Search options 2017-09-11T12:22:17+00:00
RAW file conversion

Triple-Tof

  1. AB SCIEX MS Data Converter (Beta Version 1.3)

You can download the installer for this component from the AB SCIEX:

https://sciex.com/software-support/software-downloads

  1. Msconvert : msconvert is a command line tool for converting between various file formats. For Windows users, the msConvertGUI program is also available for easy file conversion.

You can download the installer for this component from the Proteowizard:

http://proteowizard.sourceforge.net/downloads.shtml

For the GPA_TofMS Search, you have to convert into two different formats – MS1(.TXT) and MGF.

For Examples,

Orbitrap

The mass spectrometer RAW file from Orbitrap was converted to ms1 (TXT) and ms2 (MGF) files using the freeware program RawExtractor v1.9 (The Scripps Research Institute, La Jolla, CA) and MM File Conversion Tools v3.9 (http://www.massmatrix.net/mm-cgi/downloads.py).

For glycoprotein analysis, raw files were converted to ms1 and ms2 by RawConverter (Version 1.0.0.0).

Search parameters

N-glycopeptides were analyzed by high resolution mass spectrometry with HCD, CID, and HCD/CID fragmentation followed by GPA, with following search parameters: glycopeptide tolerance = 10 ppm; Fragment tolerance = 0.02 Da (HCD) and 1.5 Da (CID); Missed cleavages = 0, Modification: Carbamidomethyl cysteine (fixed), N-glycan search (Mammalian).

Glycan Database

GPA-DBs were constructed for each sample, using the software GPA-DB-Builder, by combining possible tryptic peptides and 351 N-linked glycans, where 331 retrosynthetic glycans came from references of Kronewitter, S.R. et al.[1] and 20 glycans from penta and hexa polylactosamine series of Ozohanics, O. et al.[2].

References

[1] Kronewitter, S.R. et al. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome. Proteomics 9, 2986-2994 (2009).[2] Ozohanics, O. et al. High-performance liquid chromatography coupled to mass spectrometry methodology for analyzing site-specific N-glycosylation patterns. J. Chromatogr. A 1259, 200-212 (2012).