Application of free-flow IEF to identify protein candidates changing under microgravity conditions

RESEARCH ARTICLE

Application of free-flow IEF to identify protein

candidates changing under microgravity conditions

Jessica Pietsch1,2, Richard Kussian3, Albert Sickmann4, Johann Bauer5, Gerhard Weber6,Mikkel Nissum3, Kriss Westphal7, Marcel Egli7, Jirka Grosse8, Johann Schonberger8,Robert Wildgruber3, Manfred Infanger9 and Daniela Grimm2,10

1 FU-Berlin, Division of Biology, Chemistry, Pharmacy, Berlin, Germany2 Institute of Clinical Pharmacology and Toxicology, CBF/CCM, Charite-Universit .atsmedizin Berlin, Berlin, Germany3 Becton&Dickinson, Martinsried, Klopferspitz, Martinsried, Germany4 ISAS, Institute for Analytical Sciences, Dortmund, Germany5 Max-Planck Institute of Biochemistry, Martinsried, Germany6 FFE-Service, Kirchheim, Germany7 Space Biology Group, ETH Zurich, Zurich, Switzerland8 Department of Nuclear Medicine, University of Regensburg, Regensburg, Germany9 Breast Center, Plastic Surgery, CCM, Charite-Universit .atsmedizin, Berlin, Germany10 Department of Pharmacology, Aarhus University, Aarhus, Denmark

Received: April 6, 2009

Revised: October 8, 2009

Accepted: November 16, 2009

Using antibody-related methods, we recently found that human thyroid cells express various

proteins differently depending on whether they are cultured under normal gravity (1g) or

simulated microgravity (s-mg). In this study, we performed proteome analysis in order to

identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-mg conditions

were sonicated. Proteins released into the supernatant and those remaining in the cell

fragments were fractionated by free-flow IEF. The fractions obtained were further separated

by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were deter-

mined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared

to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally

numbered free-flow IEF fractions revealed similar patterns with a number of identical bands

if proteins of a distinct cell line had been applied, irrespective of whether the cells had been

cultured under 1g or s-mg. Most of the identical band pairs contained identical proteins.

However, the concentrations of some types of proteins were different within the two pieces of

gel. Proteins that concentrated differently in such pieces of gel are considered as candidates

for further investigations of gravitational sensitivity.

Keywords:

Cell biology / Cytoskeletal proteins / Cytosolic proteins / Free-flow electrophoresis / pI /

Random positioning machine

1 Introduction

Protein separation by continuous free-flow electrophoresis

(FFE) has been considerably improved in recent years. After

instrumentation was developed which allowed segmentation

of the chamber fluid, carrier ampholytes could be used as

separation media and the FFE method became applicable for

performing IEF of proteins [1–3]. This liquid-based free-flow

IEF (FF-IEF) technique became a rather competitive

preparative protein fractionation method after the introduc-

tion of improved separation media, which comprise a

sophisticated combination of buffering substances (Prolytes as

well as novel detergents, reducing and denaturing agents [4].

According to FF-IEF protocols, proteins of body fluids,

bacteria or whole eukaryotic cells have been successfully

electrophoresed. In several studies, many soluble as well as

Abbreviations: FFE, free flow electrophoresis; FF-IEF, the free-

flow isoelectric focusing; RPM, random positioning machine; 1g,

normal gravity; s-lg, simulated microgravity

Correspondence: Dr. Johann Bauer, Max-Planck-Institut f .ur

Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany

E-mail: [email protected]

Fax: 149 89 1417931

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

904 Proteomics 2010, 10, 904–913DOI 10.1002/pmic.200900226

membrane-associated proteins were enriched and could be

identified by subsequent SDS-gel electrophoresis and MS

[5–8]. Recently, we found that FF-IEF might be more

suitable for fractionation of membrane-associated proteins

when cells are sonicated and the remaining cell fragments

are separated from the cell suspension fluid by centrifuga-

tion. Following separation from the supernatant, the cell

fragments and their proteins could be completely dissolved

in free-flow IEF media and subjected to FF-IEF analysis. We

detected interesting proteins that are expressed differently

in specific thyroid cancer cell lines [9].

