Supporting Information
Supplementary Methods
Two-step FASP
For the secretome analysis, proteins (200 g) were mixed with SDT buffer (1:1) and loaded
onto a 30-k spin filter (EMD Millipore, Billerica, MA, USA). Buffer was exchanged with UA solution
(8 M Urea in 0.1 M, pH 8.5) by centrifugation. Reduced cysteines were alkylated with IAA solution
(0.05 M iodoacetamide in UA solution) for 30 min at room temperature (RT) in the dark. Additional
buffer was exchanged with 40 mM ammonium bicarbonate. The proteins were first digested with
trypsin (enzyme-to-substrate ratio [w/w] of 1:100) at 37°C overnight, after which the peptides were
collected by centrifugation. In the second digestion, the filter units were washed sequentially with
water, UA buffer, and 40 mM ammonium bicarbonate, respectively, and the proteins were cleaved
with trypsin (enzyme-to-substrate ratio [w/w] of 1:200). For the whole-cell proteome analysis, 200 g
of proteins was mixed with UA solution and loaded onto a 30-k spin filter. The first and second
digestions were performed as for the secretome.
Desalting
Prior to peptide fractionation, all digested peptide mixtures were acidified with 1% TFA and
desalted using homemade StageTips as described [1]. Self-packed reversed-phase microcolumns were
prepared by packing POROS 20 R2 material (Applied Biosystems, Foster City, CA) into 200-l
yellow pipette tips on top of C18 Empore disk membranes. The microcolumns were washed 3 times
with 100 l 100% acetonitrile (ACN) and equilibrated 3 times with 100 l 0.1% TFA by applying air
pressure from a syringe. After the samples were loaded, the microcolumns were washed 3 times with
1
100 l 0.1% TFA, and peptides were eluted with 100 l of a series of elution buffers, containing 0.1%
TFA and 40%, 60%, and 80% ACN. All eluates were pooled and dried in a vacuum centrifuge.
StageTip-based high-pH fractionation
Desalted peptides were resolved in 200 l of loading solution (10 mM ammonium formate
solution, pH 10 and 2% acetonitrile) and separated on pipet-based reversed-phase microcolumns,
prepared by packing POROS 20 R2 material into a 200-l yellow tip with C18 Empore disk
membranes (3M, Bracknell, UK) at the bottom. The microcolumns were washed sequentially with
100 l 100% MeOH and 100% acetonitrile and equilibrated with 100 l of loading solution by
applying air pressure from a syringe. Peptides were loaded at pH 10, and 20 fractions were
subsequently eluted with buffer solution, pH 10, containing 5%, 10% 15%, 20%, 25%, 30%, 35%,
40%, 60%, and 80% acetonitrile. To improve the orthogonal fractionation of the RP-RP separation, 20
fractions were concatenated into 5 fractions by combining fractions 1, 6, 11, and 16; 2, 7, 12, and 17;
3, 8, 13, and 18; 4, 9, 14, and 19; and 5, 10, 15, and 20 (Fig. 1A). The flowthrough (FT), the elution
fraction with 100% acetonitrile, and the peptides that were obtained from the second digestion were
pooled into a sixth fraction. The fractionation and pooling took approximately 30 min. Finally, the 6
fractions were dried in a vacuum centrifuge and stored at -80°C until LC-MS/MS analysis.
LC-MS/MS analysis
The peptide samples were analyzed by LC-MS on an Easy-nLC 1000 (Thermo Fisher
Scientific, Odense, Denmark) that was coupled to a nanoelectrospray ion source (Thermo Fisher
Scientific, Bremen, Germany) on a Q Exactive mass spectrometer (all from Thermo Fisher Scientific,
Bremen, Germany). Peptides were separated on the 2-column setup with a trap column (75 m I.D. x
2 cm, 3 μm, 100 Å) and an analytic column (50 μm ID x 15 cm, 1.9 μm, 100 Å). Solvent A was 0.1%
v/v formic acid and 2% acetonitrile, and solvent B was 98% acetonitrile with 0.1% v/v formic acid.
2
In the experiments for our whole-cell proteome and secretome, a 90-min gradient from 2% to
40% acetonitrile was applied to the fractionated peptide samples. All samples were analyzed in
technical triplicates. The spray voltage was 1.8 kV in the positive ion mode, and the temperature of
the heated capillary was 325℃. Mass spectra were acquired in data-dependent mode using a top 20
method. MS spectra were acquired on an Orbitrap analyzer with a mass range of 300–1800 m/z and
70,000 resolution at m/z 200. HCD scans were acquired at a resolution of 15,000 at m/z 200. HCD
peptide fragments were acquired at a normalized collision energy (NCE) of 27. The maximum ion
injection time for the survey scan and MS/MS scan was 20 ms and 60 ms, respectively.
