TY - JOUR
T1 - 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
AU - Branca, Rui Miguel Mamede
AU - Bodó, Gabriella
AU - Bagyinka, Csaba
AU - Prokai, Laszlo
PY - 2007/12/1
Y1 - 2007/12/1
N2 - 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.
AB - 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.
KW - Bottom up
KW - Collision-induced dissociation
KW - Cytochrome c
KW - De novo protein sequencing
KW - Electron-capture dissociation
KW - Electrospray ionization
KW - Fourier-transform ion-cyclotron resonance
KW - Ion trap
KW - Liquid chromatography-mass spectrometry
UR - http://www.scopus.com/inward/record.url?scp=38149119842&partnerID=8YFLogxK
U2 - 10.1002/jms.1337
DO - 10.1002/jms.1337
M3 - Article
C2 - 18085548
AN - SCOPUS:38149119842
SN - 1076-5174
VL - 42
SP - 1569
EP - 1582
JO - Journal of Mass Spectrometry
JF - Journal of Mass Spectrometry
IS - 12
ER -