Nano-liquid Chromatography-orbitrap MS-based Quantitative Proteomics Reveals Differences Between the Mechanisms of Action of Carnosic Acid and Carnosol in Colon Cancer Cells.

Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.

Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes.

LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family. Graphical Abstract Intraspecies identification within the green frog complex.

Comprehensive Proteomic Study of the Antiproliferative Activity of a Polyphenol-Enriched Rosemary Extract on Colon Cancer Cells Using Nanoliquid Chromatography-Orbitrap MS/MS.

In this work, a proteomics strategy based on nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) using an Orbitrap high-resolution mass spectrometer together with stable isotope dimethyl labeling (DML) is applied to quantitatively examine relative changes in the protein fraction of HT-29 human colon cancer cells treated with different concentrations of a polyphenol-enriched rosemary extract over the time. The major objective of this study was to gain insights into the antiproliferative mechanisms induced by rosemary polyphenols. Using this methodology, 1909 and 698 proteins were identified and quantified in cell extracts. The polyphenol-enriched rosemary extract treatment changed the expression of several proteins in a time- and concentration-dependent manner. Most of the altered proteins are implicated in the activation of Nrf2 transcription factor and the unfolded protein response. In conclusion, rosemary polyphenols induced proteomic changes that were related to the attenuation of aggresome formation and activation of autophagy to alleviate cellular stress.

LTQ Orbitrap Velos in routine de novo sequencing of non-tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation.

RATIONALE: Mass spectrometry has shown itself to be the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most efficient methods of triggering fragmentation and combine all the possible complementary techniques. METHODS: Collision-induced dissociation (CID), high-energy collision dissociation (HCD), and electron-transfer dissociation (ETD) tandem mass spectra recorded with a LTQ Orbitrap Velos instrument were used for the elucidation of the sequence of the natural non-tryptic peptides from the skin secretion of Rana latastei. Manual interpretation of the spectra was applied. RESULTS: The combined approach using CID, HCD, and ETD tandem mass spectra of the multiprotonated peptides in various charge states, as well as of their proteolytic fragments, allowed the sequences of seven novel peptides from the skin secretion of Rana latastei to be established. CONCLUSIONS: Manual mass spectrometry sequencing of natural non-tryptic peptides from the skin secretion of Rana latastei provided the opportunity to work successfully with these species and demonstrated once again its advantage over automatic approaches.

Proteolytic degradation and deactivation of amphibian skin peptides obtained by electrical stimulation of their dorsal glands.

Amphibians are among the oldest creatures on our planet. Their only defensive weapon efficient against microorganisms and predators involves their skin secretion. The wide range of biological activities of the peptides in the skin secretion of amphibians makes these compounds rather interesting for generation of prospective pharmaceuticals. The first step in studying these molecules requires their structures to be established. Mass spectrometry is the most powerful tool for this purpose. The sampling and sample preparation stages preceding mass spectrometry experiments appear to be rather crucial. The results obtained here demonstrate that these preparation procedures might lead to partial or complete loss of the bioactive peptides in the secretion. Five minutes in water was enough to completely destroy all of the bioactive peptides in the skin secretion of the marsh frog (Rana ridibunda); even immediate addition of methanol to the water solution of the peptides did not prevent partial destruction. Concerted effort should be directed towards development of the most efficient procedure to keep the secreted peptides intact. Graphical Abstract ᅟ.

Mass spectrometric de novo sequencing of natural non-tryptic peptides: comparing peculiarities of collision-induced dissociation (CID) and high energy collision dissociation (HCD).