Since 2002, we have investigated the behavior of normal

and malignant thyroid cells exposed to simulated micro-

gravity (s-mg) [10]. First of all, it was of interest to establish

why reduced plasma thyroid hormone levels are found in

rats as well as in astronauts returning from a space mission

[11–12]. Culturing thyroid cells under different gravitational

conditions, we recognized that not only T3 and T4 secre-

tions are impaired, but numerous proteins are also differ-

ently expressed. The cells detach from the surface of the

culture flasks and are assembled three dimensionally under

s-mg.

Three-dimensional aggregates of cancer cells became a

second topic of our cellular microgravity research, because

they represent a simple model of a tumor. These resemble

the in vivo situation more than monolayer cells, but they

are not as complex as natural tumors. Therefore, they

appeared suitable for developing in vitro anti-cancer drug

test systems [13]. However, one has to be aware of normal

cell features that change when an artificial tumor is engi-

neered using gravity-annulling techniques. We investigated

several markers of apoptosis, cell adhesion and extracellular

matrix by flow cytometry and Western blotting [10].

However, both techniques require antibodies that have to be

purchased or self-developed. Therefore, exploration of

further proteins became difficult and extremely expensive,

as antibodies had to be purchased on a speculative basis.

This study was designed to establish whether the method

described above [9] could be helpful in identifying further

thyroid proteins, that are expressed differently depending on

the different gravitational conditions under which cells are

incubated.

Tumor cells may have different features and express

proteins at a different rate, even though they originate from

one distinct organ. Looking for tumor-cell and thyroid-

specific characteristics, we studied three human follicular

thyroid cancer cell lines: the T3/T4-producing ML-1 cells

[14], the rather aggressive FTC-133 [15] and the thyr-

oglobulin-negative CGTH W-1 cells [16] as well as the

normal thyroid HTU-5 cells [17]. After culturing under

normal gravity (1 g) and s-mg conditions, the cells were

analyzed in a parallel manner applying sonication, FFE

separation, SDS-PAGE analysis and MS. The results

revealed a considerable number of proteins that had not

been detected in thyroid cells before. They proved that

breaking up cells by sonication followed by centrifugation

enriches membrane-associated proteins and indicated that

certain proteins might be expressed differently under 1g and

s-mg.

2 Materials and methods

2.1 Cell culturing of the ML-1, FTC-133, CGTH W-1

and HTU-5 cell lines

The human follicular thyroid carcinoma cell lines ML-1 [14],

FTC-133 [15] and CGTH W-1 [16] were cultured in RPMI-

1640 medium containing 100mM sodium pyruvate and

2 mM L-glutamine, supplemented with 10% FCS, 100 U/mL

penicillin and 100 mg/mL streptomycin (all Invitrogen,

Eggenstein, Germany). The cell line HTU-5 derived from a

primary culture of a normal human thyroid gland. It was

grown in Coon’s F-12 medium that was modified as

described previously [17].

2.2 Cell exposure to s-lg

Subconfluent monolayers (106 cells/dish) of each of the four

thyroid cell lines (n 5 60 for each cell line) were cultured

either on a standard [18] or on a desktop [19] random posi-

tioning machine (RPM), in which both simulate micro-

gravity. At the bottom next to the machine the control cells

were incubated in a commercially available incubator [19] or

in an incubator room [10] under standard cell culture

conditions for 72 h. The standard and the desktop RPMs

were manufactured by Dutch Space, an EADS Astrium

company, Leiden, NL. Both types of RPM are laboratory

instruments enabling the position of a biological experiment

in three-dimensional space to be randomly changed under

the control of dedicated software running on a personal

computer. On the RPM, the samples were positioned as

close as possible to the center of the platform. The move-

ment of the experimental platform is realized by two inde-

pendently running engines, which are controlled by feed-

back signals from encoders, mounted on the motor-axes and

by ‘‘null position’’ sensors. The RPM was operated in a

random walk (basic mode) with a speed of 601/s. Gravity

forces were reduced below 10�2g [20].

2.3 Cell preparation

The four types of thyroid cells were harvested using cell

scrapers and then centrifuged. After determination of cellular

protein, samples of cells comprising 2 mg protein were shock-

frozen with liquid nitrogen and stored at �801C until use [9].

