ABSTRACT
An in-depth multi-omic molecular characterisation of poly(adenosine 5'-diphosphate [ADP]-ribose) polymerase (PARP) inhibitors revealed a distinct poly-pharmacology of niraparib (Zejula®) mediated by its interaction with lanosterol synthase (LSS), which is not observed with other PARP inhibitors. Niraparib, in a similar way to the LSS inhibitor Ro-48-8071, induced activation of the 24,25-epoxysterol shunt pathway, which is a regulatory signalling branch of the cholesterol biosynthesis pathway. Interestingly, the combination of a LSS inhibitor with a PARP inhibitor that does not bind to LSS, such as olaparib, had an additive effect on killing of cancer cells to levels comparable to Niraparib as single agent. In addition, the combination of PARP inhibitors and statins, inhibitors of HMGCR, an enzyme catalysing the rate-limiting step in the mevalonate pathway, had a synergistic effect on tumor cell killing in cell lines and patient-derived ovarian tumor organoids. These observations suggest that concomitant inhibition of cholesterol biosynthesis pathway and PARP activity might result in stronger efficacy of these inhibitors against tumor types highly dependent on cholesterol metabolism.
ABSTRACT
Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.
Subject(s)
Pregnenolone , Th2 Cells , Pregnenolone/metabolism , Pregnenolone/pharmacology , Th2 Cells/metabolism , Molecular Dynamics Simulation , Steroids , Carrier Proteins/metabolismABSTRACT
In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large processing bodies (P-bodies) and oogenesis arrest. Here, we show that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. We found that depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, we identify HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics we identify nutritional stress-induced changes in protein phosphorylation that act upstream of α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.
Subject(s)
Histone Deacetylase 1 , Tubulin , Animals , Acetylation , Drosophila/metabolism , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Microtubules/metabolism , Tubulin/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Protein-metabolite interactions play an important role in the cell's metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict 461 likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.
Subject(s)
Chaetomium/metabolism , Metabolomics/methods , Proteomics/methods , Chromatography , Mass SpectrometryABSTRACT
Pregnenolone (P5) promotes prostate cancer cell growth, and de novo synthesis of intratumoural P5 is a potential cause of development of castration resistance. Immune cells can also synthesize P5 de novo. Despite its biological importance, little is known about P5's mode of actions, which appears to be context dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8+ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking and in P5's non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8+ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues, and organisms.
ABSTRACT
How tissues migrate robustly through changing guidance landscapes is poorly understood. Here, quantitative imaging is combined with inducible perturbation experiments to investigate the mechanisms that ensure robust tissue migration in vivo. We show that tissues exposed to acute "chemokine floods" halt transiently before they perfectly adapt, i.e., return to the baseline migration behavior in the continued presence of elevated chemokine levels. A chemokine-triggered phosphorylation of the atypical chemokine receptor Cxcr7b reroutes it from constitutive ubiquitination-regulated degradation to plasma membrane recycling, thus coupling scavenging capacity to extracellular chemokine levels. Finally, tissues expressing phosphorylation-deficient Cxcr7b migrate normally in the presence of physiological chemokine levels but show delayed recovery when challenged with elevated chemokine concentrations. This work establishes that adaptation to chemokine fluctuations can be "outsourced" from canonical GPCR signaling to an autonomously acting scavenger receptor that both senses and dynamically buffers chemokine levels to increase the robustness of tissue migration.
Subject(s)
Cell Movement , Chemokines/metabolism , Embryo, Nonmammalian/metabolism , Receptors, CXCR4/metabolism , Receptors, CXCR/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Cell Communication , Chemokines/genetics , Embryo, Nonmammalian/cytology , Phosphorylation , Receptors, CXCR/genetics , Receptors, CXCR4/genetics , Signal Transduction , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/geneticsABSTRACT
Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.
Subject(s)
Fibroblasts/metabolism , Fusion Regulatory Protein 1, Heavy Chain/genetics , Mechanotransduction, Cellular , Multienzyme Complexes/genetics , Oxidoreductases/genetics , Sphingolipids/biosynthesis , Animals , Dermis/cytology , Dermis/metabolism , Fibroblasts/cytology , Fusion Regulatory Protein 1, Heavy Chain/deficiency , Gene Expression Regulation , Homeostasis , Lipogenesis , Mice , Mice, Transgenic , Multienzyme Complexes/metabolism , Oxidoreductases/metabolism , Primary Cell Culture , Rho Guanine Nucleotide Exchange Factors/genetics , Rho Guanine Nucleotide Exchange Factors/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.