RATIONALE: Mass spectrometry has shown itself as the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most useful methods of triggering fragmentation and combine complementary techniques. METHODS: Comparison of low-energy collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) modes for sequencing of the natural non-tryptic peptides with disulfide bonds and/or several proline residues in the backbone was achieved using an LTQ FT Ultra Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a 7 T magnet and an LTQ Orbitrap Velos ETD (Thermo Fisher Scientific, Bremen, Germany) instrument. Peptide fractions were obtained by high-performance liquid chromatography (HPLC) separation of frog skin secretion samples from ten species of Rana temporaria, caught in the Kolomna district of Moscow region (Russia). RESULTS: HCD makes the b/y series longer and more pronounced, thus increasing sequence coverage. Fragment ions due to cleavages at the C-termini of proline residues make the sequencing more reliable and may be used to detect missed cleavages in the case of tryptic peptides. Another HCD peculiarity involves formation of pronounced inner fragment ions (secondary y(n)b(m) ion series formed from the abundant primary y-ions). Differences in de novo sequencing of natural non-tryptic peptides with CID and HCD, involving thorough manual expert interpretation of spectra and two automatic sequencing algorithms, are discussed. CONCLUSIONS: Although HCD provides better results, a combination of CID and HCD data may notably increase reliability of de novo sequencing. Several pairs of b2 /a2 -ions may be formed in HCD, complicating the spectra. Automatic de novo sequencing with the available programs remains less efficient than the manual one, independently of the collision energy.

Signaling in insulin-secreting MIN6 pseudoislets and monolayer cells.

Cell-cell interactions are of fundamental importance for cellular function. In islets of Langerhans, which control blood glucose levels by secreting insulin in response to the blood glucose concentration, the secretory response of intact islets is higher than that of insulin-producing beta-cells not arranged in the islet architecture. The objective was to define mechanisms by which cellular performance is enhanced when cells are arranged in three-dimensional space. The task was addressed by making a comprehensive analysis based on protein expression patterns generated from insulin-secreting MIN6 cells grown as islet-like clusters, so-called pseudoislets, and in monolayers. After culture, glucose-stimulated insulin secretion (GSIS) was measured from monolayers and pseudoislets. GSIS rose 6-fold in pseudoislets but only 3-fold in monolayers when the glucose concentration was increased from 2 to 20 mmol/L. Proteins from pseudoislets and monolayers were extracted and analyzed by liquid-chromatography mass spectrometry, and differentially expressed proteins were mapped onto KEGG pathways. Protein profiling identified 1576 proteins, which were common to pseudoislets and monolayers. When mapped onto KEGG pathways, 11 highly enriched pathways were identified. On the basis of differences in expression of proteins belonging to the pathways in pseudoislets and monolayers, predictions of differential pathway activation were performed. Mechanisms enhancing insulin secretory capacity of the beta-cell, when situated in the islet, include pathways regulating glucose metabolism, cell interaction, and translational regulation.

Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (∼0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.

Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL.

Myeloid-derived suppressor cells (MDSCs) inhibit adaptive and innate immunity and accumulate in the blood of persons with cancer, chronic inflammation, trauma, infection, and stress. Some of the factors inducing their accumulation are known; however, mechanisms regulating their turnover have not been identified. Mass spectrometry showed prominent expression of apoptosis pathway proteins, suggesting that MDSC turnover may be regulated by Fas-FasL-mediated apoptosis. This hypothesis was confirmed by showing that blood MDSCs induced by 3 mouse tumors were Fas(+) and apoptosed in response to Fas agonist in vitro and to activated FasL(+) T cells in vivo. FasL-deficient mice contained significantly more blood MDSCs than FasL(+/+) mice, and after removal of primary tumors MDSCs regressed in STAT6(-/-) and CD1(-/-) mice but not in STAT6(-/-)FasL(-/-) or CD1(-/-)FasL(-/-) mice. Fas(+) macrophages and dendritic cells did not apoptose in response to activated T cells, indicating that Fas-FasL regulation of myeloid cells was restricted to MDSCs. These results identify a new mechanism regulating MDSC levels in vivo and show a retaliatory relationship between T cells and MDSCs in that MDSCs suppress T-cell activation; however, once activated, T cells mediate MDSC apoptosis.

