformation dependent, and it is triggered by the transition of the peptide from random coil to predominantly -sheet conformation.11,12,14 The viability assays further uncover that amylin fibril formation always accompanies amylin toxicity.15 Such findings contribute to the hypothesis that the formation of toxic amylin fibrils induces -cell death, resulting in the relative insulin deficiency in type 2 diabetes.6,15,16 Ion mobility separation (IMS) and molecular modeling studies reveal that fibril formation and the aggregation of amylin are conformation dependent.17 Nuclear magnetic resonance (NMR) measurements on this Macbecin I peptide in a membrane environment show an -helix conformation for the core residues,18,19 whereas the synthesized amylin fibrils exhibit parallel -sheet layers with the formation of steric zipper, a structural moiety common to all aggregating amyloid polypeptides.20,21 Recent studies have found that the presence of divalent copper ions reduces the cellular toxicity of hIAPP.22 This is thought to be related to the inhibition of amylin aggregation by Cu(II) ions.22C24 Copper is involved in the pathological mechanisms of certain neurodegenerative diseases, and its role has been extensively studied for amyloidogenic proteins in Alzheimer’s, Parkinson’s, and prion-protein diseases.25 However, the properties of amylinCcopper interactions, the effect of amylin conformation on its interaction with Cu(II), and the conformational changes of amylin induced by Cu(II) remain relatively unknown. Various established analytical techniques, such as fluorescence,26 surface plasmon resonance spectroscopy,27 and UVCvisible spectroscopy28,29 have been employed to identify and quantitatively assess the noncovalent peptideCprotein interactions. With high sensitivity, accuracy, and speed, mass spectrometry (MS) plays an important role in observing peptideCligand structures.30 Extensive Macbecin I reviews indicate that MS has an increasing role in gauging the composition, conformation, binding strength, and dissociation constants of noncovalent interactions.1,31C33 Native electrospray ionization (ESI) is a widely used MS technique for studying proteinCligand interactions that offers the advantages of high sensitivity, specificity, and unprecedented accuracy for the mass of the complex.1,2,30,34 However, there is an ongoing debate on the relationship between the gas phase complex ion and the corresponding species found in the solution phase. Other concerns include the need for more physiological Macbecin I buffers (most studies use ammonium acetate), the interfering effect of other macromolecules in the system, and general throughput limitations related to the need for buffer exchange that takes 1 to 2 2 h.1,35,36 Although ESI arrays can accelerate the measurement, lengthy buffer exchange is still required. As experiments often involve titration assays to determine binding constants, a rapid method.