2196 A. Dahl et al.
Mix F12 1:1) plus 2.5 mM L-glutamine, N2 supplement (N2
medium; GIBCO Invitrogen Corp., Paisley, UK), and 20 ng/
mL human basic FGF-2 (Peprotech Inc., Rocky Hill, NJ, USA).
Cells were cultured in 75-cm3 flasks coated with polyor-
nithine at a density of 3000 cells per cm2. Culture medium
was replaced 24 h after seeding, and the cells were thereafter
allowed to grow for 10 days without further medium replace-
ment, in order to maximize the concentration of secreted pro-
teins. The total volume (50 mL) of conditioned medium was
recovered from five flasks of cultured cells. Medium added to
flasks containing no cells was incubated in the same way as
the conditioned medium and used for comparison. For the
experiments confirming the secretion, AHPs were cultured
for 2 or 4 days in a similar way with initial medium volume
of 12 mL in each of the five flasks. Conditioned medium was
collected carefully to avoid contaminating cells, centrifuged
to remove any cellular material and stored at ꢀ208C until
analysis.
trifuged (10 min, 35 000 g), and the supernatants carefully
removed. The protein pellet was left to dry.
Enzymatic digestion
The fractions were dissolved in 25 mL of digestion buffer
(0.05 mM CaCl2, 0.05 M NH4HCO3). Sequencing-grade mod-
ified trypsin (5 mL, 50 mg/L; Promega, Madison, WI, USA)
dissolved in digestion buffer was added and incubated for
4 h at 378C. The samples were dried and reconstituted in
25 mL of 0.1% trifluoroacetic acid (TFA) in water.
MALDI-TOFMS
Aliquots (15 mL) of the tryptic digests were purified with Zip-
TipTM C18 (Millipore, Bedford, MA, USA) according to sup-
plier’s instructions. The samples were eluted with 3 mL
saturated a-cyano-4-hydroxycinnamic acid (Aldrich Chemie,
Steinheim, Germany) in acetonitrile/0.1% TFA in water (1:1
v/v) directly onto a matrix seed layer.13 The matrix-assisted
laser desorption/ionization (MALDI) analyses were per-
formed using an upgraded Bruker Reflex II instrument
(Bruker-Franzen Analytik, Bremen, Germany) equipped
with a two-stage electrostatic reflectron, a delayed extraction
ion source, a high-resolution detector and a 2 GHz digitizer.
The spectra were acquired in reflectron mode. Calibration
was performed externally by using a mixture of known pep-
tides or by using two auto-digestion products of trypsin if
present in the spectra. Resulting monoisotopic peaks were
compared against the NCBlnr database.14 Mass deviations
of 75 and 200 ppm were tolerated in the database search of
internal and external calibrated mass spectra, respectively.
Mammals were specified and missed cuts were between
1–3. Molecular weight was set to 0–300 kDa, but pI was not
restricted.
Liquid-phase isoelectric focusing
Liquid-phase isoelectric focusing (IEF) was performed using
a Rotofor cell (Bio-Rad Laboratories, Hercules, CA, USA)
according to the supplier’s protocol. The medium (45 mL)
was dialyzed for 2 h (MW cut-off 6000–8000 Da) against dis-
tilled water (4 L, 48C). Following dialysis, medium samples
were brought up to a volume of 55 mL with distilled
water. Thereafter octylglycoside (0.1%, Roche, Mannheim,
Germany) and Servalyt (2%, pH 3–10, Serva Electrophoresis,
Heidelberg, Germany) were added. Focusing was performed
at 48C at 15 W (constant) for approximate 4 h. Twenty frac-
tions (2.8 mL) were harvested and pH was determined using
pH indicator strips (Merck, Darmstadt, Germany). Aliquots
(100 mL) of the IEF fractions were concentrated and analyzed
by the NuPAGE system (Novex, San Diego, CA, USA), as
described below. Selected IEF fractions were pooled and
dried by vacuum centrifugation.
ESI-QTOF-MS/MS
Protein identification/verification was also obtained through
acquisition of fragment ion data using an electrospray quad-
rupole time-of-flight instrument (Q-TOF2; Micromass,
Manchester, UK). ZipTipTM C18-enriched samples in acetoni-
trile/0.1% formic acid (1:1 v/v) were sprayed from gold-
coated glass capillaries (Micromass) using a nanoflow
electrospray source. Argon was used as collision gas. The
instrument calibration was performed in positive ion
mode using fragment ions from Glu-fibrinopeptide B and a
curve fit polynomial of order 2. Peptide fragment ion spectra
were processed using MaxEnt3 (Micromass) and searched
against the NCBlnr database using MASCOT.15 Mammals
were selected as species. Molecular weight and pI were not
restricted in the search.
SDS-PAGE and electroelution
SDS-PAGE and electroelution were performed according to
the supplier’s protocol. The samples were dissolved in a total
volume of 200 mL of NuPAGE sample buffer (0.14 M tris,
0.10 M tris-HCl, 0.4 mM EDTA, pH 8.5, containing 10% gly-
cerol, 2% lithium dodecyl sulfate, 0.08% serva blue, 0.025%
phenol red, 3% dithiothreitol), boiled for 3 min and then ana-
lyzed by the NuPAGE system using one-well 10% Bis-Tris
gels and the NuPAGE MES SDS (1.0 M MES, 1.0 M trisbase,
20.5 mM EDTA, 69.3 mM SDS) running buffer. The electro-
phoresis was run for 40 min at 200 V (constant). The electroe-
lution was performed at 100 mA (constant) for 30 min using
the Mini Whole Gel eluter (Bio-Rad Laboratories) with
MOPS-histidine as elution buffer (30 mM MOPS, 25 mM his-
tidine, pH 6.5). Fourteen fractions (0.5 mL) were harvested
from the unit. Aliquots (50 mL) were concentrated, dissolved
in 15 mL of NuPAGE sample buffer, and analyzed by the
NuPAGE system followed by staining with the Novex colloi-
dal blue stain kit. The remaining part of the gel eluter samples
was dried and purified from SDS and salt by protein precipi-
tation. The samples were reconstituted in water (200 mL), ice-
cold acetone (600 mL) was added, stored at ꢀ208C for 2 h, cen-
Immunoblotting of identified proteins in
conditioned medium
Immunoblotting of conditioned medium from 2 and 4 days of
culturing was performed to confirm the secretion of immuno-
philin FK506 binding protein 12 (FKBP12) and phosphatidy-
lethanolamine binding protein (PEBP). Conditioned medium
(120 or 200 mL) was protein precipitated. Briefly, ice-cold
acetone, three times the volume of that of the conditioned
Copyright # 2003 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2003; 17: 2195–2202