L1CAM from human melanoma carries a novel type of N-glycan with Galß1-4Galß1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells.

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose ß1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and ß1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galß1-4Galß1- epitope capped with sialic acid residues A1[3]G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.

A strategy for identification of protein tyrosine phosphorylation.

To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.

Proteomic pathway analysis reveals inflammation increases myeloid-derived suppressor cell resistance to apoptosis.

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry.

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.

Two dimensional mass mapping as a general method of data representation in comprehensive analysis of complex molecular mixtures.

A recent proteomics-grade (95%+ sequence reliability) high-throughput de novo sequencing method utilizes the benefits of high resolution, high mass accuracy, and the use of two complementary fragmentation techniques collision-activated dissociation (CAD) and electron capture dissociation (ECD). With this high-fidelity sequencing approach, hundreds of peptides can be sequenced de novo in a single LC-MS/MS experiment. The high productivity of the new analysis technique has revealed a new bottleneck which occurs in data representation. Here we suggest a new method of data analysis and visualization that presents a comprehensive picture of the peptide content including relative abundances and grouping into families. The 2D mass mapping consists of putting the molecular masses onto a two-dimensional bubble plot, with the relative monoisotopic mass defect and isotopic shift being the axes and with the bubble area proportional to the peptide abundance. Peptides belonging to the same family form a compact group on such a plot, so that the family identity can in many cases be determined from the molecular mass alone. The performance of the method is demonstrated on the high-throughput analysis of skin secretion from three frogs, Rana ridibunda, Rana arvalis, and Rana temporaria. Two dimensional mass maps simplify the task of global comparison between the species and make obvious the similarities and differences in the peptide contents that are obscure in traditional data presentation methods. Even biological activity of the peptide can sometimes be inferred from its position on the plot. Two dimensional mass mapping is a general method applicable to any complex mixture, peptide and nonpeptide alike.

Oxidation versus carboxamidomethylation of S-S bond in ranid frog peptides: pro and contra for de novo MALDI-MS sequencing.

Five natural peptides isolated from ranid skin secretions of European frog species of Rana ridibunda and Rana arvalis (molecular masses 3516, 2674, 2636, 1874, and 1810 Da) were studied by MALDI-TOF/TOF to compare two procedures of disulfide bond cleavage: (1) performic oxidation and (2) reduction/carboxamidomethylation. The processes are relevant for the elucidation of the amino acid sequence inside the seven-member cystine ring at the C-terminus. The results clearly demonstrated that oxidation of the disulfide bond led to notably higher abundances of b- and y-ions, corresponding to the C-terminal peptide bonds, than reduction/carboxamidomethylation. This conclusion is true for all five peptides studied. Besides that, the oxidation procedure is simpler than carboxamidomethylation, as it is a one-step process with no purification required. The oxidation is more reproducible. The results were similar each time the peptide was subjected to the process. It was successfully applied to all five peptides while reduction/carboxamidomethylation failed in the case of brevinin-1Ra, despite all variations of reaction conditions.

Shotgun proteomic analysis to study the decrease of xenograft tumor growth after rosemary extract treatment.

The antiproliferative activity of Rosemary (Rosmarinus officinalis) has been widely studied in different in vitro and in vivo models, which demonstrate that rosemary extracts inhibit the cellular proliferation due to its ability to interact with a wide spectrum of molecular targets. However, a comprehensive proteomics study in vivo has not been carried out yet. In the present work, the effects of rosemary extract on xenograft tumor growth has been studied and, for the first time, a shotgun proteomic analysis based on nano-LC-MS/MS together with stable isotope dimethyl labeling (DML) has been applied to investigate the global protein changes in vivo. Our results show that the daily administration of a polyphenol-enriched rosemary extract reduces the progression of colorectal cancer in vivo with the subsequent deregulation of 74 proteins. The bioinformatic analysis of these proteins indicates that the rosemary extract mainly alters the RNA Post-Transcriptional Modification, the Protein Synthesis and the Amino Acid Metabolism functions and suggests the inactivation of the oncogene MYC. These results demonstrate the high utility of the proposed analytical methodology to determine, simultaneously, the expression levels of a large number of protein biomarkers and to generate new hypothesis about the molecular mechanisms of this extract in vivo.

