Proteome analysis of Sorangium cellulosum employing 2D-HPLC-MS/MS and improved database searching strategies for CID and ETD fragment spectra.
Shotgun proteome analysis of the myxobacterial model strain for secondary metabolite biosynthesis Sorangium cellulosum was performed employing off-line two-dimensional high-pH reversed-phase HPLC x low-pH ion-pair reversed-phase HPLC and dual tandem mass spectrometry with collision-induced dissociation (CID) and electron transfer dissociation (ETD) as complementary fragmentation techniques. Peptide identification using database searching was optimized for ETD fragment spectra to obtain the maximum number of identifications at equivalent false discovery rates (1.0%) in the evaluation of both fragmentation techniques. In the database search of the CID MS/MS data, the mass tolerance was set to the well-established 0.3 Da window, whereas for ETD data, it was widened to 1.1 Da to account for hydrogen-rearrangement in the radical-intermediate of the peptide precursor ion. To achieve a false discovery rate comparable to the CID results, we increased the significance threshold for peptide identification to 0.001 for the ETD data. The ETD based analysis yielded about 74% of all peptides and about 78% of all proteins compared to the CID-method. In the combined data set, 952 proteins of S. cellulosum were confidently identified by at least two peptides per protein, facilitating the study of the function of regulatory proteins in the social myxobacteria and their role in secondary metabolism.
Leinenbach
Andreas
A
Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, Saarbrucken, Germany.
Hartmer
Ralf
R
Lubeck
Markus
M
Kneissl
Benny
B
Elnakady
Yasser A
YA
Baessmann
Carsten
C
Müller
Rolf
R
Huber
Christian G
CG
D000465:Algorithms; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D030562:Databases, Protein; D016247:Information Storage and Retrieval; D009231:Myxococcales / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*