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U p d a t e s . . .
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Volume 24, Number 8
April 15, 2004
DRUG DISCOVERY
Mass-Defect Tagging for Proteomic Analysis
Tutorial: Broadened
Applicability of
Differential Display
Mass Spectrometry
Luke V. Schneider, Ph.D.,
Michael P. Hall, Ph.D., and
Michael M. Brasseur
ass spectrometry has the ability to analyze DNA, peptides,
proteins, and small molecule
drugs in automated, high-throughput
assays, enabling and/or improving such
applications as protein target identification, validation, ADMET assays, and
others. The unique advantage of MSbased assays is the ability to gain more
molecular-level information about an
analyte, such as posttranslational modifications, and thus add more clinical relevance to the assay.
Protein differential-display analysis has
proved useful for the determination of
drug mechanisms and for assessing toxicological potential during preclinical
M
Figure 1.
Mass-defect periodic table
screening studies. Protein microarray
analysis has been used to find protein biomarkers of ovarian cancer in plasma.
With the average mammalian cell
expressing anywhere from 10,000 to
40,000 proteins, however, many are only
subtly different from each other. Good
differentiation techniques are thus essential to the generation of high-quality data.
Like many measurements conducted
in biology, MS measurements are often
ratiometric rather than absolute. In MS,
quantitative comparisons between samples, or even between spectra of the same
sample, are not reliable without the use of
stable isotope techniques. Several proteomic techniques, such as [16/18O]water hydrolysis of proteins, global internal standard technology, and isotopecoded affinity tags, have been described
in the literature.
These differential display techniques
allow direct quantitative comparison of
relative protein expression between two
or more tagged samples, by comparing
the relative abundance of stable isotope
versions of the same tag. The advantage
of these differential display methods is
that they are relatively inexpensive, quick,
and accurate (<10% standard deviation).
The problem with these techniques is
that they require separation of tagged
species from untagged species prior to
MS detection, to avoid false positive and
negative results.
For this reason, differential display
mass spectrometric methods have not
been applicable to chip-based methods
(e.g., affinity protein or DNA microarrays). By digesting the proteins bound to
an antibody on a protein chip, the antibody is also digested, producing a confusing array of background peptides in the
mass spectrum. Similarly, DNA oligomers
of different lengths due to incomplete
PCR amplification reactions and/or nonstringent binding conditions in DNA
microarrays, can yield spurious signals in
the mass spectrometer that may confound the identification and quantification of the peaks of interest.
By adding a mass-defect element to a
pair of stable-isotope tags, Target Discovery (Palo Alto, CA) aims to address this
limitation. These isotope-differentiated
binding-energy–shift tags (IDBEST™)
shift the peaks of all the tagged species by
about 0.1 Da, allowing software to discriminate tagged from untagged species
directly in the mass spectrum and thus
eliminating the need for affinity cleanup
of the tagged samples.
The mass defect is related to the nuclear
binding energy released upon formation
and stabilization of the nucleus of a given
element. Chemical-noise peaks in mass
spectra predominantly arise from unlabeled peptide fragments. These fragments
are composed of combinations of C, H, N,
and O atoms, which exhibit minimal mass
defects (Figure 1), therefore, peaks resulting from biomolecules have an intrinsic
regular spacing in the mass spectrum.
Utilizing a tag with an element containing a significant mass defect (e.g., between
Br and Eu in the periodic table) shifts the
mass of the tagged biomolecules off the
intrinsic spacing of the untagged
biomolecules (Figure 1).
Bromine and iodine are
good mass-defect elements in
that they are easily incorporated into organic tags.
Bromine is particularly
advantageous since it has a
lower mass than iodine and
has a nearly equivalent natural abundance of its two stable
isotopes (79Br and 81Br.).
It is noteworthy that, in
high-resolution mass spectrometers, the space between
the single- and higher-charge
states of biomolecules (i.e.,
negligible–mass-defect
species) leave a window of
nearly 0.4 amu unoccupied in
nearly every amu of the mass
spectrum. Therefore, it is posFigure 2. Mass spectrum and deconvolved spectrum showing
sible to incorporate up to four mass-defect peaks in an IDBEST™ differential display applicamass-defect atoms into an tion
IDBEST reagent before substantial overlaps with other biomole- tides are shifted from the unlabeled pepcules are encountered. Since each mass- tides, the relative ratios of light- and heavydefect element shifts the peak of the chain IDBEST peptides can be determined,
tagged species by an additional –0.1 and, thus, differential expressional differamu, a 1–mass-defect IDBEST tag can be ences can be precisely determined.
