Regional Differences in Penetration of the Protein Stabilizer Trimethoprim (TMP) in the Rat Central Nervous System.

Regulating gene expression at the protein level is becoming increasingly important for answering basic questions in neurobiology. Several techniques using destabilizing domains (DD) on transgenes, which can be activated or deactivated by specific drugs, have been developed to achieve this goal. A DD from bacterial dihydrofolate reductase bound and stabilized by trimethoprim (TMP) represents such a tool. To control transgenic protein levels in the brain, the DD-regulating drugs need to have sufficient penetration into the central nervous system (CNS). Yet, very limited information is available on TMP pharmacokinetics in the CNS following systemic injection. Here, we performed a pharmacokinetic study on the penetration of TMP into different CNS compartments in the rat. We used mass spectrometry to measure TMP concentrations in serum, cerebrospinal fluid (CSF) and tissue samples of different CNS regions upon intraperitoneal TMP injection. We show that TMP quickly (within 10 min) penetrates from serum to CSF through the blood-CSF barrier. TMP also shows quick penetration into brain tissue but concentrations were an order of magnitude lower compared to serum or CSF. TMP concentration in spinal cord was lower than in any other analyzed CNS area. Nevertheless, effective levels of TMP to stabilize DDs can be reached in the CNS with half-lives around 2 h. These data show that TMP has good and fast penetration properties into the CNS and is therefore a valuable ligand for precisely controlling protein expression in the CNS in rodents.

JAK2-mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms.

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2-driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion.

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

Unraveling cell populations in tumors by single-cell mass cytometry.

The development of new biotechnologies for the analysis of individual cells in heterogeneous populations is an important direction of life science research. This review provides a critical overview of relevant and recent advances in the field of single-cell mass cytometry, focusing on the latest applications in the study of cell heterogeneity. New approaches for multiparameter single-cell imaging, alongside advanced computational tools for deep mining of high-dimensional mass cytometric data, are facilitating the visualization of specific cell types and their interactions in complex cellular assemblies, such as tumors, potentially revealing new insights into cancer biology.

Adenovirus composition, proteolysis, and disassembly studied by in-depth qualitative and quantitative proteomics.

Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.

Finding the same needles in the haystack? A comparison of phosphotyrosine peptides enriched by immuno-affinity precipitation and metal-based affinity chromatography.

Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal-affinity based enrichment (Ti(4+)-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling. BIOLOGICAL SIGNIFICANCE: Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways.

Toward a comprehensive characterization of a human cancer cell phosphoproteome.

Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

ZIC-cHILIC as a fractionation method for sensitive and powerful shotgun proteomics.

Multidimensional liquid chromatography (LC) combined with mass spectrometry (MS) has become a standard technique in proteomics to reduce sample complexity and to tackle the dynamic range in protein abundance. Fractionation is necessary to obtain a comprehensive analysis of complex biological samples such as tissue and mammalian cell lines. However, extensive fractionation comes at the expense of sample loss, presenting a bottleneck in the analysis of limited amounts of material. In this protocol, we describe a two-dimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography (HILIC; with a zwitterionic packing material, ZIC-cHILIC) and reversed-phase chromatography, which allows proteomic analyses with minimal sample loss. Experimental aspects related to obtaining maximum recovery are discussed, including how to optimally prepare samples for this system. Examples involving protein lysates originating from cultured cell lines and cells sorted by flow cytometry are used to show the power, sensitivity and versatility of the technique. Once the ZIC-cHILIC fractionation system has been optimized and standardized, this protocol requires ∼5-6 d, including sample preparation and fraction analysis.

Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis.

Shotgun proteomics dominates the field of proteomics. The foundations of the strategy consist of multiple rounds of peptide separation where chromatography provides the bedrock. Initially, the scene was relatively simple with the majority of strategies based on some types of ion exchange and reversed phase chromatography. The thirst to achieve comprehensivity, when it comes to proteome coverage and the global characterization of post translational modifications, has led to the introduction of several new separations. In this review, we attempt to provide a historical perspective to separations in proteomics as well as indicate the principles of their operation and rationales for their implementation. Furthermore, we provide a guide on what are the possibilities for combining different separations in order to increase peak capacity and proteome coverage. We aim to show how separations enrich the world of proteomics and how further developments may impact the field.