Immediately prior to the FFE experiments, cells were thawed

and suspended in 0.5 mL HEPES buffer (10 mM HEPES,

15 mM MgCl2, 10 mM KCl and 0.2% DTT) containing one

tablet of protease inhibitor ‘‘Complete Mini’’ (Roche, Basel,

Proteomics 2010, 10, 904–913 905


Switzerland) per 10 mL fluid. Then they were sonicated for

30 s on ice with a Soniprep 150 setting. The sonicated sample

was centrifuged at 25 000� g at 41C for 30 min. The super-

natant was collected and the HEPES buffer was exchanged for

lysis medium containing 7 M urea, 2 M thiourea, 4% CHAPS,

1% ASB-14 and 10 mM DTT by ultrafiltration, until the

concentration of the original buffer was decreased by more

than 100 times at a protein concentration of 2.5 mg/mL. The

pellet was resuspended in 1 mL HEPES buffer and washed

once in this buffer. Subsequently, the pellet was resuspended

in lysis medium as described above and centrifuged at

25 000� g at 41C for 60 min. Proteins were dissolved in lysis

medium at a concentration of approximately 1 mg/mL before

being applied to FF-IEF. The volume of each sample for FFE

separation was 100mL [9].

2.4 FF-IEF

FFE separations were conducted in the FF-IEF mode using a

BDTM FFE System (BD Diagnostics, Munich, Germany)

[21]. A stable pH gradient was created between the anodal

(pH 3) and cathodal (pH 10) edges of the separation

medium flowing continuously through the FFE separation

chamber, which is placed in horizontal position. At the one

end, separation medium was introduced into the 0.4 mm

wide gap between the two glass plates of the chamber, at the

other end the medium film was split into 96 fractions. Near

the inlet of the separation medium, the unseparated sample

was injected into the flowing medium. On the way through

the chamber the proteins were moved toward fluid zones of

their IEP by a voltage of 520 V applied perpendicularly to the

flow of the 10 cm wide separation medium film [1, 3, 21].

The separation buffers contained 7 M urea, 2 M thiourea and

250 mM mannitol in aqueous solution in addition to the

ampholytes used to create the pH gradient. The flow rates

for the separation buffers were set at 60 mL/h. A Tecan

(Maennedorf, Switzerland) liquid handling system equipped

with a pH electrode was used to measure the pH value of

each fraction. The protein separation experiments were

performed when pH gradients of several independent runs

were as stable as shown in Fig. 1. After FFE equilibration

and pI marker test, each sample was infused into the

chamber at a rate of 1 mL/h through inlet S2, which enters

the separation chamber opposite to fraction 48 [21]. Elec-

trophoresis was performed at 101C. Separated samples were

collected into 96-well plates. Collection began 25 min after

starting a run and lasted approximately 6 min.

2.5 SDS-PAGE

Protein composition was analyzed by SDS-PAGE using an

XCell SureLock Mini-Cell (Invitrogen, Carlsbad, CA, USA)

in combination with precast NuPAGE 4–12% Bis-Tris gels

(Invitrogen). Proteins were stained with a SilverQuest kit

(Invitrogen) according to the manufacturer’s instructions or

by Coomassie Brilliant Blue G-250 (Bio-Rad, Munich,

Germany).

2.6 Western Blot Analysis

SDS-PAGE, immunoblotting and densitometry were carried

out following routine protocols [22]. The following anti-

bodies were applied to quantify their antigens: Alpha-

enolase (Santa Cruz Biotechnology, CA, USA, dilution:

1:400), phosphoglycerate kinase 1 (Santa Cruz Biotechnol-

ogy, dilution: 1:2000), annexin 1 (Santa Cruz Biotechnology,

dilution: 1:200), annexin 2 (Santa Cruz Biotechnology,

dilution: 1:200), and glutathione S-transferase P (Santa

Cruz Biotechnology, dilution: 1:200). As a loading

control glyceraldehyde 3-phosphate dehydrogenase (ABR-

Affinity BioReagents, Golden, USA; dilution: 1:10 000) was

used.

