Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted ...MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1 to 100 μg starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25–100 μg peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1–25 μg range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.
Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by ...antibody-based technologies, which are limited by the availability of high-quality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)-based targeted proteomics approach, termed cLC–SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC–SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC–SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels ≥30 000 and ≥3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC–SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.
Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies ...utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries' residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide.
To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time.
All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset.
These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.
Targeted mass spectrometry is a promising technology for site-specific quantification of posttranslational modifications. However, a major constraint is the limited sensitivity for quantifying ...low-abundance PTMs, requiring the use of affinity reagents for enrichment. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometry using a sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection, and multiplexing (PRISM). PRISM provides effective enrichment of target peptides into a given fraction from complex mixture, followed by selected reaction monitoring quantification. Direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) was demonstrated from as little as 25 μg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided ∼10-fold higher signal intensities, presumably due to the better peptide recovery of PRISM. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of epidermal growth factor at both the peak activation (10 min) and steady state (2 h). The maximal ERK activation was observed with 0.3 and 3 ng/mL doses for 10 min and 2 h time points, respectively. The dose–response profiles of individual phosphorylated isoforms showed that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.
Societal Impact Statement
A future with a secure and safe food supply requires humanity to preserve and exploit the vast variation available across agricultural plant species. Apples are one of the ...most widely consumed fruits and provide significant nutritional value worldwide. Here, we characterize key agricultural traits in a diverse collection of apples to provide a foundation for future apple improvement. We show that commercially successful apple varieties capture only a small fraction of apple diversity, and demonstrate that significant improvement is possible by tapping into existing genetic diversity.
Summary
●Here we present a comprehensive evaluation of apple diversity through phenotyping of Canada's Apple Biodiversity Collection (ABC) which contains over 1000 apple accessions.
●We assessed, over a 4‐year period, more than 20,000 individual apples and quantified variation across 39 phenotypes, including phenology and fruit quality both at harvest and after 3 months of cold storage.
●We observe that apples in the ABC display a wide range of phenotypic variation that may prove useful for future apple improvement. For example, apples can differ by nearly 61‐fold in weight, 18‐fold in acidity, and 100‐fold in phenolic content. We quantified the dramatic changes to apple physiology that occur during 3 months of cold storage: on average, apples lost 39% of their firmness, 31% of their acidity, and 9% of their weight, but gained 7% in soluble solids. Harvest date, flowering date, and time to ripen were all positively correlated with firmness, which suggests that the developmental pathways that drive phenological events throughout the growing season may play a role in determining an apple's texture. Finally, we show that apple breeding has selected for a significant decline in phenolic content over the past 200 years: apple cultivars released after 1940 had a 30% lower median phenolic content than cultivars released before 1940.
●The data and analyses presented here not only provide a comprehensive quantification of the range across, and relationships among diverse apple phenotypes, but they also enable genetic mapping studies that will provide the foundation for future apple improvement via genomics‐assisted breeding.
A future with a secure and safe food supply requires humanity to preserve and exploit the vast variation available across agricultural plant species. Apples are one of the most widely consumed fruits and provide significant nutritional value worldwide. Here, we characterize key agricultural traits in a diverse collection of apples to provide a foundation for future apple improvement. We show that commercially successful apple varieties capture only a small fraction of apple diversity, and demonstrate that significant improvement is possible by tapping into existing genetic diversity.
Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of ...protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective and robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high-throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with postexcision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics and provides insights not readily obtainable from such approaches.
Impulsivity critically relates to many psychiatric disorders. Given the multifaceted construct that impulsivity represents, defining core aspects of impulsivity is vital for the assessment and ...understanding of clinical conditions. Choice impulsivity (CI), involving the preferential selection of smaller sooner rewards over larger later rewards, represents one important type of impulsivity. The International Society for Research on Impulsivity (InSRI) convened to discuss the definition and assessment of CI and provide recommendations regarding measurement across species. Commonly used preclinical and clinical CI behavioral tasks are described, and considerations for each task are provided to guide CI task selection. Differences in assessment of CI (self-report, behavioral) and calculating CI indices (e.g., area-under-the-curve, indifference point, and steepness of discounting curve) are discussed along with properties of specific behavioral tasks used in preclinical and clinical settings. The InSRI group recommends inclusion of measures of CI in human studies examining impulsivity. Animal studies examining impulsivity should also include assessments of CI and these measures should be harmonized in accordance with human studies of the disorders being modeled in the preclinical investigations. The choice of specific CI measures to be included should be based on the goals of the study and existing preclinical and clinical literature using established CI measures.
Metaproteomics conducted on soil is challenged by a low depth of protein coverage that can potentially result in an underrepresentation of the functional underpinnings of important biological ...processes and interactions. Typically, the utilization of an on-line two-dimensional chromatographic separation approach (2D LC-MS/MS) can significantly improve depth of coverage. Herein, we evaluate different fractionation modalities to determine the optimal approach for LC MS based soil metaproteomics. The first approach fractionates the digested soil proteome in 2 dimensions while coupled directly to the MS instrument (“online” approach). The second approach performs the first dimension of fractionation “offline” prior to injection to the MS (“offline” approach). While both approaches are commonly utilized for proteomic research, they have not been directly compared for soils. We rigorously compared these approaches applied to: 1) a mock community consisting of 47 different microorganisms, and 2) to natural soil. The results provide insight into protein dynamic range, the presence of mass spectrometry interfering substances, and other factors that may contribute to an observed low metaproteome depth of coverage from complex and highly diverse samples, such as soil. We observed that the “offline” approach generally resulted in the highest metaproteome coverage; however, there are advantages to using the “online” approach when dealing with limited biomass. Both approaches resulted in a larger number of protein identifications from the synthetic metaproteome rather than from the soil metaproteome, although the soil metaproteome had a significantly larger number of predicted proteins. A large dynamic range in abundances of proteins resulting from metabolically active and inactive populations within the soil metaproteome explains this observation.
•2D LC-MS/MS approaches improve soil metaproteome depth of coverage.•The prairie soil metaproteome is substantially under-sampled.•Proteins within the Kansas soil are at the lower dynamic range of detection.
There is an unmet technical challenge for mass spectrometry (MS)-based proteomic analysis of single mammalian cells. Quantitative proteomic analysis of single cells has been previously achieved by ...antibody-based immunoassays but is limited by the availability of high-quality antibodies. Herein we report a facile targeted MS-based proteomics method, termed cPRISM-SRM (carrier-assisted high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected reaction monitoring), for reliable analysis of low numbers of mammalian cells. The method capitalizes on using "carrier protein" to assist processing of low numbers of cells with minimal loss, high-resolution PRISM separation for target peptide enrichment, and sensitive SRM for protein quantification. We have demonstrated that cPRISM-SRM has sufficient sensitivity to quantify proteins expressed at ≥200,000 copies per cell at the single-cell level and ≥3000 copies per cell in 100 mammalian cells. We envision that with further improvement cPRISM-SRM has the potential to move toward targeted MS-based single-cell proteomics.
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