Primordial Black Holes as Generators of Cosmic Structures

Primordial black holes (PBHs) could provide the dark matter in various mass windows below $10^2 M_{\odot}$ and those of $30 M_{\odot}$ might explain the LIGO events. PBHs much larger than this might have important consequences even if they provide only a small fraction of the dark matter. In particular, they could generate cosmological structure either individually through through the `seed' effect or collectively through the `Poisson' effect, thereby alleviating some problems associated with the standard CDM scenario. If the PBHs all have a similar mass and make a small contribution to the dark matter, then the seed effect dominates on small scales, in which case PBHs could seed the supermassive black holes in galactic nuclei or even galaxies themselves. If they have a similar mass and provide the dark matter, the Poisson effect dominates on all scales and the first bound clouds would form earlier than in the usual scenario, with interesting observational consequences. If the PBHs have an extended mass spectrum, which is more likely, they could fulfill all three roles - providing the dark matter, binding the first bound clouds and generating galaxies. In this case, the galactic mass function naturally has the observed form, with the galaxy mass being simply related to the black hole mass. The stochastic gravitational wave background from the PBHs in this scenario would extend continuously from the LIGO frequency to the LISA frequency, offering a potential goal for future surveys.Comment: 48 pages, 3 figures, accepted for publication in Monthly Notices of Royal Astronomical Societ

Sample Collection Method Bias Effects in Quantitative Phosphoproteomics

Current advances in selective enrichment, fractionation, and MS detection of phosphorylated peptides allowed identification and quantitation of tens of thousands phosphosites from minute amounts of biological material. One of the major challenges in the field is preserving the in vivo phosphorylation state of the proteins throughout the sample preparation workflow. This is typically achieved by using phosphatase inhibitors and denaturing conditions during cell lysis. Here we determine if the upstream cell collection techniques could introduce changes in protein phosphorylation. To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, we compared different sample workflows by metabolic labeling and quantitative mass spectrometry on <i>Saccharomyces cerevisiae</i> cell cultures. We identified highly similar phosphopeptides for cells harvested in ice cold isotonic phosphate buffer, cold ethanol, trichloroacetic acid, and liquid nitrogen. However, quantitative analyses revealed that the commonly used phosphate buffer unexpectedly activated signaling events. Such effects may introduce systematic bias in phosphoproteomics measurements and biochemical analysis

Improvement of Quantitative Measurements in Multiplex Proteomics Using High-Field Asymmetric Waveform Spectrometry

Quantitative proteomics using isobaric reagent tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provides a convenient approach to compare changes in protein abundance across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC–FAIMS–MS/MS analyses of human and yeast protein digests led to significant reductions in interfering ions, which increased the number of quantifiable peptides by up to 68% while significantly improving the accuracy of abundance measurements compared to that with conventional LC–MS/MS. The improvement in quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment

Sample Collection Method Bias Effects in Quantitative Phosphoproteomics

Current advances in selective enrichment, fractionation, and MS detection of phosphorylated peptides allowed identification and quantitation of tens of thousands phosphosites from minute amounts of biological material. One of the major challenges in the field is preserving the in vivo phosphorylation state of the proteins throughout the sample preparation workflow. This is typically achieved by using phosphatase inhibitors and denaturing conditions during cell lysis. Here we determine if the upstream cell collection techniques could introduce changes in protein phosphorylation. To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, we compared different sample workflows by metabolic labeling and quantitative mass spectrometry on <i>Saccharomyces cerevisiae</i> cell cultures. We identified highly similar phosphopeptides for cells harvested in ice cold isotonic phosphate buffer, cold ethanol, trichloroacetic acid, and liquid nitrogen. However, quantitative analyses revealed that the commonly used phosphate buffer unexpectedly activated signaling events. Such effects may introduce systematic bias in phosphoproteomics measurements and biochemical analysis

Displacement of N/Q-rich Peptides on TiO₂ Beads Enhances the Depth and Coverage of Yeast Phosphoproteome Analyses

