Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Furthermore, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. We conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Further, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. Finally, we conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Infrared Photoactivation Boosts Reporter Ion Yield in Isobaric Tagging.

Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.

Autonomous submersible multiport water sampler.

Oceanography and limnology projects often require the collection of water samples for chemical analysis. Manual water sample collection is labor-intensive and often difficult, especially in remote locations or during nighttime hours. Here we describe a compact and inexpensive autonomous submersible multiport water sampler (AutoSampler) that is largely fabricated with off-the-shelf parts making it easier to build and maintain. The system can collect up to 12 discrete samples at user controllable times or intervals and is operated using open source Arduino hardware and software that can be user modified to meet deployment requirements. While the underwater pressure housing presented here is custom built from readily available materials, there are many commercially available pressure case options that can be used as a substitute. The electronic mounting plates and battery pack are designed so that they can easily be adapted to fit into other pressure case housings. Samples can be collected into bags or syringes and sample volume is set by adjusting how long the peristaltic pump is actuated. This AutoSampler allows research that would otherwise be too labor-intensive or logistically difficult to conduct, especially in remote locations.