Biomedical Engineering Reference
In-Depth Information
per molecule, the overall fragmentation pattern is indicative of the amino acid sequence
of the selected peptide ion, and this pattern can be correlated with predicted fragmenta-
tion patterns from theoretical peptide digests from selected protein databases to produce
statistically significant candidate protein identifications. Database search algorithms, such
as Sequest and Mascot, are designed to interrogate databases with either peptide mass map
and/or fragmentation data ([10], and www.matrixscience.com), and most modern ESI and
MALDI instruments are capable of performing tandem MS experiments.
The peptide mass mapping strategy for resolved proteins is quick and reliable (especially
fast using MALDI-TOF), but the power of this approach falls off when the collection
of peptides is derived from three or more proteins. For more complex protein mixtures
and more global-scale analyses, tandem MS for peptide fragmentation analysis proves to
be the most sensitive detection analysis for MS, and is the method of choice for acquiring
mass spectral data from sub-proteomes, immuno-affinity captured-complexes and even from
unfractionated proteomes. This approach, colloquially termed 'shotgun' proteomic analysis,
is described further in Section 13.2.3.
Since protein identification by MS is reliant on pattern matching to protein sequences
present in databases, both the peptide mass mapping and the peptide fragmentation approach
fail when the protein sequences are not present in the selected database. For these and
other situations where identification is ambiguous, fragmentation data from tandem MS can
sometimes be used to identify homologous regions in other proteins or to identify sequences
that are only represented in expressed sequence tag (EST) databases. In extreme cases,
fragmentation data can be interpreted
de novo
and used to direct the design of degenerate
oligonucleotides for cloning purposes.
13.2 Methods and approaches
13.2.1 Gel-based strategies
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), first described by
Laemmli [11], has long been the method of choice for resolving intact proteins (based on
apparent molecular mass, commonly referred to as molecular weight, MW) for a variety of
biochemical analyses. For more complex mixtures, 2D gel electrophoresis (2DE) is typically
the method of choice to resolve intact protein species using two orthoganol separations, the
first based on charge (isoelectric point, pI) and the second by apparent molecular mass. The
2D gel experiments are particularly powerful for visualizing protein isoforms that result from
charged post-translational modification, such as phosphorylation and sulfation (which add
charge) or acetylation (which neutralizes charge). They are also useful in detecting splice
variants and proteolytic cleavages that result in protein species with altered MW and pI.
First introduced by O'Farrell and coworkers in 1975 [12], modern 2D gel technology
makes use of first-dimension isoelectric focusing through highly reproducible immobilized
pH gradient strips (IPGs) that are commercially available from a number of vendors [13, 14].
Proteins resolved by SDS-PAGE are often directly amenable to bottom-up protein identifi-
cation strategies after gel-excision and digestion of the target protein into peptides directly
within the gel slice. Although the 2D gel approach has historically been used for cataloguing
experiments, more recently it has been employed mostly for differential expression studies
on a global scale, where proteomes are compared between multiple experimental condi-
tions. For these comparative approaches, replicate gels are required to ensure that changes