Subject(s)
Aging/genetics , Aging/pathology , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cellular Senescence/genetics , Proteome , Adult , Adult Stem Cells/cytology , Aging/metabolism , Carbon/metabolism , Female , Gene Expression Profiling , Glycolysis , Hematopoiesis , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Male , Middle Aged , Stem Cell Niche , Young AdultABSTRACT
SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.
Subject(s)
Mutation , Myelodysplastic Syndromes/genetics , RNA Splicing Factors/genetics , RNA Splicing , Spliceosomes/genetics , Cohort Studies , DNA Repair , Gene Expression Regulation , Humans , Myelodysplastic Syndromes/epidemiology , Phosphoproteins/genetics , Serine-Arginine Splicing Factors/genetics , Splicing Factor U2AF/genetics , Survival AnalysisABSTRACT
The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.
Subject(s)
Chaetomium/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Cellular Microenvironment , Cross-Linking Reagents , Cryoelectron Microscopy , Fatty Acid Synthase, Type II/chemistry , Fatty Acid Synthase, Type II/metabolism , Fatty Acid Synthase, Type II/ultrastructure , Fungal Proteins/ultrastructure , Mass Spectrometry , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Protein Interaction Mapping , Protein Interaction Maps , Proteomics , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism , Systems BiologyABSTRACT
Disruption of epithelial architecture is a fundamental event during epithelial tumorigenesis. We show that the expression of the cancer-promoting phosphatase PRL-3 (PTP4A3), which is overexpressed in several epithelial cancers, in polarized epithelial MDCK and Caco2 cells leads to invasion and the formation of multiple ectopic, fully polarized lumens in cysts. Both processes disrupt epithelial architecture and are hallmarks of cancer. The pathological relevance of these findings is supported by the knockdown of endogenous PRL-3 in MCF-7 breast cancer cells grown in three-dimensional branched structures, showing the rescue from multiple-lumen- to single-lumen-containing branch ends. Mechanistically, it has been previously shown that ectopic lumens can arise from midbodies that have been mislocalized through the loss of mitotic spindle orientation or through the loss of asymmetric abscission. Here, we show that PRL-3 triggers ectopic lumen formation through midbody mispositioning without altering the spindle orientation or asymmetric abscission, instead, PRL-3 accelerates cytokinesis, suggesting that this process is an alternative new mechanism for ectopic lumen formation in MDCK cysts. The disruption of epithelial architecture by PRL-3 revealed here is a newly recognized mechanism for PRL-3-promoted cancer progression.
Subject(s)
Cell Shape , Epithelial Cells/cytology , Epithelial Cells/metabolism , Mitosis , Neoplasm Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Animals , Caco-2 Cells , Cell Polarity , Cytokinesis , Dogs , Humans , MCF-7 Cells , Madin Darby Canine Kidney Cells , Models, BiologicalABSTRACT
We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated '-omics' data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes.
Subject(s)
Genome, Bacterial/genetics , Genomics/methods , Mycoplasma pneumoniae/genetics , Mycoplasma pneumoniae/metabolism , Proteomics/methods , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Cluster Analysis , Gene Expression Profiling/methods , Gene Expression Profiling/statistics & numerical data , Gene Expression Regulation , Genomics/statistics & numerical data , Molecular Sequence Annotation , Molecular Sequence Data , Protein Processing, Post-Translational , Proteome/genetics , Proteome/metabolism , Proteomics/statistics & numerical data , RNA, Untranslated/genetics , Systems Biology/methods , Systems Biology/statistics & numerical dataABSTRACT
BACKGROUND: The multifunctional Ca(2+)- and calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is large-scale analysis of phosphorylation events by mass spectrometry. However, current large-scale phosphoproteomics approaches have proved inadequate for high-fidelity identification of kinase-specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue. METHODS AND RESULTS: To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial-delimited expression of the specific peptide inhibitor of CaMKII (AC3-I) or an inactive control (AC3-C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of ß-adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3-I and AC3-C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high-resolution LC-MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3-I-mediated CaMKII inhibition. CONCLUSIONS: Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Myocardium/enzymology , Proteomics , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Cation Exchange Resins , Chromatography, Ion Exchange , Enzyme Activation , Isotope Labeling , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Peptides/genetics , Peptides/metabolism , Phosphorylation , Proteomics/methods , Signal Transduction , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
Saccharomyces cerevisiae τ55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (τ55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of τ55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify τ55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.
Subject(s)
Phosphoric Monoester Hydrolases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Transcription Factors, TFIII/chemistry , Crystallography, X-Ray , Molecular Docking Simulation , Phosphoric Monoester Hydrolases/genetics , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors, TFIII/geneticsABSTRACT
In order to understand cellular signaling, a clear understanding of kinase-substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome.
