The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD-diheme dissociation is far slower than monoheme dissociation at ∼0.12 min–1 and ∼0.25 s–1, respectively, indicating that MhuD has a strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

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In this study we explore the use of collision cross section distributions to allow comparability of IM-MS data for proteins on different instruments. We present measurements on seven standard proteins across three IM-MS configurations, namely an Agilent 6560 IM QToF, a Waters Synapt G2 possessing a TWIMS cell and a modified Synapt G2 possessing an RF confining linear field drift cell. Mobility measurements were taken using both He and N₂ as the drift gases. To aid comparability across instruments and best assess the corresponding gas-phase conformational landscapes of the protein ‘standards’ we present the data in the form of averaged collision cross section distributions.

Globally, food is a multi-trillion-pound industry for which proteomic analysis represents a key tool in ensuring that consumer health and rights are maintained. Here we use native mass spectrometry methodology to analyse a series of natural food products of varying complexity, namely: cow milk (liquid); chicken egg white (viscous liquid) and jack bean meal (solid). Our approach permits rapid detection (~5-30 mins) and unambiguous identification of the majority (>80%) of proteins present within milk and egg white, which are foodstuffs that between them comprise two of the most prominent sources of allergenic proteins within the food industry. Furthermore, we show that this method also enables the retention of bioactive protein complexes directly from natural sources, exemplified by the detection of three multimeric states (monomer, dimer and tetramer) of concanavalin A, naturally found in jack beans (Canavalia ensiformis). As such, we propose that native mass spectrometry methods can augment the current bottom up proteomic toolkit employed within food analyses and may prove useful for fast detection and high accuracy identification of suspected proteinaceous allergens/adulterants within sufficiently noncomplex food substances.

In this study we explore the use of collision cross section distributions to allow comparability of IM-MS data for proteins on different instruments. We present measurements on seven standard proteins across three IM-MS configurations, namely an Agilent 6560 IM QToF, a Waters Synapt G2 possessing a TWIMS cell and a modified Synapt G2 possessing an RF confining linear field drift cell. Mobility measurements were taken using both He and N₂ as the drift gases. To aid comparability across instruments and best assess the corresponding gas-phase conformational landscapes of the protein ‘standards’ we present the data in the form of averaged collision cross section distributions.