Quantitative Analysis of Nt-acetylation

siCtrl and siNatA knockdown cell lysates were prepared as described above and the samples were analyzed on a 4–16% gradient XT precast Criterion gel using XT-MOPS buffer (Bio-Rad). Gel slices corresponding to the molecular weights (∼29 kDa) of the tagged THOC7 variants (between 25 and 37 kDa) were cut from the gel and ISIL was performed as described previously (62 (link)). The resulting peptide mixtures were acidified (0.1% formic acid) and analyzed by LC–MS/MS analysis. Peptide mixtures were introduced into an Ultimate 3000 RSLC nano LC-MS/MS system (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) as described previously (30 (link)). When operating the instrument in Higher Energy Collision Dissociation (HCD), spectra were acquired in the Orbitrap with an effective FWHM resolution >7500 around m/z 400. The extent of Nt-acetylation was calculated from the peptide ion signals observed in the MS spectra (5 (link)).

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Myklebust L.M., Van Damme P., Støve S.I., Dörfel M.J., Abboud A., Kalvik T.V., Grauffel C., Jonckheere V., Wu Y., Swensen J., Kaasa H., Liszczak G., Marmorstein R., Reuter N., Lyon G.J., Gevaert K, & Arnesen T. (2014). Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects. Human Molecular Genetics, 24(7), 1956-1976.

Publication 2014

XT-MOPS buffer LTQ Orbitrap Velos

Acetylation Buffer Cell Formic acid Mops Ms ms Peptide Signals peptide

Corresponding Organization :

Other organizations : University of Bergen, VIB-UGent Center for Medical Biotechnology, Ghent University, Haukeland University Hospital, Cold Spring Harbor Laboratory, Uni Research (Norway), Stony Brook University, Caris Life Sciences (United States), University of Pennsylvania, The Wistar Institute

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Variable analysis

independent variables

SiCtrl knockdown
SiNatA knockdown

dependent variables

THOC7 variant levels (between 25 and 37 kDa)
Extent of Nt-acetylation

control variables

Molecular weights (∼29 kDa) of the tagged THOC7 variants

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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