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.
Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner.
Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.
Diseases at the molecular level are complex and patient dependent, necessitating development of strategies that enable precision treatment to optimize clinical outcomes. Organoid technology has recently been shown to have the potential to recapitulate the in vivo characteristics of the original individual's tissue in a three-dimensional in vitro culture system. Here, we present a quantitative mass-spectrometry-based proteomic analysis and a comparative transcriptomic analysis of human colorectal tumor and healthy organoids derived, in parallel, from seven patients. Although gene and protein signatures can be derived to distinguish the tumor organoid population from healthy organoids, our data clearly reveal that each patient possesses a distinct organoid signature at the proteomic level. We demonstrate that a personalized patient-specific organoid proteome profile can be related to the diagnosis of a patient and with future development contribute to the generation of personalized therapies.
Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/CFZR1 activity as an important determinant in response to CDK4/6-inhibitors.
Wnt/β‐catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt‐hypersensitive tumors that are susceptible to anti‐Wnt‐based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce β‐catenin‐mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin‐independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing β‐catenin turnover and propelling ligand‐independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche‐independent program for self‐renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti‐Wnt‐based therapy. Our data demonstrate the relevance of studying patient‐derived mutations for understanding disease mechanisms and improved applications of precision medicine.
Here, we describe an in-house built ultra-high pressure liquid chromatography (UHPLC) system, with little complexity in design and high separation power combined with convenience in operation. This system enables the use of long columns of 40 cm packed with 1.8 μm particles generating pressures below 1000 bar. Furthermore, the system could be operated at flow rates between 50 and 200 nL min(-1) while maintaining its separation power. Several gradients were optimized ranging from 23 to 458 minutes. With the longest gradient we identified over 4500 protein groups and more than 26,000 unique peptides from 1 μg of a human cancer cell lysate in a single run using an Orbitrap Velos - a level of performance often seen solely using multidimensional separation strategies. Further experiments using a mass spectrometer with faster sequencing speeds, like the TripleTOF 5600, enabled us to identify over 1400 protein groups in a 23 min gradient. The TripleTOF 5600 performed especially well, compared to the Orbitrap Velos, for the shorter gradients used. Our data demonstrate that the combination of UHPLC with high resolution mass spectrometry at increased sequencing speeds enables extensive proteome analysis in single runs.
Ultra-high pressure liquid chromatography (UHPLC) systems combined with state-of-the-art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC-MS experiment within a few hours. The recently released EASY-spray technology allows one to implement nano-UHPLC with straightforwardness. In this work we initially characterized the EASY-spray containing a 50 cm column containing <2 μm particles and found that the system allowed 3000 proteins to be identified in 90 minutes. We then asked the question whether a fast and sensitive online 2D SCX-RP UHPLC-MS/MS workflow could compete with 1D long gradient analyses, using total analysis time versus proteome coverage and sample usage as benchmark parameters. The 2D LC-MS strategy consisted of the EASY-spray system that had been augmented by the addition of an SCX column. The conversion was made facile since no additional valves were required and by the use of components containing viper fittings. We benchmarked the system using a human cell lysate digest (<10 μg). The 2D SCX-RP UHPLC-MS/MS workflow allowed the identification of almost 37,000 unique peptides and 6000 proteins in a total analysis time of ~7 hours. On the same system a 1D RP UHPLC-MS/MS workflow plateaued at only 20,000 peptides and 4400 unique proteins and required approx. 8 hours of analysis time. Furthermore, the 2D workflow could continue to increase the proteome coverage with longer analysis times, in fact with a 21 hour analysis we identified 56,600 unique peptides and >7500 proteins. We report, here, that with this fast online SCX-RP UHPLC-MS/MS workflow, the proteome coverage can be substantially extended without significantly compromising the analysis time and sample usage.
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