Differential stoichiometry among core ribosomal proteins

Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its deregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass-spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.Comment: 31 pages, 8 figure

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials

Long-Lived Electron Capture Dissociation Product Ions Experience Radical Migration via Hydrogen Abstraction

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the α-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the α-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c·, and they also show c·-1 peaks in the deuterated peptides only. The presence of the c·-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H· and −CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments

Quantifying Condition-Dependent Intracellular Protein Levels Enables High-Precision Fitness Estimates

Countless studies monitor the growth rate of microbial populations as a measure of fitness. However, an enormous gap separates growth-rate differences measurable in the laboratory from those that natural selection can distinguish efficiently. Taking advantage of the recent discovery that transcript and protein levels in budding yeast closely track growth rate, we explore the possibility that growth rate can be more sensitively inferred by monitoring the proteomic response to growth, rather than growth itself. We find a set of proteins whose levels, in aggregate, enable prediction of growth rate to a higher precision than direct measurements. However, we find little overlap between these proteins and those that closely track growth rate in other studies. These results suggest that, in yeast, the pathways that set the pace of cell division can differ depending on the growth-altering stimulus. Still, with proper validation, protein measurements can provide high-precision growth estimates that allow extension of phenotypic growth-based assays closer to the limits of evolutionary selection

Systems-Level Response to Point Mutations in a Core Metabolic Enzyme Modulates Genotype-Phenotype Relationship

Linking the molecular effects of mutations to fitness is central to understanding evolutionary dynamics. Here we establish a quantitative relation between the global effect of mutations on the E. coli proteome and bacterial fitness. We created E. coli strains with specific destabilizing mutations in the chromosomal folA gene encoding dihydrofolate reductase (DHFR) and quantified the ensuing changes in the abundances of 2000+ E. coli proteins in mutant strains using tandem mass tags with subsequent LC-MS/MS. mRNA abundances in the same E. coli strains were also quantified. The proteomic effects of mutations in DHFR are quantitatively linked to phenotype: the standard deviations of the distributions of logarithms of relative (to wild-type) protein abundances anti-correlate with bacterial growth rates. Proteomes hierarchically cluster first by media conditions, and within each condition, by the severity of the perturbation to DHFR function. These results highlight the importance of a systems-level layer in the genotype-phenotype relationship.Chemistry and Chemical Biolog

Peptidomic discovery of short open reading frame-encoded peptides in human cells

The amount of the transcriptome that is translated into polypeptides is of fundamental importance. We developed a peptidomic strategy to detect short ORF (sORF)-encoded polypeptides (SEPs) in human cells. We identified 90 SEPs, 86 of which are novel, the largest number of human SEPs ever reported. SEP abundances range from 10-1000 molecules per cell, identical to known proteins. SEPs arise from sORFs in non-coding RNAs as well as multi-cistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that non-canonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8/1866) of long intergenic non-coding RNAs (lincRNAs). Together, these results provide the strongest evidence to date that the human proteome is more complex than previously appreciated

Discovery of Human sORF-Encoded Polypeptides (SEPs) in Cell Lines and Tissue

The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation

The Cdc42-selective GAP Rich regulates postsynaptic development and retrograde BMP transsynaptic signaling

Inhibition of Cdc42 by dRich induces postsynaptic release of the BMP ligand Glass bottom boat