A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Gas vesicles across kingdoms: a comparative solid-state nuclear magnetic resonance study.

The buoyancy organelles of aquatic microorganisms have to meet stringent specifications: allowing gases to equilibrate freely across the proteinaceous shell, preventing the condensation of water vapor inside the hollow cavity and resisting collapse under hydrostatic pressures that vary with column depth. These properties are provided by the 7- to 8-kDa gas vesicle protein A (GvpA), repeats of which form all but small, specialized portions of the shell. Magic angle spinning nuclear magnetic resonance is uniquely capable of providing high-resolution information on the fold and assembly of GvpA. Here we compare results for the gas vesicles of the haloarchaea Halobacterium salinarum with those obtained previously for the cyanobacterium Anabaena flos-aquae. The data suggest that the two organisms follow similar strategies for avoiding water condensation. On the other hand, in its relatively shallow habitat, H. salinarum is able to avoid collapse with a less costly GvpA fold than is adopted by A. flos-aquae.

An amyloid organelle, solid-state NMR evidence for cross-ß assembly of gas vesicles.

Functional amyloids have been identified in a wide range of organisms, taking on a variety of biological roles and being controlled by remarkable mechanisms of directed assembly. Here, we report that amyloid fibrils constitute the ribs of the buoyancy organelles of Anabaena flos-aquae. The walls of these gas-filled vesicles are known to comprise a single protein, GvpA, arranged in a low pitch helix. However, the tertiary and quaternary structures have been elusive. Using solid-state NMR correlation spectroscopy we find detailed evidence for an extended cross-ß structure. This amyloid assembly helps to account for the strength and amphiphilic properties of the vesicle wall. Buoyancy organelles thus dramatically extend the scope of known functional amyloids.