Hydrogen-free catalytic fractionation of woody biomass.

The pulping industry could become a biorefinery if the lignin and hemicellulose components of the lignocellulose are valorized. Conversion of lignin into well-defined aromatic chemicals is still a major challenge. Lignin depolymerization reactions often occur in parallel with irreversible condensation reactions of the formed fragments. Here, we describe a strategy that markedly suppresses the undesired condensation pathways and allows to selectively transform lignin into a few aromatic compounds. Notably, applying this strategy to woody biomass at organosolv pulping conditions, the hemicellulose, cellulose, and lignin were separated and in parallel the lignin was transformed into aromatic monomers. In addition, we were able to utilize a part of the lignocellulose as an internal source of hydrogen for the reductive lignin transformations. We hope that the presented methodology will inspire researchers in the field of lignin valorization as well as pulp producers to develop more efficient biomass fractionation processes in the future.

The chaperone co-inducer BGP-15 alleviates ventilation-induced diaphragm dysfunction.

Ventilation-induced diaphragm dysfunction (VIDD) is a marked decline in diaphragm function in response to mechanical ventilation, which has negative consequences for individual patients' quality of life and for the health care system, but specific treatment strategies are still lacking. We used an experimental intensive care unit (ICU) model, allowing time-resolved studies of diaphragm structure and function in response to long-term mechanical ventilation and the effects of a pharmacological intervention (the chaperone co-inducer BGP-15). The marked loss of diaphragm muscle fiber function in response to mechanical ventilation was caused by posttranslational modifications (PTMs) of myosin. In a rat model, 10 days of BGP-15 treatment greatly improved diaphragm muscle fiber function (by about 100%), although it did not reverse diaphragm atrophy. The treatment also provided protection from myosin PTMs associated with HSP72 induction and PARP-1 inhibition, resulting in improvement of mitochondrial function and content. Thus, BGP-15 may offer an intervention strategy for reducing VIDD in mechanically ventilated ICU patients.

Proteome analysis of adenovirus using mass spectrometry.

Analysis of proteins and their posttranslational modifications is important for understanding different biological events. For analysis of viral proteomes, an optimal protocol includes production of a highly purified virus that can be investigated with a high-resolving analytical method. In this Methods in Molecular Biology paper we describe a working strategy for how structural proteins in the Adenovirus particle can be studied using liquid chromatography-high-resolving mass spectrometry. This method provides information on the chemical composition of the virus particle. Further, knowledge about amino acids carrying modifications that could be essential for any part of the virus life cycle is collected. We describe in detail alternatives available for preparation of virus for proteome analysis as well as choice of mass spectrometric instrumentation suitable for this kind of analysis.

Post translational modifications in adenovirus type 2.

We have combined 2-D SDS-PAGE with liquid chromatography-high resolving mass spectrometry (LC-MS) to explore the proteome of the adenovirus type 2 (Ad2) at the level of post translational modifications (PTMs). The experimental design included in-solution digestion, followed by titanium dioxide enrichment, as well as in-gel digestion of polypeptides after separation of Ad2 capsid proteins by 1-D and 2-D SDS-PAGE. All samples were analyzed using LC-MS with subsequent manual verification of PTM positions. The results revealed new phosphorylation sites that can explain the observed trains of protein spots observed for the pIII, pIIIa and pIV proteins. The pIIIa protein was found to be the most highly modified protein with now 18 verified sites of phosphorylation, three sites of nitrated tyrosine and one sulfated tyrosine. Nitrated tyrosines were also identified in pII. Lysine acetylations were detected in pII and pVI. The findings make the Ad2 virion much more complex than hitherto believed.