152 INSTRUMENTAL METHODS
fragment contains lys or arg, so that all fragments are suitable for positive
ionization mass spectrometric analysis. Peptides of ∼ 2.5 kDa or less in molecu-
lar mass produce the most useful data. Third, the digest mixture is analyzed
by MS, producing a complex spectrum — a peptide map — from which the
molecular masses of all the proteolytic fragments can be read. At this point,
the researcher may compare results to a peptide map database and possibly
confi rm the protein ’ s identity from this comparison. Up to this point, the
tandem mass spectrometer has been operating in the MS mode. If the protein
has not been identifi ed at this point, the operator switches to the MS – MS
mode. Using the quadrupole – TOF mass spectrometer as a model, the identi-
fi cation proceeds as follows. The digest mixture continues to spray into the
mass spectrometer. The protonated molecular fragments of each digest frag-
ment are independently selected and transmitted through the quadrupole
analyzer, now being used to submit only the ions of interest to the collision
cell lying between the fi rst and second mass analyzers. The fragment — daugh-
ter or product — ions produced by the collision cell are then analyzed by the
TOF analyzer. The MS – MS spectrum produced shows all the fragment ions
arising directly from the precursor ions for a given peptide component. MS –
MS spectra are produced for each component in the proteolytic digest. At this
point, experience in interpretation and processing of the results becomes very
important to accurately identify the protein(s) in the sample. Some peptides
from the proteolytic digest may be completely sequenced, while others may
yield only a partial sequence of four to fi ve amino acid residues. Often, this
sequence “ tag ” of four to fi ve amino acids may be suffi cient to search a protein
database and confi rm the protein ’ s identity. Only about 4 μ L of solution is
required for the digest mixture analysis if the solution concentration is suffi -
cient — that is, concentration of the original protein is ∼ 1 – 10 pmol/L. More
information on the techniques and results can be found at many websites. Two
of these are: http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm and
http://mascot.proteomics.ucdavis.edu/mascot/help/fragmentation_help.html.
Small proteins and peptides can be sequenced using MALDI – TOF – MS
with a “ ladder ” sequencing technique.^58 First, the researcher fragments the
protein or peptide using either a time - dependent or concentration - dependent
chemical degradation beginning from either the N - or C - terminus of the
protein or peptide. The resulting protein or peptide fragments differ from each
other by one amino acid residue. The mixture is mass analyzed in a single
MALDI – TOF – MS experiment with mass differences between adjacent mass
spectral peaks corresponding to a specifi c amino acid residue. This type of
analysis can be thought of as simply determining the masses of a series of
peptides or proteins that are present in a single MALDI sample. The order of
occurrence in the mass spectrum defi nes the sequence of amino acids in the
original protein or peptide.
As discussed in Section 2.2.3, proteomic investigations require powerful
analytical techniques such as those continuing to be developed using mass
spectrometry. As Aebersold and Mann declare in a 2003 review of mass spec-