Subject(s)
Cell Cycle Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Assays , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Amino Acid Motifs , Binding Sites , Cell Cycle Proteins/chemistry , Chromatography, Ion Exchange , Consensus Sequence , Cyclic AMP-Dependent Protein Kinases/chemistry , HEK293 Cells , HeLa Cells , Humans , Isotope Labeling/methods , Methylation , Molecular Sequence Data , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/chemistry , Protein-Tyrosine Kinases/chemistry , Proteomics , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
The small GTPase Rap1 is required for proper cell-cell junction formation and also plays a key role in mediating cAMP-induced tightening of adherens junctions and subsequent increased barrier function of endothelial cells. To further study how Rap1 controls barrier function, we performed quantitative global phosphoproteomics in human umbilical vein endothelial cells (HUVECs) prior to and after Rap1 activation by the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM (007-AM). Tryptic digests were labeled using stable isotope dimethyl labeling, enriched with phosphopeptides by strong cation exchange (SCX), followed by titanium(iv) immobilized metal affinity chromatography (Ti(4+)-IMAC) and analyzed by high resolution mass spectrometry. We identified 19 859 unique phosphopeptides containing 17 278 unique phosphosites on 4594 phosphoproteins, providing the largest HUVEC phosphoproteome to date. Of all identified phosphosites, 220 (â¼1%) were more than 1.5-fold up- or downregulated upon Rap activation, in two independent experiments. Compatible with the function of Rap1, these alterations were found predominantly in proteins regulating the actin cytoskeleton, cell-cell junctions and cell adhesion.
Subject(s)
Human Umbilical Vein Endothelial Cells/metabolism , Phosphoproteins/metabolism , Proteome , Signal Transduction , rap1 GTP-Binding Proteins/metabolism , Actins/metabolism , Adherens Junctions/metabolism , Amino Acid Sequence , Cell Adhesion , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Guanine Nucleotide Exchange Factors/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Intercellular Junctions/metabolism , Peptides/metabolism , Phosphoproteins/chemistry , Phosphorylation/drug effects , Position-Specific Scoring Matrices , Protein Interaction Maps , Proteomics/methods , Signal Transduction/drug effectsABSTRACT
Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or chemical isotope labeling with MS based quantification or chemical labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and chemical labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used.
Subject(s)
Algorithms , Databases, Protein , Isotope Labeling/methods , Proteome/metabolism , Proteomics/methods , HeLa Cells , Humans , Mass Spectrometry/methodsABSTRACT
Here, we describe an in-house built ultra-high pressure liquid chromatography (UHPLC) system, with little complexity in design and high separation power combined with convenience in operation. This system enables the use of long columns of 40 cm packed with 1.8 µm particles generating pressures below 1000 bar. Furthermore, the system could be operated at flow rates between 50 and 200 nL min(-1) while maintaining its separation power. Several gradients were optimized ranging from 23 to 458 minutes. With the longest gradient we identified over 4500 protein groups and more than 26,000 unique peptides from 1 µg of a human cancer cell lysate in a single run using an Orbitrap Velos - a level of performance often seen solely using multidimensional separation strategies. Further experiments using a mass spectrometer with faster sequencing speeds, like the TripleTOF 5600, enabled us to identify over 1400 protein groups in a 23 min gradient. The TripleTOF 5600 performed especially well, compared to the Orbitrap Velos, for the shorter gradients used. Our data demonstrate that the combination of UHPLC with high resolution mass spectrometry at increased sequencing speeds enables extensive proteome analysis in single runs.
Subject(s)
Escherichia coli Proteins/analysis , Neoplasm Proteins/analysis , Serum Albumin, Bovine/analysis , Animals , Cattle , Chromatography, High Pressure Liquid/instrumentation , Equipment Design , HeLa Cells , HumansABSTRACT
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.
Subject(s)
Chromatography, Liquid/methods , Peptides/isolation & purification , Proteome/chemistry , Amino Acids/analysis , Chromatography, Ion Exchange , Chromatography, Reverse-Phase , Humans , Mass Spectrometry , Online Systems , Protein Processing, Post-Translational , Proteomics/methodsABSTRACT
We present a straightforward method to enrich phosphopeptides with multiple basic residues, an under-represented class in common enrichment strategies. Our method is based on a two-dimensional strong cation exchange (SCX) strategy, operating at two different acidic pHs, enabling both separation and enrichment of different classes of phosphopeptides. The principle of enrichment is based on the change of net charge of phosphorylated peptides under strong acidic conditions in the second SCX, whereas the net charge of regular peptides remains unchanged, thus enabling separation based on net charge. Application of our tandem SCX approach to a modest amount of human cells allowed the identification of over 10,000 unique "basic" phosphopeptides of which many represent putative targets of basophilic kinases.