Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips

Rappsilber, Juri; Mann, Matthias; Ishihama, Yasushi

doi:10.1038/nprot.2007.261

Cited by 3,960 publications

(3,291 citation statements)

References 18 publications

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“…Furthermore, the PolyMAC-Ti method exhibited lower variability than the TiO 2 method in terms of the selectivity and identity of the recovered phosphopeptides. For three parallel analyses using TiO 2 beads, 38.4% of phosphopeptides were identified by all three analyses, and there was an ϳ50% overlap between every two analyses, again consistent with the recent literature report (31). In contrast, 55.2% of phosphopeptides were identified by all three analyses, and there was an ϳ70% overlap between every two analyses when using PolyMAC-Ti.…”

Section: Strategy and Design Of Polymacsupporting

confidence: 73%

“…In comparison, an average of 420 unique phosphopeptides representing 498 phosphorylation sites were identified from the same sample using the TiO 2 method. The number of identified phosphopeptides is consistent with a recent report using titania stage tips in which an average of 580 phosphopeptides were identified from 150 g of HeLa cell lysate (31). To increase the confidence in our phosphopeptide identification, we also carried out database searches using Mascot.…”

Section: Strategy and Design Of Polymacsupporting

confidence: 61%

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In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Iliuk

Martin

Alicie

et al. 2010

Molecular & Cellular Proteomics

154

180

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The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...

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Section: Strategy and Design Of Polymacsupporting

confidence: 73%

Section: Strategy and Design Of Polymacsupporting

confidence: 61%

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Iliuk

Martin

Alicie

et al. 2010

Molecular & Cellular Proteomics

154

180

View full text Add to dashboard Cite

show abstract

“…Total protein from each lane was digested according to the standard in‐gel digestion protocol (Shevchenko et al , 2006). Digested peptides were purified and concentrated on a C18 StageTip (Rappsilber et al , 2007) and were analyzed using a Q Exactive HF mass spectrometer (ThermoFisher Scientific). The peptides were separated via Easy nLC 1000 system (Thermo Scientific) via 140‐min gradient through a 15‐cm column, 75 μm inner diameter packed with 1.9 μm beads.…”

Section: Methodsmentioning

confidence: 99%

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

et al. 2016

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Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

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“…Peptide RP purification was performed with 1 mL Sep‐Pak tC18 cartridges (Sep‐Pak; Waters, Milford, MA, USA), with in‐house assembled microcolumns in 200 μL pipette tips as in ref. 31, filled with Zorbax SB‐C18 5 μm (Agilent Technologies, Santa Clara, CA, USA) or with OligoR3 50 μm (Applied Biosystems, Bedford, MA, USA) material. The resulting protein fractions were subjected to in‐solution reduction, alkylation, and digestion, and subsequently analyzed by LC‐MS with solid‐phase extraction capillary liquid chromatography (speLC) coupled to a Q‐Exactive instrument.…”

Section: Methodsmentioning

confidence: 99%

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

et al. 2018

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For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS3‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS3 detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E711–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.

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Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips

Cited by 3,960 publications

References 18 publications

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

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