Evaluation of the deuterium isotope effect in zwitterionic hydrophilic interaction liquid chromatography separations for implementation in a quantitative proteomic approach.

Quantitative methodologies for the global in-depth comparison of proteomes are frequently based on chemical derivatization of peptides with isotopically distinguishable labeling agents. In the present work, we set out to study the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC (zwitterionic hydrophilic interaction liquid chromatography) technology for quantitative proteomics. We first addressed the potential issue of isotope effects perturbing the essential coelution of differently labeled peptides under ZIC-cHILIC separation. The deuterium incorporation-induced effect can be largely eliminated by favoring the mixed-mode ZIC-cHILIC separation based on combined hydrophilic and ionic interactions. Then, we evaluated the performance and applicability of this strategy using a sample consisting of human cell lysate. We demonstrate that our approach is suitable to perform unbiased quantitative proteome analysis, still quantifying more than 2500 proteins when analyzing only a few micrograms of sample.

Highly sensitive proteome analysis of FACS-sorted adult colon stem cells.

In proteomics, multidimensional liquid chromatography combined with mass spectrometry has become a standard technique to reduce sample complexity and tackle the vast dynamic range. Such fractionation is necessary to obtain a comprehensive analysis of biological samples such as tissues and cell lines. However, extensive fractionation comes at the expense of sample losses, hampering the analysis of limited material. We previously described a highly sensitive multidimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography and reversed phase chromatography, which allows proteomic analysis with minimal sample losses. Here we apply this strategy to the analysis of a limited number of FACS-sorted colon stem cells extracted from mouse intestine, obtaining a proteome coverage comparable to current methods that generally require 100-fold more starting material. We propose that this alternative multidimensional chromatographic technology will find ample application such as in the analysis of distinct cellular populations obtained by laser microdissection.

Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC and ZIC-cHILIC) provide high resolution separation and increase sensitivity in proteome analysis.

The complexity of peptide mixtures that are analyzed in proteomics necessitates fractionation by multidimensional separation approaches prior to mass spectrometric analysis. In this work, we introduce and evaluate hydrophilic interaction liquid chromatography (HILIC) based strategies for the separation of complex peptide mixtures. The two zwitterionic HILIC materials (ZIC-HILIC and ZIC-cHILIC) chosen for this work differ in the spatial orientation of the positive and negative charged groups. Online experiments revealed a pH-independent resolving power for the ZIC-cHILIC resin while ZIC-HILIC showed a decrease in resolving power at an acidic pH. Subsequently, we extensively evaluated the performances of ZIC-HILIC and ZIC-cHILIC as first dimension in an off-line two-dimensional liquid chromatography (2D-LC) strategy in combination with reversed phase (RP), with respect to peptide separation efficiency and how the retention time correlates with a number of peptide physicochemical properties. Both resins allowed the identification of more than 20,000 unique peptides corresponding to over 3500 proteins in each experimental condition from a remarkably low (1.5 µg) amount of starting material of HeLa lysate digestion. The resulting data allows the drawing of a comprehensive picture regarding ZIC- and ZIC-cHILIC peptide separation characteristics. Furthermore, the extent of protein identifications observed from such a level of material demonstrates that HILIC can rival or surpass traditional multidimensional strategies employed in proteomics.

A novel strategy for reducing phospholipids-based matrix effect in LC-ESI-MS bioanalysis by means of HybridSPE.

A novel strategy to minimize phospholipids-based matrix effects in bioanalytical LC-MS/MS assays was evaluated. The phospholipids-based matrix effect was investigated with a commercially available electrospray ionization (ESI) source coupled with a triple quadrupole mass spectrometer. A systematic comparison approach of two sample preparation procedures was performed. In particular, the matrix effect on mass spectrometry response in rat and human plasma samples was studied by comparing sample extracts obtained by means of a conventional plasma protein precipitation with acetonitrile and the novel HybridSPE-Precipitation procedure. The HybridSPE dramatically reduced the levels of residual phospholipids in biological samples, leading to significant reduction in matrix effects. This new procedure which combines the simplicity of precipitation with the selectivity of SPE allows to obtain much cleaner extracts than with conventional procedures. The effective targeted removal of phospholipids and proteins in biological plasma samples achieved with the HybridSPE-Precipitation procedure provides significant improvement in bioanalysis and a practical and fast way to ensure the avoidance of phospholipids-based matrix effects.