Protein Protocols Protein Protocols

Nanoelectrospray MS/MS for Peptide Sequencing 703
and to detect intact proteins or oligonucleotides in contaminated samples (23). A similar
method has recently been implemented on Q-TOF type instruments (24).
Once a set of peptide m/z values has been determined by either “single MS” scans or
by precursor ion scans, high-resolution scans can be performed for selected peptide ion
signals in the “multiple ion monitoring” mode to determine the exact peptide mass and
the peptide charge state based on the isotope spacing. The reduction of sensitivity when
measuring at high resolution is compensated by adding many scans, for example, 50 or
more, to one spectrum. The latter feature further demonstrates the utility of long
measurement times that the nanoelectrospray source provides. It is advantageous to
select doubly charged tryptic peptide ions for tandem mass spectrometry experiments
because they generate relatively simple fragment ion spectra. Triply charged tryptic
peptides can also be fragmented and often allow determination of long stretches of
amino acids sequence, that is, 15–25 consecutive residues, via doubly charged fragment
ion series.
2.7. Fragmenting Peptides by Collision-Induced Dissociation
Once a set of peptide m/z values has been accurately determined each peptide is
fragmented in turn. For peptide sequencing by tandem mass spectrometry using a triple
quadrupole or a Q-TOF instrument two main instrumental parameters are adjusted to
obtain high quality amino acid sequence information. First, the resolution setting of the
first quadrupole (Q1) is adjusted according to the abundance of the peptide ion signal,
that is, the lower the ion intensity the higher the resolution setting in order to reduce the
chemical background noise in the lower half of the tandem mass spectrum for a better
signal-to-noise ratio. Second, the collision energy can be adjusted according to the
peptide mass and varied depending on the mass range scanned (Fig. 5). The collision
gas pressure is kept constant throughout the MS/MS experiment.
It may be advantageous to acquire a tandem mass spectrum in two or three segments.
The high m/z segment is acquired with a wide parent ion selection window (low
resolution) and a low collision energy to generate and detect relatively large peptide
ion fragments. The low m/z region is acquired at higher resolution and at higher
collision energies to generate and detect low m/z fragments and immonium ions. The
nanoelectrospray allow this and other types of optimization due to the stability and
long duration of the spray.
When investigating a peptide mixture by tandem mass spectrometry as many peptides
as possible should be fragmented. This motivated the development of semiautomatic
software routines to assist in data acquisition. The list of peptide m/z values is
stored by customized software that calculates the optimum hardware settings for subsequent
sequencing of each individual peptide. However, for de novo sequencing of
long stretches of amino acid sequence it is not yet advisable to use automated software
routines for data acquisition. Careful adjustments of collision energy and mass resolution
is required to obtain high-quality data for unambiguous sequence assignments.
2.8. Generation of Peptide Sequence Tags from Tandem Mass Spectra
of Peptides
Complete interpretation of tandem mass spectra of peptides can be complicated and
requires some experience. However, it is often relatively straightforward to generate