PPI
is exploring the use of a novel method (denoted SISCAPA)
for quantitation of peptides in complex digests.
In the method, anti-peptide antibodies immobilized on
100 nanoliter nanoaffinity columns are used to enrich
specific peptides along with spiked stable-isotope-labeled
internal standards of the same sequence. Upon elution
from the anti-peptide antibody supports, electrospray
mass spectrometry is used to quantitate the peptides (natural
and labeled). SISCAPA is thus limited to sequence-defined
(predetermined) analytes, but offers the possibility of
greatly increased sensitivity (by removing unwanted peptides
from the set delivered to the MS).
SISCAPA
is described in two initial papers describing 1) the overall
method
and 2) the techniques
for generating and characterizing immobilized Ab supports:
Mass Spectrometric Quantitation of Peptides and Proteins
Using Stable Isotope Standards and Capture by Anti-Peptide
Antibodies (SISCAPA). Anderson, N.L., Anderson,
N.G., Haines, L.R., Hardie, D.B., Olafson. R.W., and Pearson,
T.W. Journal of Proteome Research, in press (2004).
An
effective and rapid method for functional characterization
of immunoadsorbents using POROS® beads and flow cytometry.
N. Leigh Anderson, N.L., Haines, L.R. and Pearson,
T.W. Journal of Proteome Research, in press (2004).
In
a series of pilot experiments, tryptic test peptides were
chosen for four proteins of human plasma (hemopexin,
a1-antichymotrypsin, interleukin-6 and tumour necrosis
factor-alpha) from a pool of 10,203 in silico tryptic
peptide candidates representing 237 known plasma components.
Rabbit polyclonal antibodies raised against the
chosen
peptide sequences were affinity purified and covalently
immobilized on POROS® supports. Binding and
elution from these supports was shown to provide an average
120-fold enrichment of the antigen peptide relative to
others, as measured by selected ion monitoring (SIM) or
selected reaction monitoring (SRM) electrospray mass spectrometry.
The columns could be recycled with little loss
in binding capacity, and generated peptide ion current
measurements with cycle-to-cycle coefficients of variation
near 5%. Anti-peptide antibody enrichment will
contribute to increased sensitivity of MS-based assays,
particularly for lower abundance proteins in plasma, and
may ultimately allow substitution of a rapid bind/elute
process for the time-consuming reverse phase separation
now used as a prelude to online MS peptide assays.
The method appears suitable for rapid generation of assays
for defined proteins, and should find application in the
validation of diagnostic protein panels in large sample
sets.
To
facilitate the construction, functional characterization,
and use of immunoadsorbents, we have developed a flow
cytometry method that allows rapid assessment of large
numbers of particle-bound antibodies. Protein G derivitized
POROS beads were used to bind affinity-purified antibodies
specific for synthetic peptides designed from human plasma
proteins. The antibodies were covalently coupled to the
beads and used to capture and release synthetic peptides
that had been labeled at the C-terminus with the fluorochrome
Alexa Fluor 488. Antibody coupling and specificity of
antigen binding and release were measured by analysis
of the POROS affinity beads by flow cytometry. The affinity-capture
matrixes were also used through several antigen-binding
and release cycles without loss of peptide binding efficiency.
The ability to produce and characterize extremely small
amounts of POROS affinity matrices will facilitate their
use in protein microchemical procedures such as protein
chip technology, monoclonal antibody screening and mass
spectrometry, applications where analytes are limiting
or present in low abundance in complex mixtures.