Phosphorylation is a reversible protein modification that regulates major cellular processes such as cell division, growth, and differentiation through highly dynamic and complex signaling pathways. Large-scale phosphoproteomics analyses have been greatly facilitated using affinity chromatography such as metal oxide affinity chromatography (e.g., TiO₂), which in combination with mass spectrometry has enabled unbiased detection and quantification of thousands of phosphorylation sites in a single experiment. However, global phosphoproteome analyses do not provide comparable enrichment yields for different model organisms. While the proportion of phosphopeptides exceed 90% in mammalian cells using TiO₂, similar levels have been notoriously difficult to achieve for yeast or dictylostelium cells. In a systematic study of TiO₂ using cell extracts from different organisms, we determined that phosphopeptides are coenriched with peptides containing repetitive stretches of glutamine and asparagine residues. The proportion of these nonspecific binders can reach up to 50% in cell extracts from budding yeast and thus limit the depth and comprehensiveness of phosphoproteomics analyses. To address this limitation, we developed an effective method that used decoy amino acids to reduce the extent of nonspecific peptide binding and improve the recovery and detection of low abundance phosphopeptides that remained undetected by conventional TiO₂ enrichment protocols

Displacement of N/Q-rich Peptides on TiO₂ Beads Enhances the Depth and Coverage of Yeast Phosphoproteome Analyses

Phosphorylation is a reversible protein modification that regulates major cellular processes such as cell division, growth, and differentiation through highly dynamic and complex signaling pathways. Large-scale phosphoproteomics analyses have been greatly facilitated using affinity chromatography such as metal oxide affinity chromatography (e.g., TiO₂), which in combination with mass spectrometry has enabled unbiased detection and quantification of thousands of phosphorylation sites in a single experiment. However, global phosphoproteome analyses do not provide comparable enrichment yields for different model organisms. While the proportion of phosphopeptides exceed 90% in mammalian cells using TiO₂, similar levels have been notoriously difficult to achieve for yeast or dictylostelium cells. In a systematic study of TiO₂ using cell extracts from different organisms, we determined that phosphopeptides are coenriched with peptides containing repetitive stretches of glutamine and asparagine residues. The proportion of these nonspecific binders can reach up to 50% in cell extracts from budding yeast and thus limit the depth and comprehensiveness of phosphoproteomics analyses. To address this limitation, we developed an effective method that used decoy amino acids to reduce the extent of nonspecific peptide binding and improve the recovery and detection of low abundance phosphopeptides that remained undetected by conventional TiO₂ enrichment protocols

Displacement of N/Q-rich Peptides on TiO₂ Beads Enhances the Depth and Coverage of Yeast Phosphoproteome Analyses

Phosphorylation is a reversible protein modification that regulates major cellular processes such as cell division, growth, and differentiation through highly dynamic and complex signaling pathways. Large-scale phosphoproteomics analyses have been greatly facilitated using affinity chromatography such as metal oxide affinity chromatography (e.g., TiO₂), which in combination with mass spectrometry has enabled unbiased detection and quantification of thousands of phosphorylation sites in a single experiment. However, global phosphoproteome analyses do not provide comparable enrichment yields for different model organisms. While the proportion of phosphopeptides exceed 90% in mammalian cells using TiO₂, similar levels have been notoriously difficult to achieve for yeast or dictylostelium cells. In a systematic study of TiO₂ using cell extracts from different organisms, we determined that phosphopeptides are coenriched with peptides containing repetitive stretches of glutamine and asparagine residues. The proportion of these nonspecific binders can reach up to 50% in cell extracts from budding yeast and thus limit the depth and comprehensiveness of phosphoproteomics analyses. To address this limitation, we developed an effective method that used decoy amino acids to reduce the extent of nonspecific peptide binding and improve the recovery and detection of low abundance phosphopeptides that remained undetected by conventional TiO₂ enrichment protocols

Proteomics Analysis of Herpes Simplex Virus Type 1‑Infected Cells Reveals Dynamic Changes of Viral Protein Expression, Ubiquitylation, and Phosphorylation

