T3-Sequencing: Targeted Characterization of the N- and C-Termini of Undigested Proteins by Mass Spectrometry

Scientific Paper

Expression Proteomics

10.1021/ac034362b

2003

ultraflex TOF/TOF

Detlev Suckau* and Anja Resemann
Bruker Daltonik GmbH, Fahrenheitstrasse 4, D-28359 Bremen, Germany

Anal. Chem. 2003, 75, 5817-5824

Abstract

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/

MSsan approach referred to as T3-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 T3-sequencing, the precursor ions, which

have been generated by ISD and which included the Nor

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. T3-

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 Cterminus.

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