Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry.

Since the start of mass-spectrometry-based proteomics, proteins from non-referenced open reading frames or alternative proteins (AltProts) have been overlooked. Here, we present a protocol to identify human subcellular AltProt and decipher some interactions using cross-linking mass spectrometry. We describe steps for cell culture, in cellulo cross-link, subcellular extraction, and sequential digestion. We then detail both liquid chromatography-tandem mass spectrometry and cross-link data analyses. The implementation of a single workflow allows the non-targeted identification of signaling pathways involving AltProts. For complete details on the use and execution of this protocol, please refer to Garcia-del Rio et al.1.

Employing non-targeted interactomics approach and subcellular fractionation to increase our understanding of the ghost proteome.

Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential new immunopeptide, and the interactions between HIST1H4F and several AltProts which can play a role in mRNA transcription. Thanks to the study of the interactome and the localization of AltProts, we can reveal more of the importance of the ghost proteome.

Analytical and computational workflow for in-depth analysis of oxidized complex lipids in blood plasma.

Lipids are a structurally diverse class of biomolecules which can undergo a variety of chemical modifications. Among them, lipid (per)oxidation attracts most of the attention due to its significance in the regulation of inflammation, cell proliferation and death programs. Despite their apparent regulatory significance, the molecular repertoire of oxidized lipids remains largely elusive as accurate annotation of lipid modifications is complicated by their low abundance and often unknown, biological context-dependent structural diversity. Here, we provide a workflow based on the combination of bioinformatics and LC-MS/MS technologies to support identification and relative quantification of oxidized complex lipids in a modification type- and position-specific manner. The developed methodology is used to identify epilipidomics signatures of lean and obese individuals with and without type 2 diabetes. The characteristic signature of lipid modifications in lean individuals, dominated by the presence of modified octadecanoid acyl chains in phospho- and neutral lipids, is drastically shifted towards lipid peroxidation-driven accumulation of oxidized eicosanoids, suggesting significant alteration of endocrine signalling by oxidized lipids in metabolic disorders.

Synthesis, In Silico, and In Vitro Evaluation of Long Chain Alkyl Amides from 2-Amino-4-Quinolone Derivatives as Biofilm Inhibitors.

Infection from multidrug resistant bacteria has become a growing health concern worldwide, increasing the need for developing new antibacterial agents. Among the strategies that have been studied, biofilm inhibitors have acquired relevance as a potential source of drugs that could act as a complement for current and new antibacterial therapies. Based on the structure of 2-alkyl-3-hydroxy-4-quinolone and N-acylhomoserine lactone, molecules that act as mediators of quorum sensing and biofilm formation in Pseudomonas aeruginosa, we designed, prepared, and evaluated the biofilm inhibition properties of long chain amide derivatives of 2-amino-4-quinolone in Staphylococcus aureus and P. aeruginosa. All compounds had higher biofilm inhibition activity in P. aeruginosa than in S. aureus. Particularly, compounds with an alkyl chain of 12 carbons exhibited the highest inhibition of biofilm formation. Docking scores and molecular dynamics simulations of the complexes of the tested compounds within the active sites of proteins related to quorum sensing had good correlation with the experimental results, suggesting the diminution of biofilm formation induced by these compounds could be related to the inhibition of these proteins.