Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles and have linked their dysfunction to more than 150 distinct disorders. Still, hundreds of mitochondrial proteins lack clear functions, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved.

Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic β-cells.

β-Cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1-the activation of mitochondrial respiratory complex I-that is active in quiescent adult β-cells and hyperactive in β-cells from obese (/) mice. In WT islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition.

Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy.

The development of effective strategies for the comprehensive identification and quantification of proteoforms in complex systems is a critical challenge in proteomics. Proteoforms, the specific molecular forms in which proteins are present in biological systems, are the key effectors of biological function. Thus, knowledge of proteoform identities and abundances is essential to unraveling the mechanisms that underlie protein function.

Iron Deprivation Induces Transcriptional Regulation of Mitochondrial Biogenesis.

Mitochondria are essential organelles that adapt to stress and environmental changes. Among the nutrient signals that affect mitochondrial form and function is iron, whose depletion initiates a rapid and reversible decrease in mitochondrial biogenesis through unclear means. Here we demonstrate that, unlike the canonical iron-induced alterations to transcript stability, loss of iron dampens the transcription of genes encoding mitochondrial proteins with no change to transcript half-life.

Multiplexed quantification for data-independent acquisition.

Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS(2) spectra.

Inhibitors of bacterial tubulin target bacterial membranes .

FtsZ is a homolog of eukaryotic tubulin that is widely conserved among bacteria and coordinates the assembly of the cell division machinery. FtsZ plays a central role in cell replication and is a target of interest for antibiotic development. Several FtsZ inhibitors have been reported.

Complementary RNA and protein profiling identifies iron as a key regulator of mitochondrial biogenesis.

Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis.

Automated gas-phase purification for accurate, multiplexed quantification on a stand-alone ion-trap mass spectrometer.

Isobaric tagging enables the acquisition of highly multiplexed proteome quantification, but it is hindered by the pervasive problem of precursor interference. The elimination of coisolated contaminants prior to reporter tag generation can be achieved through the use of gas-phase purification via proton transfer ion/ion reactions (QuantMode); however, the original QuantMode technique was implemented on the high-resolution linear ion-trap-Orbitrap hybrid mass spectrometer enabled with electron transfer dissociation (ETD). Here we extend this technology to stand-alone linear ion-trap systems (trapQuantMode, trapQM).