2.7 Protease digestion

In order to identify proteins, FF-IEF-fractions of interest

were first concentrated 30- to 40-fold using Vivaspin 6

centrifugal concentrators with a cut-off of 5 kDa

(Vivascience, Hannover, Germany) according to the manu-

facturer’s instructions, and thereafter subjected to SDS-

PAGE. The proteins were stained with Coomassie Brilliant

Blue G-250. Gel bands of interest were cut out. Sample

preparation for MS was performed according to a modified

protocol of Shevchenko et al. [23, 24]. Samples were washed

twice alternately with 50 mM ammonium hydrogen carbo-

nate and 25 mM ammonium hydrogen carbonate buffer,

0

2

4

6

8

10

12

14

1 11 21 31 41 51 61 71 81 91

FFE fraction number

Figure 1. FF-IEF pH gradients were used in the experiments.

Graphs from three independent experiments were overlaid to

demonstrate the reproducibility of the separation. The flat

regions observed below pH 4 and above pH 10 represent the pH

values of the anodic and cathodic stabilization media, respec-

tively. The three experiments were conducted within a 12

months time frame with 6 months between each experiment.

Different FFE instruments were used for the experiments.

906 J. Pietsch et al. Proteomics 2010, 10, 904–913


50% v/v ACN. The gel pieces were dried in a SpeedVac

(Thermo Electron, Dreieich, Germany) and rehydrated with

4 ng of trypsin in 50 mM ammonium hydrogen carbonate

buffer. Digestion was performed overnight at 371C. The

resulting peptides were extracted by applying of 15 mL of 5%

formic acid for 15 min at 371C. The procedure was repeated

twice.

2.8 Protein identification by MS

Proteins were analyzed using nano-LC-MS/MS. In detail,

an Ultimate 3000 (Dionex, Idstein, Germany) coupled to

an ESI-linear ion trap (LTQ XL, Thermo Electron) was

employed. The LC setup consisted of an autosampler

(WPS, Dionex) and a column compartment (FLM, Dionex)

before nano-LC separation (Ultimate 3000, Dionex).

Precolumns (100 mm id� 20 mm length, Synergy Hydro-RP

C18 5 mm particle size, Phenomenex, Aschaffenburg,

Germany) and separation columns (75mm id� 150 mm

length, Synergy Hydro-RP C18 3 mm particle size,

Phenomenex) were custom-built. Samples were loaded onto

the precolumn with a flow rate of 6mL/min 0.1% TFA for

5 min. Gradient elution was performed with a linear gradi-

ent from 95% solvent A (0.1% formic acid) to 50% solvent B

(84% ACN, 0.1% formic acid) during a time period of

33 min. Solvent A was 0.1% formic acid in water. Separation

was followed by rinsing the column with 95% B for 5 min

before equilibration to 5% solvent B prior to the next

separation.

Peptides were directly eluted into the ESI-linear iontrap

(LTQ XL) using distal-coated fused silica tips (New Objec-

tives, Woburn, MA, USA) with spray voltage set to 1800 V. A

survey scan (m/z 400–2000) was followed by five MS/MS

scans that fragmented the five most intensive peptide

signals (1000 cps, 30 ms). Duplicate detection of one mass

within 30 s led to dynamic exclusion for 180 s.

Mass spectra obtained from LC-MS/MS analysis were

used to identify the corresponding peptides by the

MASCOTTM algorithm (version 2.1.6) [25]. The raw data

were converted with the LCQ-DTA.EXE as plug-in to

MASCOT Daemon with the following parameters: (i)

minimum mass: 400, (ii) maximum mass 3000, (iii)

grouping tolerance 1.4, (iv) min. scans/group: 1, (v) inter-

mediate scans: 1, (vi) precursor charge: auto. Searches were

conducted against the current FASTA database of Homosapiens using the following parameter set: (i) fixed modifi-

cation: carbamidomethyl (C); (ii) variable modification:

oxidation (M); (iii) peptide and MS/MS tolerance: 70.5 Da;

(iv) ion score cut-off: 35, (v) significance threshold po0.05

and (vi) enzyme trypsin with miss cleavage: max. 1. After

manual validation, a protein was to have been identified

when at least two different peptides with a score 435 were

found and the cumulative score was 4100. The exponen-

tially modified protein abundance index (emPAI) was

calculated according to Ishihama et al. [26].

