Highlights d BraInMap is a global proteomic survey of over 1,000 multiprotein brain complexes d Near-native complex identification by CF-MS and reconstruction by computer learning d Technique interrogates complexes in normal and pathophysiological context d Allows study of functional modules that are adversely affected in neurological diseases

A new approach to assess the orthogonality of two-dimensional (2-D) separation systems based on conditional entropy is developed. It considers the quantitative distribution of peaks in the entire separation space such that the orthogonality obtained is independent of the number of peaks observed for each separation technique. Therefore, it can be used to compare the orthogonality of different 2-D separation protocols for a given sample. Herein, the developed method has been employed to estimate the orthogonality of peptide separation by off-gel electrophoresis (OGE) hyphenated to capillary zone electrophoresis (CZE).

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein–protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans . In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules.

Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) are two techniques highly suitable for the separation and detection of intact proteins. Herein, based on the use of a recently introduced iontophoretic fraction collection interface for the coupling of CE and MALDI-MS, the potential of the combination of both techniques for the analysis of intact proteins is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Under optimized conditions, more than 3200-and 4800-fold improvement, respectively in terms of peak height and peak area, as well as LODs ranging from 5 to 10 nM, has been achieved.

A microchip electrospray emitter with a magnetic bead trap has been designed for solid-phase extraction-gradient elution-mass spectrometry (SPE-GEMS). The goal of this method is the detection of analytes at low concentrations and it is here demonstrated using reverse phase coated magnetic beads (Mbs) for the preconcentration and detection of the peptides. The sample is passed through the chip, and the peptides are retained and enriched in the trap. After washing, the peptides are released sequentially by stepwise gradient elution and electrosprayed for mass spectrometry analysis. This approach allows effective sample desalting, enrichment, sequential elution, and MS detection without the introduction of an additional separation step after SPE. Efficient preconcentration of model peptides by SPE and sequential release and analysis of peptides by GEMS were demonstrated for diluted sample solutions within the range of 1 μM to 10 nM. Fortified human blood serum, protein digest and fractions collected after protein digest OFFGEL separation were analyzed by SPE-GEMS allowing the detection of low abundance peptides usually not observed by direct mass spectrometry analysis. A mathematical model for gradient elution is proposed.