Biomedical Engineering Reference
In-Depth Information
expression levels and in post-translational alterations, including biologically significant pro-
teolysis and modification (via phosphorylation, acetylation and glycosylation, among many
others) that are completely hidden in the static DNA code.
Many proteomic strategies contain protein identification as a major component, and this is
routinely performed using MS followed by a statistical comparison of the mass spectral data
with theoretical data generated from protein sequence databases that themselves are mostly
generated from the genomic sequences. In some cases, quantification of protein abundance
changes can also be made using MS (described in more detail in Sections 13.2.2 and
13.2.3). For identification purposes, MS is used to produce data characteristic of individual
proteins, usually at the level of amino acid sequence or peptides that are generated after
digestion with a site-specific protease, and sometimes include accurate measurements of
the intact protein mass. Powerful bioinformatics algorithms can then be applied to search
ever-expanding databases for proteins that match these experimentally derived mass spectral
signatures [2-9].
The implementation of new ionization sources for MS in the mid 1980s allowed for
dramatic increases in sensitivity, resolution and mass accuracy for peptide and protein MS.
These ionization sources are electrospray ionization (ESI) and MALDI, both of which can
be coupled to a wide variety of mass analyzers. Many instrument configurations are avail-
able that have complementary and overlapping capabilities, and it is easy for a novice to
get lost in the technical jargon. Typical configurations for proteomics experiments include
ESI sources coupled with a variety of medium-high-resolution mass analyzers, including
linear quadrupoles, time-of-flight (TOF), quadrupolar ion traps (three-dimensional or linear
ion traps) and ultra-high resolution orbitrap and Fourier-transform ion cyclotron resonance
(FTICR) mass analyzers. MALDI sources can also be coupled to this variety of mass
analyzers, but are more typically in line with TOF mass analyzers.
Strategies for protein characterization using MS come in two basic forms, colloqui-
ally termed a 'top-down' and a 'bottom-up' approach. Top-down strategies are named so
because they begin with the acquisition of the intact mass of the protein, typically using a
high-resolution instrument such as a MALDI-TOF or ESI-FTICR mass spectrometer, fol-
lowed by subsequent fragmentation analysis. This approach is considerably difficult to adapt
to a global-scale discovery-phase proteomics experiment, but can be extremely effective for
targeted studies. The bottom-up approach begins by digesting the target protein(s) or pro-
teome into much smaller fragments. This approach works because the peptides are very easy
to acquire mass spectral information on, and the resulting peptide masses and fragmentation
patterns can be extremely predictive for protein identification using software search tools.
In a typical bottom-up protein identification strategy, individual proteins are first digested
with a site-specific protease (such as trypsin) to produce a discrete set of peptides that can
be collectively mass analyzed. This is easily performed on biochemically- or gel-resolved
proteins. The collection of peptide ion masses, which can be measured under conditions of
high mass accuracy (less than 10 ppm using MALDI-TOF MS), can be used to interrogate
protein databases directly for statistically significant candidate protein matches, a process
referred to as peptide mass mapping or fingerprinting [6-9]. In many cases, peptide mass
mapping is more than sufficient to generate unambiguous matches.
Additional mass spectral data can be generated from individual peptide ions from the mix-
ture to reveal information about the amino acid sequence. In such a tandem MS experiment,
a fragmentation step is incorporated where individual ions, selected in a first-stage mass
analyzer, are now fragmented about the amide bonds (between the amino acids), and the
fragment ions are subsequently mass analyzed. As ions are fragmented about once or twice