2.9 Statistics

Statistical analysis was performed using SPSS 12.0 (SPSS,

Chicago, IL, USA). All data were expressed as mean7SD.

We tested all parameters for deviations from Gaussian

distribution by means of the Kolmogorov–Smirnov test and

cases were compared using the independent samples t-test

or the Mann–Whitney U-test (depending on the results of

the normality test). Differences at the level of po0.05 were

considered significant.

3 Results and discussion

3.1 FF-IEF

After cells of each cell line had been sonicated, the proteins

which had been released into the cell suspension fluid

as well as the proteins which remained linked to the

cell fragments were solubilized in separation medium.

Then they were applied to FFE and fractionated according

to the regime of FF-IEF. In each run, 96 fractions

were collected. Every second fraction of an FF-IEF separa-

tion experiment was applied to SDS gels. Silver-stained

gels revealed that irrespective of the originating cell

line, proteins solubilized by sonication could be collected

in FFE fractions ranging from number 27 with a pH of

4.4 up to number 71 with a pH of 10.3 (Figs. 1 and 2A).

A number of protein bands were observed on each lane.

The FFE fractions 33–35 (pH 5.5–5.9) and 49–53 (pH

7.0–7.4) appeared to contain enhanced quantities of

protein. Such a pattern was generated by proteins from

each type of thyroid cells, if proteins liberated by sonica-

tion were applied to a gel, irrespective of whether the

analyzed cells had been cultured under 1g or s-mg prior to

analysis.

In addition, we electrophoresed proteins dissolved from

cell fragments that had already been sonicated. These

proteins could also be separated by FF-IEF and were

collected in FFE fractions ranging from number 29 (pH 4.7)

up to fraction 69 with a pH of 10.2 (Figs. 1 and 2B).

However, another gel band pattern that was specific for

proteins remaining within the cell fragments during soni-

cation was observed (Fig. 2B). The highest protein concen-

trations were found in FFE fractions 33–35 (pH 5.5–5.9).

Less protein was seen in the alkaline range of FFE fractions

51–69 (pH 7.0 and above). Again, this pattern was obtained

irrespective of the type of human thyroid cells analyzed and

independently of the mode of cell culturing prior to soni-

cation. Comparing silver-stained gels as shown in Fig. 2, it

became obvious that proteins obtained from the cell frag-

ments (Fig. 2B) generated different patterns of bands during

SDS-gel electrophoresis than those released into the cell

suspension fluid during sonication (Fig. 2A). This conclu-

sion is consistent with the earlier findings described by

Obermaier et al. [9].

Proteomics 2010, 10, 904–913 907


3.2 Identification of selected polypeptide bands

In order to define which proteins are included in which gel

band, various FFE fractions of each cell line were selected

and subjected to an SDS-gel electrophoresis again, but this

time at higher concentration so that they could be stained by

means of Coomassie blue (Fig. 3) and used for subsequent

MS. We applied protein samples of comparable FFE frac-

tions to gels in neighboring lanes. Comparable FFE frac-

tions had been obtained by independent runs from protein

solutions of one distinct cell line cultured either under 1g or

under s-mg (�) and collected in equally numbered FFE

fractions. Clear protein bands became visible on the gels and

neighboring lanes such as 33 and 33� looked rather

similar (Fig. 3). The experiments proved that FFE fractions

33, 35, 49 and 53 contained more proteins than fractions 31,

41 and 67, when proteins liberated during sonication

had been subjected to FF-IEF. Only fractions 33

and 35 contained the most proteins when the proteins

remaining in cell fragments had been electrophoresed

(Fig. 3).

We excised a total of 250 gel pieces from the Coomassie

blue-stained SDS-PAGE gels prepared in this study. The

positions of 105 of these gel bands are indicated by squares

and numbered in Fig. 3. Preferentially, we selected pairs of

gel pieces such as piece 6 and 6� or 14 and 14� (Fig. 3). Both

contained proteins from the same type of cells. However,

the left piece of a pair contained proteins originating from

cells that had been cultured under 1g and the right one

contained proteins of cells cultured under s-mg. Distinct

proteins could be identified in 204 of the gel pieces and a

total of 1210 proteins were determined. Their SDS-PAGE

migration behavior corresponded to their known molecular

weights. Many of the proteins were detected two, three or

four times, because they were found in several cell lines

used. A total of 235 unique types of proteins were identified.