Figure 2 shows an example in which
resolved from a 2–mass-defect IDBEST
BSA
samples were labeled separately with
tag, and so on.
light and heavy versions of an IDBEST
Protein Differential Display
tag and mixed in equal portions in a pool
The mass defect in combination with of unlabeled E. Coli cytosolic proteins.
isotope-paired reagents can be used to The entire mixture was trypsinized and
determine expressional differences between analyzed on an Applied Biosystems 4700
control and perturbed samples (e.g., healthy MALDI-TOF MS Proteomics Analyzer.
versus diseased). In this case, the complex
Panel A shows the raw mass spectrum
protein mixtures are labeled separately in the region surrounding the [286-97]
with light or heavy chains of IDBEST. The tryptic fragment (labeled monoisotopic
samples are mixed, and the target pro- mass of 1,682.64 Da). The peaks corretein(s) of interest enriched by affinity sponding to the IDBEST peptide are seen
purification, such as on a microarray.
as shoulders shifted to the left of the abunThe resulting spot is digested to peptides dant chemical noise.
and directly analyzed by MALDI mass specPanel B shows the mass-defect spectrometry. Since the IDBEST-labeled pep- trum after deconvolution by the Target
sequencing by a method
called inverted mass ladder
sequencing (IMLS™). IMLS is
a variant of top-down sequencing, in which an N- or
C-terminal PST is generated
from the intact protein by
fragmentation in-source in an
inexpensive MS. IMLS involves
labeling the terminus of a protein with a mass-defect tag
that allows assembly of a PST
by mass addition of fragment
ions starting with the unique
mass of the chemical tag.
As described above, the fragmentation of whole proteins
generates a multitude of fragFigure 3. N-Terminal top-down protein-sequencing application
ment ions (“chemical noise”),
of mass-defect tags showing the raw protein-fragmentation
giving rise to peaks at nearly
spectrum, the deconvolved mass-defect spectrum showing the
Br-doublets, and the final peak-paired sequencing spectrum
every mass position in the
spectrum with an average
Discovery software. The chemical noise sequence-dependent peak spacing of
has been effectively eliminated and the 1.000464 amu. For example, myoglobin
isotope series of IDBEST-labeled tryptic was labeled with a bromine-containing
peptide are evident. Peak 1 corresponds to IDBEST tag, and the published sequence
the monoisotopic (12C/79Br) peptide. of myoglobin (GLSDGE) through six
Peak 2 corresponds to an overlap of both residues was recovered from the IMLS
the second 13C isotope of the 79Br light spectrum generated in an Applied BiosysIDBEST tag, the 12C isotope of the 81Br tems Mariner ESI-TOF MS (Figure 3).
light IDBEST tag, and the 79Br version of The IMLS software both deconvolves the
the heavy IDBEST tag. Peak 3 corresponds mass-defect spectrum and automatically
predominantly to the 81Br heavy IDBEST- determines the protein sequence by mass
tagged peptide.
addition to the known mass of the tag.
While visually confusing, these isotopic
Conclusion
distributions are easily resolved by the
software into simple light and heavy
Mass-defect tags provide a powerful
IDBEST-tagged contributions. The ratio method for enhancing the utility of MS in
of light to heavy chain in this example is proteomic analysis, both for protein differ1.04, which is very close to the expected ential display in affinity microarrays and
value of 1 of the known 50:50 mixture.
protein sequencing by IMLS. This
approach may also have utility in many
Mass-Defect Tags in Protein
other biomolecular mass spectrometric
Sequencing
Target Discovery has also successfully applications, such as identification of
utilized mass-defect tags for protein splice variants from DNA microarrays and
the sequencing of oligosaccharides.
The key advantage of a differential display strategy based on the mass defect is
preservation of the relative abundance of
each isotope peak, because these are
shifted away from any chemical noise. In
addition, this shift potentially eliminates
the need for prior separation (e.g., affinity purification of the tagged species).
Incorporating a nonextendable base
containing one or more mass-defect elements into DNA sequencing methodologies should allow discrimination of the
resulting mass spectral sequence ladders
from exogenous DNA in the sample. Furthermore, it should be possible to analyze
the sequencing ladders for all four bases
simultaneously if a different number of
mass-defect elements are incorporated
into each terminal base.
Another advantage of mass-defect tagging is that it potentially allows for the
simultaneous discrimination of more
than two molecules. Just as a single massdefect element tag can be discriminated
from unlabeled chemical noise, tags containing different numbers of mass-defect
elements can be discriminated from one
another within an amu.
Therefore, mass-defect tags may allow
up to five different species with the same
nominal mass to be discriminated in the
mass spectrometer before hitting the
double– charge-state limit. This suggests
that mass-defect tags may also extend
the number of possible tags that can be
discriminated simultaneously in combinatorial chemistry and high-throughput
screening applications where mass tags
are used.
GEN
Luke V. Schneider, Ph.D., is CSO, Michael P. Hall, Ph.D., is
senior scientist, and Michael M. Brasseur is director of
business development, at Target Discovery (Palo Alto).
For more information, contact Michael Brasseur. Phone:
(650) 812-8132. E-mail: [email protected].
Website: www.targetdiscovery.com.