Data independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. Here, we introduce an experimental workflow ...that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in a proteomics experiment. These coordinates calibrate protein databases or spectrum libraries to a specific mass spectrometer and chromatography setup, facilitating DIA-only pipelines and the reuse of global resource libraries. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect 20-25% more peptides from DIA experiments than with data dependent acquisition-based spectrum libraries alone.
Proteins can be phosphorylated at neighboring sites resulting in different functional states, and studying the regulation of these sites has been challenging. Here we present Thesaurus, a search ...engine that detects and quantifies phosphopeptide positional isomers from parallel reaction monitoring and data-independent acquisition mass spectrometry experiments. We apply Thesaurus to analyze phosphorylation events in the PI3K/AKT signaling pathway and show neighboring sites with distinct regulation.
Post-translational modification (PTM) of proteins allows cells to regulate protein functions, transduce signals and respond to perturbations. PTMs expand protein functionality and diversity, which ...leads to increased proteome complexity. PTM crosstalk describes the combinatorial action of multiple PTMs on the same or on different proteins for higher order regulation. Here we review how recent advances in proteomic technologies, mass spectrometry instrumentation, and bioinformatics spurred the proteome-wide identification of PTM crosstalk through measurements of PTM sites. We provide an overview of the basic modes of PTM crosstalk, the proteomic methods to elucidate PTM crosstalk, and approaches that can inform about the functional consequences of PTM crosstalk.
Display omitted
•Description of basic modules and different modes of PTM crosstalk.•Overview of current proteomic methods to identify and infer PTM crosstalk.•Discussion of large-scale approaches to characterize functional PTM crosstalk.•Future directions and potential proteomic methods for elucidating PTM crosstalk.
We provide an overview of current experimental and computational proteomic methods, as well as a perspective on emerging technologies to study PTM crosstalk.
The success in profiling the phosphoproteome by mass spectrometry-based proteomics has been intimately related to the availability of methods that selectively enrich for phosphopeptides. To this end, ...we describe a protocol that combines two sequential enrichment steps. First, strong cation exchange (SCX) chromatography separates peptides by solution charge. Phosphate groups contribute to solution charge by adding a negative charge at pH 2.7. Therefore, at that pH, phosphopeptides are expected to elute earlier than their nonphosphorylated homologs. Second, immobilized metal affinity chromatography (IMAC) takes advantage of phosphate's affinity for metal ions such as Fe(3+) to uniformly enrich for phosphopeptides from the previously collected SCX fractions. We have successfully employed the SCX/IMAC enrichment strategy in the exploration of phosphoproteomes from several systems including mouse liver and Drosophila embryos characterizing over 5,500 and 13,000 phosphorylation events, respectively. The SCX/IMAC enrichment protocol requires 2 days, and the entire procedure from cells to a phosphorylation data set can be completed in less than 10 days.
Determining the correct localization of post-translational modifications (PTMs) on peptides aids in interpreting their effect on protein function. While most algorithms for this task are available as ...standalone applications or incorporated into software suites, improving their versatility through access from popular scripting languages facilitates experimentation and incorporation into novel workflows. Here we describe pyAscore, an efficient and versatile implementation of the Ascore algorithm in Python for scoring the localization of user defined PTMs in data dependent mass spectrometry. pyAscore can be used from the command line or imported into Python scripts and accepts standard file formats from popular software tools used in bottom-up proteomics. Access to internal objects for scoring and working with modified peptides adds to the toolbox for working with PTMs in Python. pyAscore is available as an open source package for Python 3.6+ on all major operating systems and can be found at pyascore.readthedocs.io.
Recent developments in proteomics have enabled signaling studies where > 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, ...reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase–substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid‐robotic phosphoproteomics (R2‐P2), an end‐to‐end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry‐ready phosphopeptides. R2‐P2 is rapid, robust, versatile, and high‐throughput. To showcase the method, we applied it, in combination with data‐independent acquisition mass spectrometry, to study signaling dynamics in the mitogen‐activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high‐osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large‐scale signaling studies involving hundreds of perturbations opening the door to systems‐level studies aiming to capture signaling complexity.
Synopsis
The study presents R2‐P2, an automated end‐to‐end phosphoproteomic sample preparation method. Application of R2‐P2 to study phosphorylation temporal dynamics in yeast stimulated to perturb MAPK signaling reveals treatment‐specific responses and pathway crosstalks.
R2‐P2 is a novel workflow for proteomic and phosphoproteomic sample preparation that compares favorably to common methods.
R2‐P2 is automated, high‐throughput, rapid, robust, reproducible and yields high phosphopeptide enrichment efficiency.
R2‐P2 facilitates large‐scale cell signaling studies.
R2‐P2 in combination with DIA‐MS reveals broad and specific signaling events along the different branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors.
The study presents R2‐P2, an automated end‐to‐end phosphoproteomic sample preparation method. Application of R2‐P2 to study phosphorylation temporal dynamics in yeast stimulated to perturb MAPK signaling reveals treatment‐specific responses and pathway crosstalks.
Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on ...a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.
Protein turnover maintains the recycling needs of the proteome, and its malfunction has been linked to aging and age-related diseases. However, not all proteins turnover equally, and the factors that ...contribute to accelerate or slow down turnover are mostly unknown. We measured turnover rates for 3,160 proteins in exponentially growing yeast and analyzed their dependence on physical, functional, and genetic properties. We found that functional characteristics, including protein localization, complex membership, and connectivity, have greater effect on turnover than sequence elements. We also found that protein turnover and mRNA turnover are correlated. Analysis under nutrient perturbation and osmotic stress revealed that protein turnover highly depends on cellular state and is faster when proteins are being actively used. Finally, stress-induced changes in protein and transcript abundance correlated with changes in protein turnover. This study provides a resource of protein turnover rates and principles to understand the recycling needs of the proteome under basal conditions and perturbation.
Display omitted
•Functional properties have greater impact on protein turnover than sequence traits•Protein turnover is faster when proteins are actively used•mRNA stability and protein turnover are correlated in exponentially growing yeast•Protein turnover is highly sensitive to changes in cellular state
Turnover measurements of the yeast proteome reveal co-regulation with mRNA stability and dependence on protein functional attributes, including protein active state.
MicroRNAs (miRNAs) regulate target mRNAs through a combination of translational repression and mRNA destabilization, with mRNA destabilization dominating at steady state in the few contexts examined ...globally. Here, we extend the global steady-state measurements to additional mammalian contexts and find that regardless of the miRNA, cell type, growth condition, or translational state, mRNA destabilization explains most (66%–>90%) miRNA-mediated repression. We also determine the relative dynamics of translational repression and mRNA destabilization for endogenous mRNAs as a miRNA is induced. Although translational repression occurs rapidly, its effect is relatively weak, such that by the time consequential repression ensues, the effect of mRNA destabilization dominates. These results imply that consequential miRNA-mediated repression is largely irreversible and provide other insights into the nature of miRNA-mediated regulation. They also simplify future studies, dramatically extending the known contexts and time points for which monitoring mRNA changes captures most of the direct miRNA effects.
Display omitted
•In diverse cell types and conditions, mRNA decay is the dominant miRNA effect•Just after miRNA induction, translational repression contributes more of the repression•However, by the time substantial repression is observed, decay is the dominant effect•A mathematical model provides a framework for miRNA-mediated repression
miRNAs repress gene expression via mRNA decay and translational repression. Eichhorn et al. characterize the impact and dynamics of mRNA decay and translational repression in numerous contexts, showing that decay is the predominant mode of action whenever there is substantial repression.
Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment ...methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.
PDF