Data analysis
The MS data from the Q Exactive were processed in MaxQuant, version 1.3.0.5 [2] using the
Andromeda search engine [3]. Precursor MS signal intensities were determined, and HCD MS/MS
spectra were de-isotoped and filtered, such that only the 10 most abundant fragments per 100-m/z
range were retained. Protein groups were identified by searching the MS and MS/MS data of peptides
against the IPI mouse database (v3.78, 59,534 entries), containing both forward and reverse protein
sequences. Data were searched for Trypsin/P specificity.
The database search parameters were as follows: the initial precursor and HCD fragment
mass tolerances were set to 7 ppm and 20 ppm, respectively; up to 2 missed cleavages were allowed;
carbamidomethylation of Cys was set as a fixed modification; oxidation of Met; and acetylation of
protein N-term. Minimum peptide length was set to 6 residues. All peptides, modification sites, and
protein identifications were filtered at a false discovery rate (FDR) < 1%. To specify the FDR
independently for peptides and proteins, peptides that belonged to proteins that did not meet the FDR
threshold were removed from the dataset. Peptides were assigned to protein groups, rather than
proteins.
3
Bioinformatics analysis
The gene ontologies of the whole proteome and secretome were annotated using the DAVID
bioinformatics
resource
(http://www.uniprot.org/).
tool
(http://david.abcc.ncifcrif.gov/)[4]
Pathway
analysis
was
and
performed
UniprotKB
using
database
the
KEGG
(http://www.genome.jp/kegg). Secretory protein prediction and functional annotation were performed
using
SignalP
4.1
(http://www.cbs.dtu.dk/services/SignalP)[5],
SecretomeP
2.0
(http://www.cbs.dtu.dk/services/SecretomeP)[6], TargetP (http://www.cbs.dtu.dk/services/TargetP)[7],
the
Exocarta
database
(http://www.exocarta.org)[8],
(http://www.cbs.dtu.dk/services/TMHMM).
4
and
TMHMM,
server
2.0
Supplementary Figures
Supplementary Figure 1. Detailed flowchart of the proteomic approach and data analysis
5
Supplementary Figure 2. Global feature of astrocyte proteome
(A) Sequence coverage of the identified proteins in our astrocyte proteome, including whole-cell
lysate (WCL) and conditioned media (CM). (B) Relative proportions for the number of peptides used
for identification. (C) Distribution of Andromeda score for the identified peptides. (D) Relative
molecular weights of the identified proteins.
6
Supplementary Figure 3. Reproducibility between proteomics analysis
Reproducibility between 3 technical and 3 biological replicates is represented by the correlation value,
R2. With regard to the abbreviations of the replicates, for example, CM_BR1_tech1 is the technical
replicate 1 of biological replicate 1 in conditioned media (CM). Red boxes represents the comparison
between technical replicates.
7
Supplementary Figure 4. Precision of proteomic approaches
Label-free quantitation intensities of each protein were calculated using Maxquant and transformed
into base-2 logarithms. Coefficient of variation (CV) values of the technical replicates from the
proteins identified in biological replicate 1 (BR1), biological replicate 2 (BR2), and biological
replicate 3 (BR3) of conditioned media (CM) are shown as box plots (A) and histograms (B). CV
values of whole-cell lysate (WCL) are also shown as box plots (C) and histograms (D). CVs across
biological replicates are described.
8
Supplementary Figure 5. Characterization of C8-D1A proteome
Distribution of protein abundance with selected proteins in conditioned media (A) and whole-cell
lysate (B). Common astrocyte markers are labeled red. Proteins closely related to astrocyte function
are in black. (C) GO term analysis of all identified proteins. GO terms enriched for cellular
component, molecular function, and biological process are shown. The number of protein groups
according to each GO term is indicated on the right of the bars.
9
Supplementary Figure 6. Label-free quantitation of identified secreted proteins
Total proteins identified from conditioned media (CM) were filtered by label-free quantitation (2-fold
change), classical secretion, nonclassical secretion, Exocarta, and Gene ontology (GO) analysis.
Average number of proteins secreted via the classical secretion pathway (Classical), nonclassical
secretion pathway (Non-Classical), and exosomes (Exocarta) and localized to membrane or
extracellular space (GO annotation) is displayed as bar graphs. Error bars indicate SD (asterisk, p <
0.05; double asterisk, p < 0.01; compared between 2-fold up and 2-fold down; student’s t-test).
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Supplementary Figure 7. Comparison between current astrocyte proteome and previous studies
(A) Comparison between our mouse astrocyte cell proteome and those of 4 proteomic studies using
mouse primary astrocyte cells [9-12]. (B) Comparison between current CM proteome and astrocyte
secretome by Skorupa et al. [12]. Accession number for proteins identified in the current study have
been changed to gene symbols.
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Supplementary References
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[2] Cox, J., Mann, M., MaxQuant enables high peptide identification rates, individualized p.p.b.-range
mass accuracies and proteome-wide protein quantification. Nature biotechnology 2008, 26, 13671372.
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engine integrated into the MaxQuant environment. Journal of proteome research 2011, 10, 1794-1805.
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