Generation of Antibodies Targeting Cleavable Cross-Linkers.

Chemical cross-linking has become a powerful tool for the analysis of protein structures and interactions by mass spectrometry. A particular strength of this approach is the ability to investigate native states in vivo, investigating intact organelles, cells, or tissues. For such applications, the cleavable cross-linkers disuccinimidyl sulfoxide (DSSO) and disuccinimidyl dibutyric urea (DSBU) are gaining increasing popularity, as they allow for the analysis of complex mixtures. It is inherently difficult to follow the reaction of cross-linkers with proteins in intact biological structures, stalling the optimization of in vivo cross-linking experiments. We generated polyclonal antibodies targeting DSSO- and DSBU-modified proteins, by injection of cross-linked bovine serum albumin (BSA) in rabbits. We show that the cross-linker-modified BSA successfully triggered an immune response, and that DSSO- and DSBU-specific antibodies were generated by the animals. Using affinity-purified antibodies specific for the individual cross-linkers, we demonstrate their application to the detection of cross-linker-modified proteins in Western blot and immunocytochemistry experiments of intact and permeabilized cells. Furthermore, we show their ability to immunoprecipitate DSSO/DSBU-modified proteins and provide evidence for their affinity toward water-quenched dead-links. These antibodies provide a valuable tool for the investigation of proteins modified with the cross-linkers DSSO and DSBU.

Systematic Comparison of Strategies for the Enrichment of Lysosomes by Data Independent Acquisition.

In mammalian cells, the lysosome is the main organelle for the degradation of macromolecules and the recycling of their building blocks. Correct lysosomal function is essential, and mutations in every known lysosomal hydrolase result in so-called lysosomal storage disorders, a group of rare and often fatal inherited diseases. Furthermore, it is becoming more and more apparent that lysosomes play also decisive roles in other diseases, such as cancer and common neurodegenerative disorders. This leads to an increasing interest in the proteomic analysis of lysosomes for which enrichment is a prerequisite. In this study, we compared the four most common strategies for the enrichment of lysosomes using data-independent acquisition. We performed centrifugation at 20,000 × g to generate an organelle-enriched pellet, two-step sucrose density gradient centrifugation, enrichment by superparamagnetic iron oxide nanoparticles (SPIONs), and immunoprecipitation using a 3xHA tagged version of the lysosomal membrane protein TMEM192. Our results show that SPIONs and TMEM192 immunoprecipitation outperform the other approaches with enrichment factors of up to 118-fold for certain proteins relative to whole cell lysates. Furthermore, we achieved an increase in identified lysosomal proteins and a higher reproducibility in protein intensities for label-free quantification in comparison to the other strategies.

Toxicological and analytical assessment of e-cigarette refill components on airway epithelia.

There are over 2.6 million users of e-cigarettes in the United Kingdom alone as they have been promoted as a safer alternative to traditional cigarettes. The addition of flavours and aromas has also proven to be popular with younger generations. In this review, we survey the range of studies in the short timeframe since e-cigarettes reached the market to draw attention to the health associated risks and benefits of their introduction. We complement this review with a case study reporting on the composition of selected e-cigarette refills with particular emphasis on the toxicological activity of its components on lung cells.

The mitochondrial Hsp70 controls the assembly of the F1FO-ATP synthase.

The mitochondrial F1FO-ATP synthase produces the bulk of cellular ATP. The soluble F1 domain contains the catalytic head that is linked via the central stalk and the peripheral stalk to the membrane embedded rotor of the FO domain. The assembly of the F1 domain and its linkage to the peripheral stalk is poorly understood. Here we show a dual function of the mitochondrial Hsp70 (mtHsp70) in the formation of the ATP synthase. First, it cooperates with the assembly factors Atp11 and Atp12 to form the F1 domain of the ATP synthase. Second, the chaperone transfers Atp5 into the assembly line to link the catalytic head with the peripheral stalk. Inactivation of mtHsp70 leads to integration of assembly-defective Atp5 variants into the mature complex, reflecting a quality control function of the chaperone. Thus, mtHsp70 acts as an assembly and quality control factor in the biogenesis of the F1FO-ATP synthase.

Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo.

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyze lysosomes and early endosomes. Based on the identification of 5376 cross-links, we investigate protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identify >300 putative cargo proteins and confirm eleven substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

Targeted Quantification of the Lysosomal Proteome in Complex Samples.

In eukaryotic cells, lysosomes play a crucial role in the breakdown of a variety of components ranging from small molecules to complex structures, ascertaining the continuous turnover of cellular building blocks. Furthermore, they act as a regulatory hub for metabolism, being crucially involved in the regulation of major signaling pathways. Currently, ~450 lysosomal proteins can be reproducibly identified in a single cell line by mass spectrometry, most of which are low-abundant, restricting their unbiased proteomic analysis to lysosome-enriched fractions. In the current study, we applied two strategies for the targeted investigation of the lysosomal proteome in complex samples: data-independent acquisition (DIA) and parallel reaction monitoring (PRM). Using a lysosome-enriched fraction, mouse embryonic fibroblast whole cell lysate, and mouse liver whole tissue lysate, we investigated the capabilities of DIA and PRM to investigate the lysosomal proteome. While both approaches identified and quantified lysosomal proteins in all sample types, and their data largely correlated, DIA identified on average more proteins, especially for lower complex samples and longer chromatographic gradients. For the highly complex tissue sample and shorter gradients, however, PRM delivered a better performance regarding both identification and quantification of lysosomal proteins. All data are available via ProteomeXchange with identifier PXDD023278.

Comprehensive draft of the mouse embryonic fibroblast lysosomal proteome by mass spectrometry based proteomics.

Lysosomes are the main degradative organelles of cells and involved in a variety of processes including the recycling of macromolecules, storage of compounds, and metabolic signaling. Despite an increasing interest in the proteomic analysis of lysosomes, no systematic study of sample preparation protocols for lysosome enriched fractions has been performed to date. In the current study, we used samples enriched for lysosomes by paramagnetic nanoparticles and systematically evaluated experimental parameters for the analysis of the lysosomal proteome. This includes different approaches for the concentration of lysosome-containing fractions; desalting of samples by solid phase extraction; fractionation of peptide samples; and different gradient lengths for LC-MS/MS analyses of unfractionated samples by data dependent and data independent acquisition. Furthermore, we evaluated four different digestion methods including filter aided sample preparation (FASP), in-gel digestion, and in-solution digestion using either RapiGest or urea. Using the combined data, we generated a benchmark lysosomal proteome data set for mouse embryonic fibroblasts as well as a spectral library for the analysis of lysosomes by data independent acquisition.