Download New astrocyte cell surface markers

New astrocyte cell surface markers
Mass-spectrometric identification of the
astrocyte cell surface proteome reveals
new classification markers
Melanie Jungblut1, Andrea Jacobs1,2, Thomas Bock 2, Andreas Bosio1, and
Bernd Wollscheid2
1
Miltenyi Biotec GmbH, 51429 Bergisch Gladbach, Germany
2
Institute of Molecular Sytems Biology, NCCR Neuro Center for
Proteomics, ETH Zurich, Switzerland
4.
1.
5.
Introduction
Astrocytes are the most abundant cell type among cells of
the central nervous system. They are involved in the control
of synaptogenesis, synaptic transmission, neurogenesis,
and maintenance of neuronal metabolism.
Despite the importance of astrocytes, little is known about
their phenotype at the cell surface protein level and this
limits our understanding of the cell’s interactions with its
environment.
The isolation of astrocytes and their different subpopulations for functional studies is currently hindered mainly due
to a lack of specific cell surface protein markers. Magnetic
cell sorting by MACS® Technology is a convenient approach
for the fast and easy separation of a large amount of specific
cells from a mixed cell suspension. However, a prerequisite
for the selective isolation of a particular cell type via MACS
Technology is the identification of specific cell surface
proteins and availability of suitable antibodies.
Therefore, we used the mass spectrometry–based cell
surface capturing (CSC) technology to generate a snapshot
of the astrocyte cell surface glycoproteome (fig. 1).1–3 The
CSC technology enables phenotyping of cells without
antibodies and allows the specific analysis of a wide variety
of cell surface glycoproteins, leading to identification of
previously unknown astrocyte cell surface markers.
2.
6.
3.
Figure 1 Identification of cell surface glycoproteins using the
CSC technology. The CSC technology was used to detect cell surface
N-glycoproteins of astrocytes. The steps involve specific labeling of
oxidized cell surface polysaccharides using the bi-functional linker
molecule biocytin hydrazide (1), followed by cell homogenization and
protein digestion (2), affinity enrichment of biocytin hydrazide–labeled
peptides (3), enzymatic peptide release (4), peptide analysis by
tandem mass spectrometry (LC-MS/MS) (5), and peptide or protein
identification (6).
Results
Figure 2 Astrocyte cultures. Astrocytes were cultured for 21 days
after plating and then immunostained using an anti-GFAP antibody
(green). Nuclei were visualized with DAPI (blue).
GLAST
25%
8,5%
8%
Marker 1
Conclusion
• The identified markers should represent ideal targets for
the ongoing development of novel astrocyte-specific
cell separation reagents.
References
1. Wollscheid, B. et al. (2009) Nat. Biotechnol. 27: 378–386.
2. Doerr, A. (2009) Nat. Methods 6: 401.
3. Gundry, R.L. et al. (2008) Proteomics Clin. Appl. 2: 892–903.
4. Pennartz, S. et al. (2009) J. Vis. Exp. 29 pii:1267;
doi: 10.3791/1267.d
CD45
21%
4%
Marker 2
Marker 2
• The CSC analysis identified 482 astrocyte cell surface
glycoproteins and revealed a high complexity in the
astrocyte cell surface proteome.
14%
GLAST
• New astrocyte cell surface markers were identified and
antibody generation, as well as functional studies, are
ongoing.
2%
22%
12%
CD45
The antibody-independent experimental approach
created a qualitative ‘snapshot’ of the astrocyte cell
surface proteome used as the basis for MACS Cell
Separation and quantitative studies.
Marker 1
GLAST
•
0.5%
CD45
GLAST
As astrocytes cannot be isolated from primary brain tissue
of wild-type mice, astrocytes in vitro were used for mass
spectrometric analysis of the surface proteome. Therefore,
cortical tissue obtained from P1 mice was dissociated using
the Neural Tissue Dissociation Kit (P) and single-cell suspensions were plated onto poly-D-lysine–coated culture flasks.
After three weeks, the cultures consisted predominantly of
astrocytes, which was confirmed by immunocytochemical
staining of the intracellular astrocyte marker glial fibrillary
acidic protein (GFAP) (fig. 2).
The astrocyte cultures were then analyzed by the CSC
technology to identify cell-specific surface markers. This
experimental approach revealed 482 astrocyte cell surface–
exposed glycoproteins.
To test whether some of these antigens could serve as
useful astrocyte markers, brain tissue derived from P7
mice was dissociated using a Neural Tissue Dissocation
Kit (T or P) and single-cell suspensions were labeled with
commercially available antibodies specific for the identified
proteins and analyzed by flow cytometry. A monoclonal
antibody directed against the astrocyte-specific glutamate
transporter (GLAST, sometimes referred to as EAAT1) was
used for co-staining, as well as an antibody specific for the
leukocyte marker CD45 (fig. 3). We found that Markers 1 and
2, for example, are expressed by GLAST+ astrocytes but not
by CD45+ leukocytes and may serve as astrocyte-specific
surface markers. Marker 3, in contrast, is also expressed by
CD45+ leukocytes.
23%
20%
Marker 3
Marker 3
Figure 3 Validation of promising astrocyte cell surface markers.
Brain tissue from P7 mice was dissociated using a Neural Tissue
Dissociation Kit and single-cell suspensions were labeled with
antibodies specific for astrocyte cell surface antigens identified by
CSC technology, as well as with antibodies against either the astrocyte
marker GLAST or the leukocyte marker CD45.
MACS® Product
Order no.
Neural Tissue Dissociation Kit (P)
130-092-628
Neural Tissue Dissociation Kit (T)
130-093-231
Anti-Astrocyte (GLAST) MicroBeads
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