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Cellular functions are performed and regulated at a molecular level by the coordinated action of intricate protein assemblies, and hence the study of protein folding, structure, and interactions is vital to the appreciation and understanding of complex biological problems. In the past decade, continued development of chemical cross-linking methodologies combined with mass spectrometry has seen this approach develop to enable detailed structural information to be elucidated for protein assemblies often intractable by traditional structural biology methods. In this review article, we describe recent advances in reagent design, cross-linking protocols, mass spectrometric analysis, and incorporation of cross-linking constraints into structural models, which are contributing to overcoming the intrinsic challenges of the cross-linking method. We also highlight pioneering applications of chemical cross-linking mass spectrometry approaches to the study of structure and function of protein assemblies.

We report the decomposition of curcumin due to reduction of Cu(ii) to Cu(i). Cu(ii) binds tightly with curcumin to form a complex which exhibits a high stability in methanol, but it decomposes readily in acetonitrile and in SDS micelles in the presence of ascorbic acid, coincident with reduction of Cu(ii) to Cu(i). In this study, the UV-Vis absorption of the Cu-curcumin complex shows a monotonic decrease as a function of time, consistent with the decomposition of curcumin. At a high copper : curcumin molar ratio of 10 : 1, the UV-Vis absorption spectrum of the Cu(ii)-curcumin complex in acetonitrile exhibits a substantial blue shift of the absorption maximum from 420 nm to 350 nm, which is indicative of a significant decrease in conjugation length of curcumin in the presence of Cu(ii). Time-dependent mass spectrometry and high performance liquid chromatography (HPLC) data are also consistent with the decomposition of curcumin as a consequence of reduction of Cu(ii) to Cu(i).

This study demonstrates the ability of travelling wave ion mobility-mass spectrometry to measure collision cross-sections of ions in the negative mode, using a calibration based approach. Here, negative mode ion mobility-mass spectrometry was utilised to understand structural transitions of calmodulin upon Ca2+ binding and complexation with model peptides melittin and the plasma membrane Ca2+ pump C20W peptide. Coexisting calmodulin conformers were distinguished on the basis of their mass and cross-section, and identified as relatively folded and unfolded populations, with good agreement in collision cross-section to known calmodulin geometries. Titration of calcium tartrate to physiologically relevant Ca2+ levels provided evidence for intermediately metalated species during the transition from apo- to holo-calmodulin, with collision cross-section measurements indicating that higher Ca2+ occupancy is correlated with more compact structures. The binding of two representative peptides which exemplify canonical compact (melittin) and extended (C20W) peptide-calmodulin binding models has also been interrogated by ion mobility mass spectrometry. Peptide binding to calmodulin involves intermediates with metalation states from 1–4 Ca2+, which demonstrate relatively collapsed structures, suggesting neither the existence of holo-calmodulin or a pre-folded calmodulin conformation is a prerequisite for binding target peptides or proteins. The biological importance of the different metal unsaturated calmodulin complexes, if any, is yet to be understood.

Ion mobility-mass spectrometry (IM-MS) is emerging as an important biophysical technique for the structural analysis of proteins and their assemblies, in particular for structurally heterogeneous systems such as those on the protein misfolding and aggregation pathway. Using IM-MS we have monitored amyloid fibril formation of A53T α-synuclein, a mutant synuclein protein associated with Parkinson’s disease, and identified that a conformational change towards a more compact structure occurs during the initial stages of aggregation. Binding of A53T α-synuclein to a flavenoid based amyloid fibril inhibitor, (–)-epigallocatechin-3-gallate, has been observed with a 1:1 stoichiometry. By analysis of ion collision cross-sections, we show epigallocatechin gallate binding prevents protein conformational change, and in turn decreases the formation of fibrillar aggregates.

Knowledge of protein structure and protein–protein interactions is vital for appreciating the elaborate biochemical pathways that underlie cellular function. While many techniques exist to probe the structure and complex interplay between functional proteins, none currently offer a complete picture. Mass spectrometry and associated methods provide complementary information to established structural biology tools, and with rapidly evolving technological advances, can in some cases even exceed other techniques by its diversity in application and information content. This is primarily because of the ability of mass spectrometry to precisely identify protein complex stoichiometry, detect individual species present in a mixture, and concomitantly offer conformational information. This review describes the attributes of mass spectrometry for the structural investigation of multiprotein assemblies in the context of recent developments and highlights in the field.

The host defence peptides from the skin secretions of the Australian Glandular Frog (Litoria subglandulosa) are similar to those of the closely related species Litoria citropa. Both species produce several potent caerulein neuropeptides and antimicrobial- and anticancer-active citropin peptides. The major neuropeptides from Litoria subglandulosa are caerulein 1.1 [pGlu Gln Asp Tyr(SO3) Thr Gly Trp Met Asp Phe–NH2], caerulein 1.2 [pGlu Gln Asp Tyr(SO3) Thr Gly Trp Phe Asp Phe–NH2], and caerulein 2.1 [pGlu Gln Asp Tyr(SO3) Thr Gly Ala His Met Phe–NH2], all of which are smooth muscle active. The major peptide, citropin 1.2 [Gly Leu Phe Asp Ile Ile Lys Lys Val Ala Ser Val Val Gly Gly Leu–NH2], is a wide-spectrum antibiotic and anticancer agent at the micromolar concentration. Citropin 1.2 also inhibits the formation of nitric oxide by the enzyme neuronal nitric oxide synthase (nNOS) at the micromolar concentration. Another peptide, citropin 2.2 [Gly Leu Ile Ser Ile Gly Lys Ala Leu Gly Gly Leu Ile Val Asp Val Leu Lys Pro Lys Ser–OH], also inhibits nNOS.