An analysis of exhaled breath enables specialists to noninvasively monitor biochemical processes and to determine any pathological state in the human body. Breath analysis holds the greatest potential to remold and personalize diagnostics; however, it requires a multidisciplinary approach and collaboration of many specialists. Despite the fact that breath is considered to be a less complex matrix than blood, it is not commonly used as a diagnostic and prognostic tool for early detection of disordered conditions due to its problematic sampling, analysis, and storage. This review is intended to determine, standardize, and marshal experimental strategies for successful, reliable, and especially, reproducible breath analysis

Molecular machines from the AAA+ family play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in conformation that underlie their specialized functions. Furthermore, complexes between these machines and substrates begin to explain their mechanism of activity. Here we explore how crosslinking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP and how substrates are associated. We applied a panel of crosslinking reagents to produce high-resolution crosslinking maps and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify crosslinks that break the asymmetry that are only present when ATP is bound and are absent in an ATP-binding deficient mutant. Finally, we identify contacts between Hsp104 and a model substrate to identify contacts on the central channel of Hsp104 across the length of the substrate indicating that we have trapped interactions consistent with translocation of the substrate. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to substrates even in the context of homo-oligomeric assemblies.

Molecular machines from the AAA+ family play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in conformation that underlie their specialized functions. Furthermore, complexes between these machines and substrates begin to explain their mechanism of activity. Here we explore how crosslinking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP and how substrates are associated. We applied a panel of crosslinking reagents to produce high-resolution crosslinking maps and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify crosslinks that break the asymmetry that are only present when ATP is bound and are absent in an ATP-binding deficient mutant. Finally, we identify contacts between Hsp104 and a model substrate to identify contacts on the central channel of Hsp104 across the length of the substrate indicating that we have trapped interactions consistent with translocation of the substrate. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to substrates even in the context of homo-oligomeric assemblies.