Spatiochemically Profiling Microbial Interactions with Membrane Scaffolded Desorption Electrospray Ionization-Ion Mobility-Imaging Mass Spectrometry and Unsupervised Segmentation.

Imaging the inventory of microbial small molecule interactions provides important insights into microbial chemical ecology and human medicine. Herein we demonstrate a new method for enhanced detection and analysis of metabolites present in interspecies interactions of microorganisms on surfaces. We demonstrate that desorption electrospray ionization-imaging mass spectrometry (DESI-IMS) using microporous membrane scaffolds (MMS) enables enhanced spatiochemical analyses of interacting microbes among tested sample preparation techniques. Membrane scaffolded DESI-IMS has inherent advantages compared to matrix-assisted laser desorption ionization (MALDI) and other IMS methods through direct IMS analyses of microbial chemistry in situ. This rapid imaging method yields sensitive MS analyses with unique m/z measurements when compared to liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) via unmediated sampling by MMS DESI-IMS. Unsupervised segmentation imaging analysis of acquired DESI-IMS data reveals distinct chemical regions corresponding to intermicrobial phenomenon such as predation and communication. We validate the method by linking Myxovirescin A and DKxanthene-560 to their known biological roles of predation and phase variation, respectively. In addition to providing the first topographic locations of known natural products, we prioritize 54 unknown features using segmentation within the region of predation. Thus, DESI-IMS and unsupervised segmentation spatially annotates the known biology of myxobacteria and provides functional exploration of newly uncharacterized small molecules.

Multi-mode acquisition (MMA): An MS/MS acquisition strategy for maximizing selectivity, specificity and sensitivity of DIA product ion spectra.

In proteomics studies, it is generally accepted that depth of coverage and dynamic range is limited in data-directed acquisitions. The serial nature of the method limits both sensitivity and the number of precursor ions that can be sampled. To that end, a number of data-independent acquisition (DIA) strategies have been introduced with these methods, for the most part, immune to the sampling issue; nevertheless, some do have other limitations with respect to sensitivity. The major limitation with DIA approaches is interference, i.e., MS/MS spectra are highly chimeric and often incapable of being identified using conventional database search engines. Utilizing each available dimension of separation prior to ion detection, we present a new multi-mode acquisition (MMA) strategy multiplexing both narrowband and wideband DIA acquisitions in a single analytical workflow. The iterative nature of the MMA workflow limits the adverse effects of interference with minimal loss in sensitivity. Qualitative identification can be performed by selected ion chromatograms or conventional database search strategies.

The chromosome-centric human proteome project: a call to action.

The grand vision of the human proteome project (HPP) is moving closer to reality with the recent announcement by HUPO of the creation of the HPP consortium in charge of the development of a two-part HPP, one focused on the description of proteomes of biological samples or related to diseases (B/D-HPP) and the other dedicated to a systematic description of proteins as gene products encoded in the human genome (the C-HPP). This new initiative of HUPO seeks to identify and characterize at least one representative protein from every gene, create a protein distribution atlas and a protein pathway or network map. This vision for proteomics can be the roadmap of biological and clinical research for years to come if it delivers on its promises. The Industrial Advisory Board (IAB) to HUPO shares the visions of C-HPP. The IAB will support and critically accompany the overall project goals and the definitions of the critical milestones. The member companies are in a unique position to develop hardware and software, reagents and standards, procedures, and workflows to ensure a reliable source of tools available to the proteomics community worldwide. In collaboration with academia, the IAB member companies can and must develop the tools to reach the ambitious project goals. We offer to partner with and challenge the academic groups leading the C-HPP to define both ambitious and obtainable goals and milestones to make the C-HPP a real and trusted resource for future biology.