Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography
TL;DR: A detailed protocol for the preparation of monodisperse microsphere–based Ti4+-IMAC adsorbents and the subsequent enrichment process is presented and it is demonstrated how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes.
Abstract: Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere-based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO(2)). Here we present a detailed protocol for the preparation of monodisperse microsphere-based Ti(4+)-IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ∼2 weeks to complete the protocol: 1 week to prepare the Ti(4+)-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.
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TL;DR: An enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed, and the largest phosphoproteome dataset of human liver was generated.
224 citations
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...[43] Zhou H, Ye M, Dong J, Corradini E, Cristobal A, Heck AJ, et al....
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TL;DR: This study highlights the importance of knowing the carrier and removal status of canine coronavirus, as a source of infection for other animals, not necessarily belonging to the same breeds.
Abstract: Profiling Nicholas M. Riley†,‡ and Joshua J. Coon*,†,‡,§ †Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ‡Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ■ CONTENTS Sampling the Phosphoproteome 74 Generating Phosphopeptides 75 Enrichment Strategies 75 Chromatographic Separations 77 Mass Spectrometry Instrumentation 78 Data Acquisition Strategies 79 Balancing Throughput and Depth 79 Quantifying the Phosphoproteome 81 Stable Isotope Labeling 81 Label-Free Strategies 83 Confident Phosphosite Assignment 84 Tandem MS Approaches 84 Post-Acquisition Processing and Informatics 85 Biological Insights via Phosphoproteomics 87 PTM Cross-Talk 88 Phosphorylation beyond Serine, Threonine, and Tyrosine 88 Looking Forward 89 Author Information 89 Corresponding Author 89 Notes 89 Biographies 89 Acknowledgments 89 References 89
210 citations
TL;DR: Applied to glioblastoma cells acutely treated with epidermal growth factor, EasyPhos quantified 20,132 distinct phosphopeptides from 200 μg of protein in less than 1 d of measurement time, revealing thousands of EGF-regulated phosphorylation events.
Abstract: Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation ('phosphoproteomics'). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol, including digestion, in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and a small sample size requirement (≤200 μg of protein starting material). After cell culture or tissue collection, the protocol takes 1 d, whereas mass spectrometry measurements require ~1 h per sample. Applied to glioblastoma cells acutely treated with epidermal growth factor (EGF), EasyPhos quantified 20,132 distinct phosphopeptides from 200 μg of protein in less than 1 d of measurement time, revealing thousands of EGF-regulated phosphorylation events.
209 citations
TL;DR: A peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites is presented to facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.
Abstract: We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.
159 citations
TL;DR: This work systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of Ti4+-IMAC enrichedosphopeptides from a tryptic human cell line digest.
Abstract: We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissociation (termed EThcD), for improved peptide ion fragmentation and identification. We reasoned that phosphosite localization, one of the major hurdles in high-throughput phosphoproteomics, could also highly benefit from the generation of such EThcD spectra. Here, we systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of Ti4+-IMAC enriched phosphopeptides from a tryptic human cell line digest. In combination with a modified version of phosphoRS, we observed that in the majority of cases EThcD generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. Our data demonstrates the distinctive potential of EThcD for PTM localization, also beyond protein phosphorylation.
144 citations
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TL;DR: A new computer program, Mascot, is presented, which integrates all three types of search for protein identification by searching a sequence database using mass spectrometry data, and the scoring algorithm is probability based.
Abstract: Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
8,195 citations
TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Abstract: Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
6,597 citations
TL;DR: The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.
6,317 citations
TL;DR: SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system and is applied to the relative quantitation of changes in protein expression during the process of muscle cell differentiation.
5,653 citations
TL;DR: An approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures based on isotope-coded affinity tags and tandem mass spectrometry is described.
Abstract: We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we com- pared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively glob- al protein expression in cells and tissues.
4,893 citations