Generating protein sequence tags by combining cone and conventional collision induced dissociation in a quadrupole time-of-flight mass spectrometer.
The goal of proteomics research is to be able to identify and quantify the vast numbers of proteins within an organism or tissue. "Top-down" methods address this goal without the need for proteolytic digestion prior to mass analysis. We report here an approach for top-down protein identification that has been implemented on a commercially available, unmodified Qq-TOF mass spectrometer. Intact protein molecular ions first undergo cone fragmentation in the electrospray inlet. Conventional MS/MS is then performed on a mass selected cone fragment using CID in the Qq interface of the Qq-TOF mass spectrometer to generate a sequence tag through a pseudo-MS3 experiment. Seven proteins varying in molecular weight between 11 and 66 kDa were chosen to demonstrate applicability of method. After the molecular weight of the intact protein was determined, the cone voltage was varied to induce fragmentation. Cone fragment ions were then further dissociated using conventional CID, and the resulting MS/MS spectra were processed and analyzed for sequence tags. Sequence tags were easily identified from a MS/MS spectrum of a cone induced fragment ion both manually and through a de novo sequencing program included in the software associated with the mass spectrometer. Sequence tags were subjected to database searching using the PeptideSearch program of EMBL, and all protein sequence tags gave unambiguous search results. In all cases, sequence tags were found to originate from the n- and/or c-termini of the proteins.
Ginter
Joy M
JM
Department of Chemistry and Biochemistry, University of Delaware, 19716, Newark, DE, USA.
Zhou
Feng
F
Department of Chemistry and Biochemistry, University of Delaware, 19716, Newark, DE, USA.
Johnston
Murray V
MV
Department of Chemistry and Biochemistry, University of Delaware, 19716, Newark, DE, USA. mvj@udel.edu.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006736:Horses; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D013194:Staining and Labeling
D011506:Proteins