Herpesviruses are among the most complex and widespread human viruses and cause a number of diseases ranging from cold sores to genital infections and encephalitis. While the composition of viral particles has been studied, less is known about the expression of the whole viral proteome in infected cells. Here, we analyzed the proteome of the prototypical Herpes Simplex Virus type 1 (HSV1) in infected cells by mass spectrometry. Using a high sensitivity LTQ-Orbitrap, we achieved a very high level of protein coverage and identified a total of 67 structural and nonstructural viral proteins. We also identified 90 novel phosphorylation sites and 10 novel ubiquitylation sites on different viral proteins. Ubiquitylation was observed on nine HSV1 proteins. We identified phosphorylation sites on about half of the detected viral proteins; many of the highly phosphorylated ones are known to regulate gene expression. Treatment with inhibitors of DNA replication induced changes of both viral protein abundance and modifications, highlighting the interdependence of viral proteins during the life cycle. Given the importance of expression dynamics, ubiquitylation, and phosphorylation for protein function, these findings will serve as important tools for future studies on herpesvirus biology

Proteomics Analysis of Herpes Simplex Virus Type 1‑Infected Cells Reveals Dynamic Changes of Viral Protein Expression, Ubiquitylation, and Phosphorylation

Herpesviruses are among the most complex and widespread human viruses and cause a number of diseases ranging from cold sores to genital infections and encephalitis. While the composition of viral particles has been studied, less is known about the expression of the whole viral proteome in infected cells. Here, we analyzed the proteome of the prototypical Herpes Simplex Virus type 1 (HSV1) in infected cells by mass spectrometry. Using a high sensitivity LTQ-Orbitrap, we achieved a very high level of protein coverage and identified a total of 67 structural and nonstructural viral proteins. We also identified 90 novel phosphorylation sites and 10 novel ubiquitylation sites on different viral proteins. Ubiquitylation was observed on nine HSV1 proteins. We identified phosphorylation sites on about half of the detected viral proteins; many of the highly phosphorylated ones are known to regulate gene expression. Treatment with inhibitors of DNA replication induced changes of both viral protein abundance and modifications, highlighting the interdependence of viral proteins during the life cycle. Given the importance of expression dynamics, ubiquitylation, and phosphorylation for protein function, these findings will serve as important tools for future studies on herpesvirus biology

Occurrence and Detection of Phosphopeptide Isomers in Large-Scale Phosphoproteomics Experiments

The past decade has been marked by the emergence of selective affinity media and sensitive mass spectrometry instrumentation that facilitated large-scale phosphoproteome analyses and expanded the repertoire of protein phosphorylation. Despite these remarkable advances, the precise location of the phosphorylation site still represents a sizable challenge in view of the labile nature of the phosphoester bond and the presence of neighboring phosphorylatable residues within the same peptide. This difficulty is exacerbated by the combinatorial distribution of phosphorylated residues giving rise to different phosphopeptide isomers. These peptides have similar physicochemical properties, and their separation by LC is often problematic. Few studies have described the frequency and distribution of phosphoisomers in large-scale phosphoproteomics experiments, and no convenient informatics tools currently exist to facilitate their detection. To address this analytical challenge, we developed two algorithms to detect separated and co-eluting phosphopeptide isomers and target their subsequent identification using an inclusion list in LC–MS/MS experiments. Using these algorithms, we determined that the proportion of isomers present in phosphoproteomics studies from mouse, rat, and fly cell extracts represents 3–6% of all identified phosphopeptides. While conventional analysis can identify chromatographically separated phosphopeptides, targeted LC–MS/MS analyses using inclusion lists provided complementary identification and expanded the number of phosphopeptide isomers by at least 52%. Interestingly, these analyses revealed that the occurrence of phosphopeptides isomers can also correlate with the presence of extended phosphorylatable amino acids that can act as a “phosphorylation switch” to bind complementary domains such as those present in SR proteins and ribonucleoprotein complexes