T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry.
A novel extension of the "top-down" approach is introduced for the selective characterization of protein termini that does not involve proteolytic digestion steps. N- and C-terminal peptides were generated from intact proteins in the mass spectrometer and further analyzed by MS/MS-an approach referred to as T(3)-sequencing. N-terminal and C-terminal fragment ion series were obtained by the pseudo-MS/MS technique in-source decay (ISD) on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). These ions provided near-terminal sequence tags from the undigested protein in the ISD spectrum acquired in reflector mode and allowed to screen for the proper processing state of the terminus with respect to a reference sequence. In the second step of T(3)-sequencing, the precursor ions, which have been generated by ISD and which included the N- or C-terminal sequence, were selected in the timed ion gate of a MALDI-TOF/TOF mass spectrometer for MS/MS analysis. These spectra allowed identification of the protein, the proper definition of both termini, and allowed confirmation of suspected terminal modifications. T(3)-Sequencing appears to be an alternative to classical Edman sequencing, which is fast and even permits the analysis of N-terminally blocked proteins and their C-terminus.
Bruker Daltonik GmbH, Fahrenheitstrasse 4, D-28359 Bremen, Germany. Dsu@bdal.de
Detlev
D
Suckau
Anja
A
Resemann
D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D030562:Databases, Protein; D001330:Electronic Data Processing; D007477:Ions / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012260:Ribonucleases / Q000032:analysis / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D007477:Ions; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; D012260:Ribonucleases; C029600:ribonuclease B; D002256:Carbonic Anhydrases