For some of the proteins, subunits or variants could be

identified so that a total of 356 different polypeptides were

found. Some of the proteins were determined with high

scores, some with rather low scores. Since high MASCOT

score means that an MS result indeed indicated a protein

being included in the relevant piece of gel, the proteins with

MASCOT scores above 500 were also counted. There were

631, including 128 unique proteins or 193 different poly-

peptides. The greater part of all the 235 proteins identified is

known to be expressed in thyroid cells. However, 59 proteins

were detected which, to our knowledge, have not yet been

described as being expressed in human thyroid cells,

although several proteome analyses of human thyroid cells

have already been performed [27–32]. Thirty-five of these

proteins were detected after at least two independent FFE

runs, whereas 24 were identified with a MASCOT

Score higher than 500. Taken together, there were 37

proteins (Table 1) that strongly suggest that our technology

enabled us to identify proteins in thyroid cells for the first

time.

A M 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 M

BM 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 M

18898

6249

3828

1714

6

3

18898

6249

3828

1714

6

3

kDa

kDa

Figure 2. Silver-stained gels

obtained when proteins of the

cancer cell line FTC-133

cultured under gravity were

analyzed. The proteins of (A)

had been obtained after the

cells had been sonicated and

the proteins released into the

cell medium fluid were subjec-

ted to FFE separation prior to

SDS gel analysis. The proteins

of (B) had been obtained after

the cells had been sonicated

and the proteins remaining

within the cell fragments were

subjected to FFE separation

prior to SDS gel analysis.



3.3 Effect of sonication

The proteins with scores above 500 were analyzed. We inves-

tigated to what extent the various proteins reacted to sonica-

tion. Three types of proteins were found. One type was

completely dissolved within the fluid after sonication. This type

comprises protease inhibitors such as serpin-6 [33], or regula-

tory proteins like 14-3-3 proteins [34] or cofilin [35] as well as

soluble glycolytic enzymes such as fructose-bisphosphate

aldolase and glyceraldehyde-3-phosphate dehydrogenase [36].

Another type of proteins had remained within the cell frag-

ments during sonication. This second group contained the

cytoskeletal proteins spectrin and vimentin. Both of these

proteins are known to possess relatively stable polymeric

properties [37, 38] as well as lamin and integrin-a, which

strongly interact with the cytoskeleton [39, 40].

Furthermore, we detected a third group of proteins such

as annexins, which were partly released into the fluid during

sonication, whereas another part remained in the cell frag-

ments. Annexins are calcium-dependent phospholipid-

binding proteins. They not only interact with cell-membrane

components, but may also form complexes with other

proteins such as the S-100 protein [41, 42]. A similar beha-

vior showed tropomyosin and cytoplasmic actin. Tropo-

myosin interacts with actin [43]. Cytoplasmic actin is known

to build up intracellular filaments [44]. These filaments are

permanently subject to polymerization and depolimeriza-

tion. There is thus a distribution of actin between the

polymer and the monomer phases [45]. A very interesting

observation was made regarding tubulin, which is also

included in cytoskeletal structures [46]. After sonication of

ML-1 and FTC-133 cells, tubulin was found in cell frag-

ments only at a MASCOT score above 500. But analysis of

CGTH W-1 proteins revealed tubulin molecules released

into the fluid during sonication. This could be an indication

that the molecular properties of tubulin have changed

during the carcinogenesis of CGTH W-1 cells [47].

