P74-M Identification and Site Determination of PTM in FMIP Peptides Using Electron Capture Dissociation on a FTICR Mass Spectrometer

Abstract

The Fms-interacting protein (FMIP, MW 78 kDa) acts as a shuttling protein during macrophage differentiation in the human immune system.¹

Previous work on an enzymatic digest of this protein has revealed a peptide with a precursor mass corresponding to the FMIP peptide YTCQELQR with the addition of 80Da. This mass difference may be attributable to either phosphorylation or sulfonation. The type of post-translational modification (PTM) could not be determined by detailed analysis of the resulting MS/MS spectrum obtained under typical collision-induced dissociation conditions.

In order to distinguish the PTM, we analyzed the sample on a Thermo Fisher LTQ-FT Ultra mass spectrometer making use of its very high and reliable mass accuracy. In addition, electron capture dissociation (ECD) was performed on the peptide in an attempt to assign unambiguously the site of modification.

The sample was separated using a nanoLC setup (ThermoFisher Scientific Micro Autosampler and Surveyor MS Plus pump) equipped with a C18 trapping column and a C18 100 × 0.075 mm analytical column (both: www.nanoseparations.com). Standard HPLC solvents (MS grade) were used for the analysis: water/acetonitrile (98:2 v/v, 0.1% formic acid) as solvent A and acetonitrile/water (80:20 v/v, 0.1% formic acid) as solvent B.

The applied method used a FTMS full scan and three data-dependent ECD scans per cycle, picking the top three signals from the full scan and subsequently excluding these for 20 sec.

The analysis clearly reveals the type of modification as well as the site. The poster will present the data as well as their interpretation.