| Metadata |
| datasetIdentifier | PASS00280 |
| datasetType | SRM |
| submitter | Benjamin Parker <b.parker@garvan.org.au> |
| submitter_organization | The Garvan Institute of Medical Research |
| lab_head_full_name | David E. James |
| lab_head_email | d.james@garvan.org.au |
| lab_head_organization | The Garvan Institute of Medical Research |
| lab_head_country | Australia |
| datasetTag | CKM_AcetylK_Phospho |
| datasetTitle | Investigation of proximal phosphorylation and lysine acetylation crosstalk on muscle-type creatine kinase using synthetic peptide substrates. |
| publicReleaseDate | 2013-07-25 00:00:00 |
| finalizedDate | 2013-11-27 00:18:45 |
| summary | Investigation of the rates of phosphorylation and lysine acetylation in response to proximal modifications. In vitro kinase, phosphatase, acetyltransferase and deacetylase reactions were performed using synthetic peptide variants with or without modification of phosphorylation and/or lysine acetylation. Substrates and products were monitored by nano-liquid chromatography - selected reaction monitoring (nLC-SRM) |
| contributors | Benjamin L. Parker, Nicholas E. Shepherd, Sophie Trefely, Nolan J. Hoffman, Melanie Y. White, Kasper Engholm-Keller, Brett D. Hambly, Martin R. Larsen, David E. James, Stuart J. Cordwell |
| publication | unpublished |
| growth | |
| treatment | Synthetic peptides were spiked into myocardial lysates or reacted with recombinant kinases or phosphatases, and the rates of (de)phosphorylation and (de)acetylaiton were measured. |
| extraction | Myocardial lysates were extracted in 20 mM Tris, 7.5 mM MgCl2, Protease Inhibitor Cocktail, pH 7.4 at 4蚓. For kinase assays, hearts were homogenized in lysis buffer supplemented with 5 mM ATP, Phosphatase Inhibitor Cocktail III (Calbiochem), and deacetylase inhibitor cocktail containing 10 然 Trichostatin A, 10 mM nicotinamide, and 50 mM butyric acid. For acetyltransferase assays, hearts were homogenized in lysis buffer supplemented with 1 mM acetyl-CoA containing phosphatase and deacetylase inhibitor cocktails. For phosphatase / deacetylase assays, hearts were homogenized in lysis buffer supplemented with 1 mM NAD. All lysates were adjusted to pH 7.4, kept at 4蚓 and assays performed within 30 min of lysis. |
| separation | Reactions were performed at 28蚓 and quenched with the addition of 20 無 of 20% trichloroacetic acid (TCA) to precipitate the lysate following 0, 10, 20, 30 and 60 min. The reactions were centrifuged at 16,000 x g for 15 min at 4蚓, and the supernatants removed. The supernatant was diluted 1:1 with 0.2% TFA and desalted with C18/R2/R3 microcolumns. Peptides were resuspended in 100 無 of 0.1% FA and 5 無 analyzed by nLC-SRM in technical triplicate. Peptides were trapped on a 0.5 cm x 300 痠 Zorbax C18 column (5 痠; Agilent Technologies, Palo Alto, CA) at 5 無/min for 5 min using an nanoLC 2Dplus system (Eksigent, Dublin CA). Peptides were separated by RP chromatography on an in-house packed 30 cm x 75 痠 Reprosil-Pur C18-AQ (3 痠; Dr. Maisch) PicoFrit column (New Objective, Woburn MA). The HPLC gradient was 0-40% solvent B (A = 0.1% FA; B = 90% ACN, 0.1% FA) over 10 min at a flow of 250 nL/min. |
| digestion | |
| acquisition | Mass spectrometric detection was achieved using a 5500 QTRAP hybrid triple quadrupole / linear ion trap mass spectrometer (AB SCIEX, Foster City CA) operated in MRM mode. Parameters for acquisition were as follows: GS1 20 psi, curtain gas 20 psi, interface temperature 150蚓, ionspray voltage 2200 V, unit-resolution. All 4 peptides were monitored (unscheduled) for each reaction. |
| informatics | Areas under the curve obtained using skyline (MacLean et al, 2010b) and expressed as % substrate conversion [Areaproduct/(Areasubstrate + Areaproduct)]. Transitions were Savitzky Golay transformed and integration boundaries manually verified. All reactions were analyzed in technical triplicate, and triplicates with a CV > 20% were removed. |
| instruments | AB Sciex QTRAP 5500 |
| species | Rattus |
| massModifications | variable: K+42.010565, S+79.966331 |
