Isobaric labeling using tandem mass tags (TMTs) is increasingly applied for deep-scale proteomic studies in a multitude of organisms and addressing diverse research questions. The cost of labeling ...reagents represents a substantial proportion of the total expenses for conducting such experiments. Here, Zecha et al. present an economically optimized TMT labeling approach that reduces the quantity of required labeling reagent by a factor of eight and reproducibly achieves complete labeling.
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Highlights
•TMT labeling protocol with excellent intra- and interlaboratory reproducibility.•Complete in-solution labeling of peptides using 1/8 of recommended TMT quantities.•Demonstration of utility for deep-scale (phospho)proteome analysis.
Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.
Patient-derived xenograft (PDX) tumor models represent a valuable platform for identifying new biomarkers and novel targets, to evaluate therapy response and resistance mechanisms. This study aimed ...at establishment, characterization and therapy testing of colorectal carcinoma-derived PDX. We generated 49 PDX and validated identity between patient tumor and corresponding PDX. Sensitivity of PDX toward conventional and targeted drugs revealed that 92% of PDX responded toward irinotecan, 45% toward 5-FU, 65% toward bevacizumab, and 61% toward cetuximab. Expression of epidermal growth factor receptor (EGFR) ligands correlated to the sensitivity toward cetuximab. Proto-oncogene B-RAF, EGFR, Kirsten rat sarcoma virus oncogene homolog gene copy number correlated positively with cetuximab and erlotinib sensitivity. The mutational analyses revealed an individual mutational profile of PDX and mainly identical profiles of PDX from primary tumor vs corresponding metastasis. Mutation in PIK3CA was a determinant of accelerated tumor doubling time. PDX with wildtype Kirsten rat sarcoma virus oncogene homolog, proto-oncogene B-RAF, and phosphatidylinositol-4,5-bisphosphate 3-kinaseM catalytic subunit alfa showed higher sensitivity toward cetuximab and erlotinib. To study the molecular mechanism of cetuximab resistance, cetuximab resistant PDX models were generated, and changes in HER2, HER3, betacellulin, transforming growth factor alfa were observed. Global proteome and phosphoproteome profiling showed a reduction in canonical EGFR-mediated signaling via PTPN11 (SHP2) and AKT1S1 (PRAS40) and an increase in anti-apoptotic signaling as a consequence of acquired cetuximab resistance. This demonstrates that PDX models provide a multitude of possibilities to identify and validate biomarkers, signaling pathways and resistance mechanisms for clinically relevant improvement in cancer therapy.
Display omitted Three-phase methanol–water–chloroform extraction for biological samples. Examples of components available from each phase are shown. These different phases can be then used for a ...variety of different analysis methods on different levels of cellular regulation.
Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified.
Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology.
Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine ...exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammation-relevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols.
The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required ...for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function.
Ship diesel combustion particles are known to cause broad cytotoxic effects and thereby strongly impact human health. Particles from heavy fuel oil (HFO) operated ships are considered as particularly ...dangerous. However, little is known about the relevant components of the ship emission particles. In particular, it is interesting to know if the particle cores, consisting of soot and metal oxides, or the adsorbate layers, consisting of semi- and low-volatile organic compounds and salts, are more relevant. We therefore sought to relate the adsorbates and the core composition of HFO combustion particles to the early cellular responses, allowing for the development of measures that counteract their detrimental effects. Hence, the semi-volatile coating of HFO-operated ship diesel engine particles was removed by stepwise thermal stripping using different temperatures. RAW 264.7 macrophages were exposed to native and thermally stripped particles in submersed culture. Proteomic changes were monitored by two different quantitative mass spectrometry approaches, stable isotope labeling by amino acids in cell culture (SILAC) and dimethyl labeling. Our data revealed that cells reacted differently to native or stripped HFO combustion particles. Cells exposed to thermally stripped particles showed a very differential reaction with respect to the composition of the individual chemical load of the particle. The cellular reactions of the HFO particles included reaction to oxidative stress, reorganization of the cytoskeleton and changes in endocytosis. Cells exposed to the 280 °C treated particles showed an induction of RNA-related processes, a number of mitochondria-associated processes as well as DNA damage response, while the exposure to 580 °C treated HFO particles mainly induced the regulation of intracellular transport. In summary, our analysis based on a highly reproducible automated proteomic sample-preparation procedure shows a diverse cellular response, depending on the soot particle composition. In particular, it was shown that both the molecules of the adsorbate layer as well as particle cores induced strong but different effects in the exposed cells.
The yeast plasma membrane H+‐ATPase Pma1p is a P‐type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here ...we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H+‐ATPases are autoinhibited.
The yeast plasma membrane H+‐ATPase Pma1p was isolated and reconstituted into lipid vesicles in both an autoinhibited and uncoupled basal state and a coupled activated state. Acetylated tubulin is shown not be required for keeping Pma1p in the autoinhibited basal state.
Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a ...detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.
Background
The Muscle‐specific RING‐finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated ...muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo.
Methods
MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real‐time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass‐spectrometry were used for mechanistic analyses.
Results
DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3‐deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts.
Conclusions
The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo.
Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16
..., p21
and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.
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