Electron Transfer Dissociation in Conjunction with Collision Activation To Investigate the <i>Drosophila melanogaster</i> Phosphoproteome

Domon, Bruno; Bodenmiller, Bernd; Carapito, Christine; Hao, Zhiqi; Huehmer, Andreas; Aebersold, Ruedi

doi:10.1021/pr800834e

Cited by 21 publications

(27 citation statements)

References 28 publications

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“…This is in line with our previous observations and those made by others and confirm the benefit of combining these two fragmentation techniques. 20,24,[45][46][47][48] Although the advantage of CID-MSA has been debated, 49,50 it does give a large contribution (39%) to the highest scoring identified unique phosphopeptides in the main phosphofractions in our experimental setup, thereby performing on par with HCD (42%). Possibly, the high phosphopeptide content of the main phosphofractions and the increased sequencing speed of the LTQ Velos allow the CID-MSA method to be more beneficial.…”

Section: Cid-msa Etd and Hcd Have Complementary Contributions To Phomentioning

confidence: 97%

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

et al. 2013

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The small GTPase Rap1 is required for proper cell-cell junction formation and also plays a key role in mediating cAMP-induced tightening of adherens junctions and subsequent increased barrier function of endothelial cells. To further study how Rap1 controls barrier function, we performed quantitative global phosphoproteomics in human umbilical vein endothelial cells (HUVECs) prior to and after Rap1 activation by the Epac-selective cAMP analog 8-pCPT-2 0 -O-Me-cAMP-AM (007-AM). Tryptic digests were labeled using stable isotope dimethyl labeling, enriched with phosphopeptides by strong cation exchange (SCX), followed by titanium(IV) immobilized metal affinity chromatography (Ti 4+ -IMAC) and analyzed by high resolution mass spectrometry. We identified 19 859 unique phosphopeptides containing 17 278 unique phosphosites on 4594 phosphoproteins, providing the largest HUVEC phosphoproteome to date. Of all identified phosphosites, 220 (B1%) were more than 1.5-fold up-or downregulated upon Rap activation, in two independent experiments. Compatible with the function of Rap1, these alterations were found predominantly in proteins regulating the actin cytoskeleton, cell-cell junctions and cell adhesion.

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Section: Cid-msa Etd and Hcd Have Complementary Contributions To Phomentioning

confidence: 97%

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

et al. 2013

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“…Not only did ETD contributed to more identifications, it also resulted in a higher specificity being calculated for positive phosphopeptide enrichment by the Ti 4ϩ -IMAC a method. An observation that can be rationalized by the known higher efficiency of ETD in fragmenting phosphopeptides with a higher charge where the higher charge is a product of the presence of multiple basic residues (38,39,45).…”

Section: Fig 2 Multipronged Phosphoproteomics Analysis Of a Hela Lymentioning

confidence: 99%

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment

Zhou

Low

Hennrich

et al. 2011

Molecular & Cellular Proteomics

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Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti 4؉ -immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO 2 enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improves the enrichment of phosphopeptides containing multiple basic residues. We found that Ti 4؉ -IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 g of a HeLa cell lysate digest. In comparison, ϳ2000 unique phosphopeptides could be identified by Ti 4؉ -IMAC with HFP and close to 3000 by TiO 2 . We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti 4؉ -IMAC methodology alongside the use of collision-induced dissocia- Reversible protein phosphorylation widely regulates cellular functions through protein kinases and phosphatases (1, 2). Determination and a quantitative analysis of phosphorylation sites are a prerequisite for unraveling regulatory processes and signaling networks (3-6). The analytical methods of choice for characterizing protein phosphorylation have shifted from traditional methods such as radioactive labeling and gel electrophoresis to advanced mass spectrometry, a highthroughput technology (7). It has been estimated that ϳ30% of cellular proteins are phosphorylated during the life cycle of the cell (8). There has been a continuing intense focus on developing enrichment and phosphopeptide sequencing strategies to facilitate the large-scale profiling of phosphorylation events. Currently, one of the most commonly adopted strategies is the use of two sequential steps of chromatographic based separations; an initial fractionation step for reducing sample complexity and, subsequently, a more specific enrichment of phosphopeptides.Typically, low-pH strong cation exchange (SCX) 1 chromatography is used as the first step where peptides are fraction- 1 The abbreviations used are: SCX, strong cation exchange chromatography; Ti 4ϩ -IMAC, immobilized titanium (IV) ion affinity ...

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“…Importantly, this resulted in the characterization of significantly more phosphorylation sites than DDA LC-MS/MS. More recently, Domon et al applied these approaches to enhance their characterization of the Drosophila melanogaster phosphoproteome [20].…”

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confidence: 99%

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

Hart‐Smith

Low

Erce

et al. 2012

J. Am. Soc. Mass Spectrom.

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When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites-17 of which fall within a glycine-arginine-rich (GAR) domain spanning G120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95 % more (P=0.030) methylargininecarrying peptides are selected for MS/MS than DDA, leading to an 86 % increase (P=0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities G6×10 4 cps) are selected for MS/MS using this approach relative to DDA (50 % more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586 % increase (P=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTM of known mass.

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Electron Transfer Dissociation in Conjunction with Collision Activation To Investigate the Drosophila melanogaster Phosphoproteome

Cited by 21 publications

References 28 publications

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

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