Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Köcher, Thomas; Pichler, Peter; Swart, Remco; Mechtler, Karl

doi:10.1038/nprot.2012.036

Cited by 107 publications

(107 citation statements)

References 38 publications

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“…Notably, running replicate of the enriched K562 sample from Step 42 helps increase the number of phosphopeptide identifications by another 30-40%, as also shown in other reports 58,59 , and generally increases the coverage of the phosphoproteome. Similar to previous reports 59, 75 , we also observed a marked increase of phosphopeptide identifications with increasing separation time (Fig. 8e).…”

Section: Anticipated Resultssupporting

confidence: 80%

“… crItIcal step The MS analysis of a standard complex sample tests the LC separation paramaters such as chromatography, peptide elution time and LC system pressure, as well as the performance of MS such as instrumental sensitivity and peptide fragmentation. We highly recommend the recently published protocol by Kocher et al 75 , in which the authors present an excellent protocol to benchmark the nanoLC-MS/MS setup.…”

Section: | the Obtained Samples Can Be Analyzed By Ms For Maldi-tomentioning

confidence: 99%

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Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

et al. 2013

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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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Section: Anticipated Resultssupporting

confidence: 80%

Section: | the Obtained Samples Can Be Analyzed By Ms For Maldi-tomentioning

confidence: 99%

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

et al. 2013

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“…Finally, we sought to identify the Rpa43 residues targeted by Rio1 using mass spectrometry. After repeating the in vitro kinase assay in the presence and absence of unlabelled ATP, nanoLC-MS/MS analysis 44 Fig. 8f and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

et al. 2015

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The conserved protein kinase Rio1 localizes to the cytoplasm and nucleus of eukaryotic cells. While the roles of Rio1 in the cytoplasm are well characterized, its nuclear function remains unknown. Here we show that nuclear Rio1 promotes rDNA array stability and segregation in Saccharomyces cerevisiae. During rDNA replication in S phase, Rio1 downregulates RNA polymerase I (PolI) and recruits the histone deacetylase Sir2. Both interventions ensure rDNA copy-number homeostasis and prevent the formation of extrachromosomal rDNA circles, which are linked to accelerated ageing in yeast. During anaphase, Rio1 downregulates PolI by targeting its subunit Rpa43, causing PolI to dissociate from the rDNA. By stimulating the processing of PolI-generated transcripts at the rDNA, Rio1 allows for rDNA condensation and segregation in late anaphase. These events finalize the genome transmission process. We identify Rio1 as an essential nucleolar housekeeper that integrates rDNA replication and segregation with ribosome biogenesis. A t anaphase onset, the replicated chromosomes separate and then segregate along the mitotic spindle into the daughter cells. In the budding yeast Saccharomyces cerevisiae, the locus containing the genes that encode the ribosomal RNAs (rDNA) segregates after the rest of the genome, in late anaphase [1][2][3][4] . The rDNA locus exists as a tandem-repeat array comprising B150 rDNA units containing the 35S and 5S genes, which are transcribed by RNA polymerase I (PolI) and PolIII, respectively. Processing of the 35S pre-rRNA generates 5.8S, 18S and 25S rRNA that, together with the 5S rRNA, become the catalytic backbones of each ribosome 5,6 . Only in anaphase does yeast repress rDNA transcription 4 , which allows the sister rDNA loci to condensate and segregate. PolI downregulation in anaphase is mediated by the Cdc14 phosphatase acting on PolI subunit Rpa43 (ref. 4), resulting in PolI dissociating from the 35S rDNA. The removal of PolI and the local resolution of its transcripts allow the condensin complex to bind. The latter compacts the rDNA array and recruits the DNA decatenating enzyme topoisomerase II (refs 1,3,4,7) resulting in the physical separation and subsequent segregation of the sister rDNA loci.S. cerevisiae Rio1 belongs to the atypical RIO protein kinase family whose members lack the activation loop and substrate recognition domain present in canonical eukaryotic protein kinases [8][9][10][11] . Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 . Roles in the nucleus are unknown for any RIO member, either in yeast or eukaryotes beyond. Using S. cerevisiae, we now describe the first activities of Rio1 in the nucleus. Foremost, Rio1 downregulates PolI transcription through the cell cycle. In G...

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“…21 Advantages are that, for peptides, (1) separation by chromatography, (2) ionization, and (3) fragmentation are more efficient, (4) their masses are less heterogeneously distributed, and (5) their identification through database searches is more straightforward. Complex peptide mixtures can be either directly analyzed by liquid chromatography-MS using long gradients 22 or fractionated by hyphenation of different separation techniques beforehand 23 to reduce sample complexity and increase depth and coverage of the proteome analysis. Peptide sequences are usually identified by comparing the molecular masses of the peptide (parent) ion and corresponding fragment ions, as determined in MS and MS/MS scans, with the predicted peptide/fragment masses obtained from in silico digestion of a protein database.…”

Section: Text Box 1 Liquid Chromatography-mass Spectrometry Analysismentioning

confidence: 99%

What Can Proteomics Tell Us About Platelets?

Burkhart

Gambaryan

Watson

et al. 2014

Circulation Research

105

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More than 130 years ago, it was recognized that platelets are key mediators of hemostasis. Nowadays, it is established that platelets participate in additional physiological processes and contribute to the genesis and progression of cardiovascular diseases. Recent data indicate that the platelet proteome, defined as the complete set of expressed proteins, comprises >5000 proteins and is highly similar between different healthy individuals. Owing to their anucleate nature, platelets have limited protein synthesis. By implication, in patients experiencing platelet disorders, platelet (dys)function is almost completely attributable to alterations in protein expression and dynamic differences in post-translational modifications. Modern platelet proteomics approaches can reveal (1) quantitative changes in the abundance of thousands of proteins, (2) post-translational modifications, (3) protein–protein interactions, and (4) protein localization, while requiring only small blood donations in the range of a few milliliters. Consequently, platelet proteomics will represent an invaluable tool for characterizing the fundamental processes that affect platelet homeostasis and thus determine the roles of platelets in health and disease. In this article we provide a critical overview on the achievements, the current possibilities, and the future perspectives of platelet proteomics to study patients experiencing cardiovascular, inflammatory, and bleeding disorders.

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Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Cited by 107 publications

References 38 publications

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

What Can Proteomics Tell Us About Platelets?

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