Biology Reference
In-Depth Information
subsequent MS analysis. Furthermore, endoproteinases
work optimally in a detergent-free environment. The first
MS sample preparation methods successfully employed on
biological samples used detergent-mediated solubilization
followed by SDS polyacrylamide gel electrophoresis and
in-gel enzymatic digestion of proteins
[19]
. 'In-solution'
digestion methods employed detergent-free protein
extraction using strong chaotropic agents such as urea, and
digestion of proteins under denaturing conditions. In the
early days of applying MS to protein identification, stained
protein bands were excised from one-dimensional gel
electrophoresis runs, in-gel digested and analyzed directly
in the mass spectrometer by MALDI or electrospray. For
samples containing peptides from only one or a few
proteins, the combination of several peptide masses may be
sufficient for identification. This technique is called 'mass
fingerprinting' and it is still often used in conjunction with
two-dimensional gel electrophoresis (2D-GE). However,
both mass fingerprinting and 2D-GE have serious analytical
limitations in the dynamic range of protein abundances that
they can handle, as well as many other issues, and they are
no longer generally used in proteomics. Today the inherent
complexity of proteomic samples is being addressed by
a combination of fractionation techniques as well as fast
and sensitive mass spectrometers, but it remains a major
challenge when the goal is to define complete proteomes
[20]
. For these very complex mixtures, electrospray, and
not MALDI, is the ionization method of choice. This is
because electrospray handles analytes in solution, which
allows it to be coupled directly or 'on-line' to liquid
chromatography (LC) by applying the spray voltage to the
end of the chromatographic column. LC is arguably the
most powerful separation technique available for peptides,
which can then be analyzed sequentially as they elute from
the column. Current developments in peptide LC aim at
further improvements in separation as well as decreasing
flow rates and column diameters, which increases sensi-
tivity
[21]
.
In addition, a multitude of gel-based and gel-free frac-
tionation techniques have been developed that are applied
on either the protein or the peptide level prior to the liquid
chromatography step
[22
'image' analytes in situ, e.g. on tissue slices treated with
appropriate MALDI matrices
[27, 28]
.
Once peptides have been transferred into the vacuum of
the mass spectrometer, their mass-to-charge ratio (m/z) and
intensity have to be measured. For unambiguous identifi-
cation, it is additionally necessary to fragment each peptide
in turn and to record the resulting mass spectrum, a tech-
nique called MS/MS, MS
2
or tandem mass spectrometry. In
the data-dependent 'shotgun' approach, the most abundant
peptide species eluting from the LC column at a given time
are isolated one at a time and activated in the mass spec-
trometer, usually by collisions at low pressure of an inert
gas. Peptides mainly dissociate at the amide bonds,
generating overlapping series of N-terminal and C-terminal
fragments (called b-ions and y-ions, respectively)
[29]
.In
principle, the peptide sequence can be reconstructed 'de
novo' from a complete fragment ion series. In practice, it is
much easier to match uninterpreted fragment information
to a comprehensive protein sequence database of the
organism under investigation. There are many different
algorithms and search engines for this (see section
Computational Proteomics), but virtually all are based on
the comparison of measured masses with the theoretical
masses of expected peptides and their fragments.
Determining accurate masses is a key step in this
procedure, and advances in mass spectrometric technology
in recent years have made significant contributions to the
achievable depth of analysis. Key characteristics of a high-
performance mass spectrometer are resolution, mass
accuracy, speed, sensitivity and dynamic range
[30]
. High
resolution is the ability to distinguish two peaks of only
slightly different m/z ratio, while mass accuracy describes
the difference between measured and theoretical mass
[31]
.
Sensitivity is the capacity to detect low abundant analytes
whereas dynamic range of an instrument denotes the
difference between the lowest and highest abundant species
that are detected. Together, the aforementioned properties
should allow a high-performance instrument to perform
peptide sequencing at sufficiently high speed to obtain
adequate coverage of the complexity of the sample within
the timeframe of analysis. The Orbitrap mass analyzer is
a particularly powerful instrument in proteomics
[32
26]
. While increasing the
number of separation steps generally increases the depth of
coverage of the proteome, it also increases the sample
processing and MS-measurement time, as well as require-
ments for sample amount. Therefore, proteomics experi-
ments should be planned with the minimum number of
fractionation steps possible. This is especially important
when several conditions are to be measured and compared.
Although online coupling of LC with MS via electro-
spray is clearly the method of choice for complex protein
mixtures, the MALDI method still offers advantages in
specific situations. For instance, in principle the spatial
resolution of the MALDI laser spot makes it possible to
e
34]
,
but modern TOF-based analyzers are also popular
[35,36]
.
However, even today's best mass spectrometers are
technically unable to isolate and sequence all peptide
species present in an LC-MS run, resulting in extensive
undersampling of the observable peptides
[37]
. This leads
to a certain degree of stochastic behavior between shotgun
runs, which can complicate analysis, especially in systems
biology applications. In such cases, it is often attractive to
measure only a subset of peptides
e
such as those of a few
e
key proteins
but to ensure that they are measured in each
of multiple conditions. This requirement has led to so-
called targeted approaches, where the mass spectrometer is
e
