Automated and High Confidence Protein Phosphorylation Site Localization Using Complementary Collision-Activated Dissociation and Electron Transfer Dissociation Tandem Mass Spectrometry

Hansen, Thomas Aarup; Sylvester, Marc; Jensen, Ole N.; Kjeldsen, Frank

doi:10.1021/ac302364r

Cited by 17 publications

(28 citation statements)

References 30 publications

(47 reference statements)

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“…9 Also methods of advanced data interpretation have been applied to obtain sitelocalization of the phosphate group in CID spectra via scoring algorithms. [10][11][12][13][14][15][16][17] Fragmentation techniques relying on electron-driven ion excitation such as electron capture dissociation (ECD) 18 and electron transfer dissociation (ETD) 19 do not induce significant detachment of labile modifications due to a high rate of bond cleavage and low amount of excess electronic energy. [20][21][22] Obtaining similar protection against phosphate loss in CID would be a significant gain to phosphoproteomics.…”

Section: Introductionmentioning

confidence: 99%

Effect of Metals in Biomimetic Dimetal Complexes on Affinity and Gas-Phase Protection of Phosphate Esters

et al. 2015

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Although the biomimetic dimetal complex [LGa2(OH)2(H2O)2](3+) [L = 2,6-bis((N,N'-bis(2-picolyl)amino)methyl)-4-tertbutylphenolate] provides efficient protection against phosphate loss in phosphopeptides upon collision-induced dissociation tandem mass spectrometry (CID MS/MS), the underlying mechanism remains unknown. Here, we explored the mechanism in detail and investigated the selective binding to phosphate groups in solution. Dimetal complexes containing combinations of Ga(3+), In(3+), Fe(3+), Co(3+), Zn(2+), Cu(2+), and V(2+) were reacted with HPO4(2-), phosphoserine, and a phosphopeptide (FQpSEEQQQTEDELQDK, abbreviated "βcas") and studied with isothermal titration calorimetry (ITC), CID MS/MS, and density functional theory (DFT). Ka for HPO4(2-) binding scaled with the metal charge and was 35-fold larger for [LGa2(OH)2(H2O)2](3+) (3.08 ± 0.31 × 10(6) M(-1)) than for [LZn2(HCOO)2](+). CID MS/MS of [LGa2(βcas)](n+) revealed protection against phosphate detachment (<3% of the total ion intensity). Phosphate detachment from βcas was 22-40% and increased to 42-71% when bound to dimetal complexes of lower charge than {LGa2}(5+). CID data suggests that facile metal-phosphate dissociation is associated with proton transfer from the intermediate oxazoline ring formed in the phosphopeptide to the metal-phosphate complex. The observed phosphate stabilization was attributed to a significant reduction in the gas-phase basicity (GB) of the phosphate group when bound to {LGa2}(5+)/{LIn2}(5+) complex cores. Absence of proton transfer results in formation of an ion-zwitterion intermediate with a greater dissociation threshold. This hypothesis is supported by DFT calculations for [LGa2(PO4)](2+), [LGaZn(PO4)](+), [LZn2(PO4)], and 2,4-dimethyl-3-oxazoline showing that [LGa2(PO4)](2+) is the only compound with a substantial lower GB (321 kJ/mol less) than 2,4-dimethyl-3-oxazoline.

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Section: Introductionmentioning

confidence: 99%

Effect of Metals in Biomimetic Dimetal Complexes on Affinity and Gas-Phase Protection of Phosphate Esters

et al. 2015

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“…In general, ETD fragmentation of parental ions with a charge state of 3 or higher provide better spectra than those with charge state of 2. Therefore, the combination of CID and ETD has been found advantageous to provide complete information for PTM identification (32,33).…”

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confidence: 99%

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

Wang

Yuan

Fan

et al. 2016

Molecular & Cellular Proteomics

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Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13 C-labeled UDP-GlcNAc from 13 C 6 -glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein OGlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time. Molecular &

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“…The combination of ECD/CID and ETD/CID data has been proposed and utilized for more reliable sequence identification or phosphorylaton site assignments [30, 31]. Since the HCD data are rich in carbohydrate-related fragments, some information can be gained about the modifying glycan, at least the presence of certain saccharide units can be confirmed, while some structures can be excluded.…”

Section: Discussionmentioning

confidence: 99%

Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

Darula

Medzihradszky

2014

J. Am. Soc. Mass Spectrom.

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Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common ‘capping’ unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in ‘beam-type’ CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was ‘neutralized’—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

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Automated and High Confidence Protein Phosphorylation Site Localization Using Complementary Collision-Activated Dissociation and Electron Transfer Dissociation Tandem Mass Spectrometry

Cited by 17 publications

References 30 publications

Effect of Metals in Biomimetic Dimetal Complexes on Affinity and Gas-Phase Protection of Phosphate Esters

Effect of Metals in Biomimetic Dimetal Complexes on Affinity and Gas-Phase Protection of Phosphate Esters

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

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