Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms.

The mitochondrial inner membrane glycerophospholipid cardiolipin (CL) associates with mitochondrial proteins to regulate their activities and facilitate protein complex and supercomplex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated. Trypanosoma brucei bloodstream forms use an unconventional ETC composed of glycerol-3-phosphate dehydrogenase and alternative oxidase (AOX), while the mitochondrial membrane potential (ΔΨm) is generated by the hydrolytic action of the Fo F1 -ATP synthase (aka Fo F1 -ATPase). We now report that the inducible depletion of cardiolipin synthase (TbCls) is essential for survival of T brucei bloodstream forms. Loss of CL caused a rapid drop in ATP levels and a decline in the ΔΨm. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of Fo F1 -ATPase subunits and AOX, resulting in a strong decline of glycerol-3-phosphate-stimulated oxygen consumption. The changes in cellular respiration preceded the observed decrease in Fo F1 -ATPase stability, suggesting that the AOX-mediated ETC is the first pathway responding to the decline in CL. Select proteins and pathways involved in glucose and amino acid metabolism were upregulated to counteract the CL depletion-induced drop in cellular ATP.

A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.

Proteomic profiling of human cancer pseudopodia for the identification of anti-metastatic drug candidates.

Cancer metastasis causes approximately 90% of all cancer-related death and independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis implies cancer cell migration and invasion throughout the body. Migration requires the formation of pseudopodia in the direction of movement, but a detailed understanding of this process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human cancer pseudopodia to examine this mechanisms essential to metastasis formation, and identify potential candidates for pharmacological interference with the process. We demonstrate that Prohibitins (PHBs) are significantly enriched in the pseudopodia fraction derived from cancer cells, and knockdown of PHBs, as well as their chemical inhibition through Rocaglamide (Roc-A), efficiently reduces cancer cell migration.

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. SIGNIFICANCE: Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.

The arc of Mass Spectrometry Exchange Formats is long, but it bends toward HDF5.

The evolution of data exchange in Mass Spectrometry spans decades and has ranged from human-readable text files representing individual scans or collections thereof (McDonald et al., 2004) through the official standard XML-based (Harold, Means, & Udemadu, 2005) data interchange standard (Deutsch, 2012), to increasingly compressed (Teleman et al., 2014) variants of this standard sometimes requiring purely binary adjunct files (Römpp et al., 2011). While the desire to maintain even partial human readability is understandable, the inherent mismatch between XML's textual and irregular format relative to the numeric and highly regular nature of actual spectral data, along with the explosive growth in dataset scales and the resulting need for efficient (binary and indexed) access has led to a phenomenon referred to as "technical drift" (Davis, 2013). While the drift is being continuously corrected using adjunct formats, compression schemes, and programs (Röst et al., 2015), we propose that the future of Mass Spectrometry Exchange Formats lies in the continued reliance and development of the PSI-MS (Mayer et al., 2014) controlled vocabulary, along with an expedited shift to an alternative, thriving and well-supported ecosystem for scientific data-exchange, storage, and access in binary form, namely that of HDF5 (Koranne, 2011). Indeed, pioneering efforts to leverage this universal, binary, and hierarchical data-format have already been published (Wilhelm et al., 2012; Rübel et al., 2013) though they have under-utilized self-description, a key property shared by HDF5 and XML. We demonstrate that a straightforward usage of plain ("vanilla") HDF5 yields immediate returns including, but not limited to, highly efficient data access, platform independent data viewers, a variety of libraries (Collette, 2014) for data retrieval and manipulation in many programming languages and remote data access through comprehensive RESTful data-servers. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:668-673, 2017.

Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Quantitative proteomic approaches in mouse: stable isotope incorporation by metabolic (SILAC) or chemical labeling (reductive dimethylation) combined with high-resolution mass spectrometry.

Mass spectrometry-based quantitative proteomics is a powerful method for in-depth exploration of protein expression, allowing researchers to probe its regulation and study signal-transduction networks, protein turnover, secretion, and spatial distribution, as well as post-translational modification and protein-protein interaction, on a large scale. Precise protein quantitation may be achieved by incorporation of stable isotopes, which introduce a mass shift detectable by mass spectrometry, allowing multiplexing of several samples and therefore relative quantification. Stable isotope incorporation into proteins or peptides can be attained either by metabolic labeling (e.g., SILAC) or by chemical labeling (e.g., reductive dimethylation). Both labeling approaches are presented here. They are straightforward and robust and can be applied to murine samples. While both SILAC and reductive dimethylation offer similar multiplexing capabilities and quantitative accuracy, reductive dimethylation is more versatile and can be used with any sample type.

Genome-wide identification and functional analysis of Apobec-1-mediated C-to-U RNA editing in mouse small intestine and liver.

BACKGROUND: RNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoprotein B mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1-mediated C-to-U RNA editing remain incompletely explored. RESULTS: Deep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and Apobec-1-deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs, all within 3' untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites are unique to liver. Changes in RNA editing lead to corresponding changes in intestinal mRNA and protein levels for 11 genes. Analysis of RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression reveals that a subset of targets identified in wild-type mice are restored in Apobec-1-deficient mouse intestine and liver following Apobec-1 rescue. We find distinctive polysome profiles for several RNA editing targets and demonstrate novel exonic editing sites in nuclear preparations from intestine but not hepatic apolipoprotein B RNA. RNA editing is validated using cell-free extracts from wild-type but not Apobec-1-deficient mice, demonstrating that Apobec-1 is required. CONCLUSIONS: These studies define selective, tissue-specific targets of Apobec-1-dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific.

