Biology Reference
In-Depth Information
using MS
2
requires the digestion of the proteins
into peptides. In MS
2
, peptide are collided with
an inert gas and fragmented into a series of peptide
ladders via a process known as collision-induced
dissociation (CID). Fortunately, CID results in
the fragmentation of peptide ions primarily along
its backbone.
Figure 2
illustrates a MS
2
spectrum
for a short peptide. As indicated in this
peptide mapping data (
Table 1
). At a minimum,
the programs require an input that includes the
experimental peptide ion list and database to
compare this list to. Some programs allow addi-
tional information, such as isoelectric point (pI),
molecular weight (MW), and source organism
that can be used to identify the protein. Some of
this additional information (e.g., pI and MW) is
determined using other protein chemistry tech-
niques primarily one- or two-dimensional gel
electrophoresis. Although all of the programs
listed in
Table 1
are suf
gure,
the distances between various y ions is equal to
the molecular mass of the speci
c amino acids
within the peptide. That fragmentation occurs
primarily across amide bonds and has allowed
“
cient for analyzing
peptide mapping data, Mascot
2
stands out as
the most popular, owing to its longevity, and
many MS users have built a familiarity with this
software. Probably the primary reasons for select-
ing a speci
”
to be devised for creating software
programs for analyzing MS
2
data. Most mass
spectrometers yield b and y ions, which corre-
spond to fragmentation of the amide bonds with
the charge retained at theNH
2
andCOOHtermini,
respectively. Other bonds are fragmented during
CID (i.e., a, c, x, and z ions); however, these are
generally less intense. As with peptide mapping,
the experimental MS
2
spectra are compared to
a database of protein sequences using the known
rules for CID fragmentation of peptides. A subtle,
but important, point is that the energy put into the
peptides during CID is insuf
rules
c peptide mapping software program
include it being part of the MS purchase, its ease
of use, and its integration with other software
programs used in the analysis of MS data.
Tandem Mass Spectrometry
Peptide mapping is useful only for identifying
isolated proteins or maybe a simple mixture of
2 ot 3 proteins. For complex mixtures, MS
2
is
required.
3
This method can identify isolated
proteins or proteins within mixtures containing
up to 100,000 different species. As with peptide
mapping, bottom-up identi
cient to completely
dissociate every amide bond. If every amide
bond fragmented, the MS spectrum would
primarily contain masses equal to those of the
constituent amino acids. The resultant CID frag-
mentation creates
of amino acid resi-
dues originating from the peptide produced,
enabling the sequence of the peptide to be read
much like a DNA sequencing ladder is produced
by Sanger sequencing.
4
Although MS
2
identi
“
ladders
”
cation of a protein
TABLE 1
Software Available for Protein Identi
cation
Using Peptide Mass Fingerprinting
ca-
tion is required for
identifying peptides in
Software Program
URL
complex mixtures,
it also provides higher
MultiIdent
con
cations for isolated proteins
than can be achieved using peptide mapping.
The reason for the greater con
dent
identi
Mascot
dence afforded
usingMS
2
is that themost distinctive characteristic
that identi
MS-Fit
PepMAPPER
es a protein is its amino acid sequence,
which is the type of data provided by MS
2
.
The rawMS
2
data produced by the mass spec-
trometer is quite complicated. Although it is
possible to determine part of
MassWiz
Protein Lynx
ProFound
the peptide
'
s
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