Anna E. Merrill

Neutron-encoded mass signatures for multiplexed proteome quantification

We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spectrometry analysis with high mass resolution (>200,000) reveals the isotopologue-embedded peptide signals, permitting quantification. Neutron encoding will enable highly multiplexed proteome analysis with excellent dynamic range and accuracy.

Proteome sequencing goes deep

Advances in mass spectrometry (MS) have transformed the scope and impact of protein characterization efforts. Identifying hundreds of proteins from rather simple biological matrices, such as yeast, was a daunting task just a few decades ago. Now, expression of more than half of the estimated ∼20,000 human protein coding genes can be confirmed in record time and from minute sample quantities. Access to proteomic information at such unprecedented depths has been fueled by strides in every stage of the shotgun proteomics workflow-from sample processing to data analysis-and promises to revolutionize our understanding of the causes and consequences of proteome variation.

NeuCode Labels for Relative Protein Quantification

We describe a synthesis strategy for the preparation of lysine isotopologues that differ in mass by as little as 6 mDa. We demonstrate that incorporation of these molecules into the proteomes of actively growing cells does not affect cellular proliferation, and we discuss how to use the embedded mass signatures (neutron encoding (NeuCode)) for multiplexed proteome quantification by means of high-resolution mass spectrometry. NeuCode SILAC amalgamates the quantitative accuracy of SILAC with the multiplexing of isobaric tags and, in doing so, offers up new opportunities for biological investigation. We applied NeuCode SILAC to examine the relationship between transcript and protein levels in yeast cells responding to environmental stress. Finally, we monitored the time-resolved responses of five signaling mutants in a single 18-plex experiment.

Amine-reactive neutron-encoded labels for highly plexed proteomic quantitation.

We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between 13C and 15N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference—relative to its nearest neighbor—so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration.

Pathway connectivity and signaling coordination in the yeast stress‐activated signaling network

Stressed cells coordinate a multi‐faceted response spanning many levels of physiology. Yet knowledge of the complete stress‐activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho‐proteome changes in cells responding to salt stress, to infer the salt‐responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress‐defense genes with reduction of growth‐related transcripts. We find that the orthologous human network is enriched for cancer‐causing genes, underscoring the importance of the subnetworks predictions in understanding stress biology.

Neutron-encoded mass signatures for quantitative top-down proteomics.

The ability to acquire highly accurate quantitative data is an increasingly important part of any proteomics experiment, whether shotgun or top-down approaches are used. We recently developed a quantitation strategy for peptides based on neutron encoding, or NeuCode SILAC, which uses closely spaced heavy isotope-labeled amino acids and high-resolution mass spectrometry to provide quantitative data. We reasoned that the strategy would also be applicable to intact proteins and could enable robust, multiplexed quantitation for top-down experiments. We used yeast lysate labeled with either 13C615N2-lysine or 2H8-lysine, isotopologues of lysine that are spaced 36 mDa apart. Proteins having such close spacing cannot be distinguished during a medium resolution scan, but upon acquiring a high-resolution scan, the two forms of the protein with each amino acid are resolved and the quantitative information revealed. An additional benefit NeuCode SILAC provides for top down is that the spacing of the isotope peaks indicates the number of lysines present in the protein, information that aids in identification. We used NeuCode SILAC to quantify several hundred isotope distributions, manually identify and quantify proteins from 1:1, 3:1, and 5:1 mixed ratios, and demonstrate MS2-based quantitation using ETD.

Neutron-Encoded Protein Quantification by Peptide Carbamylation

AbstractWe describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet. Figureᅟ

A Calibration Routine for Efficient ETD in Large-Scale Proteomics

AbstractElectron transfer dissociation (ETD) has been broadly adopted and is now available on a variety of commercial mass spectrometers. Unlike collisional activation techniques, optimal performance of ETD requires considerable user knowledge and input. ETD reaction duration is one key parameter that can greatly influence spectral quality and overall experiment outcome. We describe a calibration routine that determines the correct number of reagent anions necessary to reach a defined ETD reaction rate. Implementation of this automated calibration routine on two hybrid Orbitrap platforms illustrate considerable advantages, namely, increased product ion yield with concomitant reduction in scan rates netting up to 75% more unique peptide identifications in a shotgun experiment. Graphical Abstractᅟ

Quantifying proteomes and their post-translational modifications by stable isotope label-based mass spectrometry.

Stable isotope labeling coupled with mass spectrometry has revolutionized the scope and impact of protein expression studies. Label incorporation can occur metabolically or chemically, and each method bears specific strengths and weaknesses. Quantitative proteomics confidently identifies specific interactions between proteins and other biological species, such as nucleic acids and metabolites. Extending label-based methods to phosphorylation-modified forms of proteins enables the construction of signaling networks and their temporal responses to stimuli. The integration of multiple data types offers systems-level insight on coordinated biological processes. Finally, the development of methods applicable to tissue quantification suggests the emerging role of label-based, quantitative mass spectrometry in translational science.

NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.