Heterolytic N-Cα bond cleavage in electron capture and transfer dissociation of peptide cations.

Mass spectrometry techniques employing electron capture and electron transfer dissociation represent powerful approaches for the analysis of biological samples. Despite routine employment in analytical fields, the underlying physical processes dictating peptide fragmentation remain less understood. Among the most accepted mechanisms, the Cornell proposal of McLafferty postulates that the homolytic cleavage of N-C(α) bonds located in the peptide backbone occurs on the right (C-terminal) side of a hydrogen acceptor carbonyl group. Here, we illustrate that an alternative "enol" mechanism, based on a heterolytic N-C(α) bond cleavage located on the left (N-terminal) side of an acceptor carbonyl group, not only is thermodynamically viable but also often represents the energetically preferred cleavage route.

On the utility of predictive chromatography to complement mass spectrometry based intact protein identification.

The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, "reading" a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop "predictive" HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using "predictive" HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments.

Repeatability and reproducibility of product ion abundances in electron capture dissociation mass spectrometry of peptides.

Site-specific reproducibility and repeatability of electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) are of fundamental importance for product ion abundance (PIA)-based peptide and protein structure analysis. However, despite the growing interest in ECD PIA-based applications, these parameters have not yet been investigated in a consistent manner. Here, we first provide a detailed description of the experimental parameters for ECD-based tandem mass spectrometry performed on a hybrid linear ion trap (LTQ) FT-ICR MS. In the following, we describe the evaluation and comparison of ECD and infrared multiphoton dissociation (IRMPD) PIA methodologies upon variation of a number of experimental parameters, for example, cathode potential (electron energy), laser power, electron and photon irradiation periods and pre- irradiation delays, as well as precursor ion number. Ranges of experimental parameters that yielded an average PIA variation below 5% and 15% were determined for ECD and IRMPD, respectively. We report cleavage site-dependent ECD PIA variation below 20% and correlation coefficients between fragmentation patterns superior to 0.95 for experiments performed on three FT-ICR MS instruments. Overall, the encouraging results obtained for ECD PIA reproducibility and repeatability support the use of ECD PIA as a complementary source of information to m/z data in radical-induced dissociation applied for peptide and protein structure analysis.

Structural preferences of gas-phase M2TMP monomers upon sequence variations.

The conformations of a number of M2TMP(22-46) sequence variants have been investigated using ion mobility spectrometry (IMS). Substantial conformational changes were evidenced by IMS upon the variation of a single amino acid in the peptide sequence, with two main drift time signatures. Replica-exchange molecular dynamics simulations were used to help assign the structures of the different identified conformers. Even though one-on-one agreement with experiment was found for only two variants, the simulations generally confirmed the existence of two structural families. Based on these results, most of the triply protonated variants, including the wild-type peptide, were found to display collision cross sections in agreement with compact conformations in the gas phase, whereas they tend to form extended α-helices in the condensed phase, as confirmed by circular dichroism and previously reported NMR measurements. The destabilization of α-helices in vacuo upon amino acid substitution is interpreted as being driven by the solvation pattern of the charges.

Radical stability directs electron capture and transfer dissociation of ß-amino acids in peptides.

We report on the characteristics of the radical-ion-driven dissociation of a diverse array of ß-amino acids incorporated into α-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for ß-amino acids than for their α-form counterparts. In particular, only aromatic (e.g., ß-Phe), and to a substantially lower extent, carbonyl-containing (e.g., ß-Glu and ß-Gln) amino acid side chains, lead to N-Cß bond cleavage in the corresponding ß-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α-amino acids, ECD of peptides composed mainly of ß-amino acids reveals a shift in cleavage priority from the N-Cß to the Cα-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven ß-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.

Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability.

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. The systematic arrangement of amino acids reported here indicates a possible route toward development of a predictive model for quantitative electron capture/transfer dissociation tandem mass spectrometry, with possible applications in proteomics.

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins.

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as alpha-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an alpha-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of alpha-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure.

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels.

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.

High resolution mass spectrometry for studying the interactions of cisplatin with oligonucleotides.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to probe the interaction of the anticancer drug cisplatin with oligonucleotides. The binding kinetics, the nature of the adducts formed, and the location of the binding site within the specifically designed double-stranded DNA oligonucleotides, ds(GTATTGGCACGTA) and ds(GTACCGGTGTGTA), were determined by recording mass spectra over time and/or employing tandem mass spectrometry (MS/MS). The FT-ICR MS studies show that binding to DNA takes place via a [Pt(NH 3) 2Cl] (+) intermediate prior to formation of bifunctional [Pt(NH 3) 2] (2+) adducts. Tandem MS reveals that the major binding sites correspond to GG and GTG, the known preferred binding sites for cisplatin, and demonstrates the preference for binding to guanosine within the oligonucleotide. The obtained results are discussed and compared to published data obtained by other mass spectrometric techniques, NMR spectroscopy and X-ray crystallography.