Fast and Simple Protocols for Mass Spectrometry-Based Proteomics of Small Fresh Frozen Uterine Tissue Sections.

Human tissues are an important link between organ-specific spatial molecular information, patient pathology, and patient treatment options. However, patient tissues are uniquely obtained by time and location, and limited in their availability and size. Currently, little knowledge exists about appropriate and simplified protocols for routine MS-based analysis of the various types and sizes of tissues. Following standard procedures used in pathology, we selected small fresh frozen uterine tissue samples to investigate how the tissue preparation protocol affected the subsequent proteomics analysis. First, we observed that protein extraction with 0.1% SDS followed by extraction with a 30% ACN/urea resulted in a decrease in the number of identified proteins, when compared to extraction with 30% ACN/urea only. The decrease in the number of proteins was approximately 55% and 20%, for 10 and 16 µm thick tissue, respectively. Interestingly, the relative abundance of the proteins shared between the two methods was higher when SDS/ACN/urea was used, compared to the 30% ACN/urea extraction, indicating the role of SDS to be beneficial for protein solubility. Second, the influence of tissue thickness was investigated by comparing the results obtained for 10, 16, and 20 µm thick (1 mm2) tissue using urea/30% ACN. We observed an increase in the number of identified proteins and corresponding quantity with an increase in the tissue thickness. Finally, by analyzing very small amounts of tissues (∼0.2 mm2) of 10, 16, and 20 µm thickness, we observed that the increase in tissue thickness resulted in a higher number of protein identifications and corresponding quantitative values.

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein-Protein Interactions at the Peptide Level.

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and ß' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.