Autoantibodies and Biomarker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 358

Cellular Fractionation and
Immunoblotting
A primary concern with the use of phage
display, as well as solution-based display, is
that proteins are not expressed in native form
and post-translational modi

antigens are displayed in the physiologically
relevant form that initially elicited the humoral
immune response. However, proteins in cell/
tissue lysates exist in varying abundance that
differs by orders of magnitude, and the dynamic
range of analysis is limited. The signals from low
abundance proteins can be buried by high abun-
dant housekeeping proteins. The identi

cations are not rep-
resented. An alternative approach uses relevant
tissue lysates as the source of candidate antigens,
which are then probed with patient sera.
Initially, autoantigens were observed directly in
cell lysates using immunodiffusion, but the
complexity of protein content within lysates
limited this approach until the innovation of
techniques in protein separation by column
chromatography and gel electrophoresis per-
mitted suf

cation

of target antigens within a speci c spot or frac-

tion requires deconvolution by mass spectrom-
etry and often requires further targeted

separation to con rm the identity of the anti-

gens. Translation of these targets to clinical
biomarker assays is also a major challenge;
high-throughput serologic validation assays
require recombinant protein production that
may not display the antigenic PTMs.

cient separation of protein content.
Currently, lysate preparation requires two-
dimensional and three-dimensional protein
separation coupled with target identi

Protein Microarrays
The development of protein microarrays has
now enabled the simultaneous analysis of thou-
sands of different proteins on the footprint of
a microscopic slide or in bead-array format.
With the development of high-throughput
protein production, it is now possible to produce
proteins that are limited only by ORFeome
content. In the near term, protein arrays display-
ing the entire human proteome will soon be
available, although protein arrays that re

cation by

mass spectrometry.
Two-dimensional immunoblotting (2D-
Western) is well established for the assessment
of antibody reactivity and has been applied to
autoantigen discovery. Two-dimensional gel
electrophoresis separates proteins in complex
biological samples based on isoelectric point
and molecular weight. Lysate preparation and
gel composition can be customized to improve
the c protein classes.

After addition of sera, immunogenic spots are
excised and sequenced for protein identi

separation of speci

ect
the genetic variability of tumors and the splice
variation of tissues will take longer to develop.
One natural advantage of protein microarrays
is the rapid, multiplexed measurement of anti-
body levels in sera.
There are three primary methods for
producing protein microarrays. First, proteins
are expressed in model organisms such as
E. coli, yeast, or insect cells, puri

cation
by mass spectrometry. 36,37 Because immunoblot-
ting favors the detection of denatured epitopes,
solution-based methods of antigen detection
were developed. Multidimensional separation
in liquid phase by reverse phase/ion exchange
chromatography and chromatofocusing in
conjunction with arraying thousands of protein
factions prepared from cell/tissue lysates results
in native protein arrays that contain relevant
post-translational modi

ed, and printed
on the arrays. 39 e 44 In vitro protein production is
a challenging process that can be hindered by
protein purity, batch-to-batch variation, and
limited stability of puri

cations (PTMs). 38 For
both 2D-Western and native protein arrays, the
autoantigens can be prepared with relative ease
without prior cloning and puri

ed protein products.
The second approach is to use printed antibody
arrays to detect antigen/antibody complexes cation. The