De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry

• Published in: Journal of mass spectrometry Vol. 42; no. 12; pp. 1569 - 1582
• Main Authors: Branca, Rui Miguel Mamede, Bodó, Gabriella, Bagyinka, Csaba, Prokai, Laszlo
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.12.2007; Wiley
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; bottom up; collision-induced dissociation; Cyclotrons; Cytochrome c Group - chemistry; cytochrome c4; de novo protein sequencing; electron-capture dissociation; electrospray ionization; Fourier Analysis; Fourier-transform ion-cyclotron resonance; Fundamental and applied biological sciences. Psychology; Hemoproteins; ion trap; liquid chromatography-mass spectrometry; Metalloproteins; Methylation; Molecular Weight; Nanotechnology; Oxidation-Reduction; Peptide Hydrolases - chemistry; Peptides - analysis; Peptides - chemistry; Proteins; Sequence Analysis, Protein; Spectrometry, Mass, Electrospray Ionization; Thiocapsa - chemistry; Tandem mass spectrometry; Hemoprotein; Fourier transformation; Peptides; Thiocapsa roseopersicina; HPLC chromatography; Fourier-transform ion-cyclotron resonance; electrospray ionization; Electrospray; Electron capture; Rhodospirillales; Ion trap; collision-induced dissociation; Basic protein; bottom up; Bacteria; Aminoacid sequence; Collisional activation; electron-capture dissociation; Coupled method; liquid chromatography-mass spectrometry; Primary structure; de novo protein sequencing; Ion cyclotron resonance spectrometry; cytochrome c4; De novo; Cytochrome c; Electron capture dissociation; Peptide map; Analysis method; Microbial origin; Peptide fragment; Thiocapsaceae; Structural analysis
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/jms.1337

We have determined the primary structure of cytochrome c4 from Thiocapsa roseopersicina by de novo protein sequencing using the ‘bottom up’ approach. Three different enzymes (trypsin, endoproteinase Lys‐C, and endoproteinase Glu‐C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier‐transform ion‐cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision‐induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination. Copyright © 2007 John Wiley & Sons, Ltd.