Recent advances in gas phase unfolding: Instrumentation and applications.

Broader adoption of native mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS) has propelled the development of several techniques which take advantage of the selectivity, sensitivity, and speed of MS to make measurements of complex biological molecules in the gas phase. One such method, collision induced unfolding (CIU), has risen to prominence in recent years, due to its well documented capability to detect shifts in structural stability of biological molecules in response to external stimuli (e.g., mutations, stress, non-covalent interactions, sample conditions etc.). This increase in reported CIU measurements is enabled partly due to advances in IM-MS instrumentation by vendors, and also innovative method development by researchers. This perspective highlights a few of these advances and concludes with a look forward toward the future of the gas phase unfolding field.

A lethal mitonuclear incompatibility in complex I of natural hybrids.

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

Enhancing Data Reliability in TOMAHAQ for Large-Scale Protein Quantification.

The analytical scale of most mass-spectrometry-based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post-acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.