3.4 Protein candidates altered by s-lg conditions

The objective of this study was to find further thyroid

proteins whose expression depends on gravity conditions

[10]. After separation of protein samples according to the FF-

IEF regime, comparable FFE fractions were applied to SDS

gels as described in Section 3.2 (Fig. 3). Then pairs of gel

pieces such as gel pieces 30 and 30� (Fig. 3), which were

located side by side on the SDS gel, were excised at the same

molecular weight level and prepared for MS. Table 2 lists

proteins found in gel pieces of the three pairs 30, 30�; 40,

A

188

98

6249

38

28

1714

6

3

M 67 67* 49 49* 35 35* 31 31* kDa

M 33 33* 59 59* M 33 33* 43 43* M 35 35* 53 53*

188

98

6249

38

28

1714

6

3

kDa

C D E

M 33 33* 41 41* 67 67* 53 53* kDaB

188

98

6249

38

28

1714

6

3

Figure 3. Coomassie blue-stained SDS

gels indicating fractionated proteins

either released during sonication from

CGTH W-1 cells (A) and from FTC-133

cells (B) or retained in cell fragments

during sonication by HTU-5 cells (C),

by FTC-133 cells (D) and by CGTH W-1

cells (E). Proteins of selected FFE

fractions were applied in pairs. The

numbers above the lanes designate

the FF-IEF fractions and the asterisk

indicates that the cell had been

cultured under s-mg gravity prior to

sonication.

Proteomics 2010, 10, 904–913 909


40� and 41, 41�. Each pair contained equal types of proteins,

respectively. However, the emPAI values determined for

each protein suggested variations in the protein concentra-

tions. Some proteins such as pyruvate kinase isozymes M1/

M2 (Table 2, upper panel) or Ras-related protein Rab-1B

(Table 2, lower panel) showed rather similar emPAI values,

irrespectively whether the proteins had been enriched from

1 g or s-mg cultured FTC-133 cells. Other proteins such as a-

enolase (Table 2, upper panel) or annexin A2 (Table 2,

middle panel) or translationally controlled tumor protein

(Table 2, lower panel) showed significantly different emPAI

values, depending on whether the proteins had been enri-

ched from 1 g or s-mg cultured FTC-133 cells.

In order to investigate whether different emPAI values

determined for one kind of protein indicate different

expression of this protein, we performed Western blotting

using antibodies against annexin A1, annexin A2 as well as

a-enolase, phosphoglycerate kinase 1 and glutathione S-

transferase. The tests prove that the emPAI values listed in

Table 2 indicate a tendency. Also Western blot analyses

showed that a-enolase, phosphoglycerate kinase 1, annexin

1 and annexin 2 were significantly reduced in cultures

under s-mg, whereas glutathione S-transferase was enhanced

in FTC-133 cells grown under conditions of s-mg (Fig. 4).

Therefore, it seems to be worthwhile to apply Western

blotting to quantify also proteins such as plastin-3, ferritin,

Rab GDP dissociation inhibitor, phosphoglucomutase, dihy-

dropyrimidinase-related protein 2, sialic acid synthase or

deoxyhypusine synthase, which showed different emPAI

values, when they were determined in comparable pieces of

Table 1. List of proteins, which were either detected after at least two independent FFE runs or had received MASCOT scores greater than500

Proteins Number of peptides observedMASCOT score

Cell line(s) isolatedfrom

26S protease regulatory subunit 7 (4–202, 15–714) FTC-133CAP-Gly domain-containing linker protein 1 (21–693, 35–965) FTC-133, CGTH W-1Copine-1 (10–368, 7–235) FTC-133Cytoplasmic dynein 1 intermediate chain 2 (11–634) ML-1D-3-phosphoglycerate dehydrogenase (12–431, 12–410) CGTH W-1Deoxyhypusine synthase (14–1000, 5–309) CGTH W-1Fumarylacetoacetase (4–121, 8–296) HTU-5Glucosamine-6-phosphate isomerase 1 (12–839, 10 487) FTC-133Hypoxia upregulated protein 1 (10–304, 15–310) CGTH W-1Hydroxymethylglutaryl-CoA lyase, mitochondrial (10–605, 5–425) FTC-133Interferon-induced 17 kDa protein precursor (8–955, 8–610) ML-1Multisynthetase complex auxiliary component p43 (10–443, 10–379) CGTH W-1Nucleolin (7–207, 3–135) CGTH-W1Nucleophosmin (3–132, 4–112) FTC-133Nucleoredoxin (12–867, 7–343) CGTH W-1Plastin-2 (18–896, 3–165) FTC-133Polymerase delta-interacting protein 2 (8–234, 7–274) CGTH W-1Programmed cell-death protein 6 (7–170, 7–265) FTC-133Protein AHNAK2 (19–640, 29–1194) FTC-133Reticulocalbin-1 (8–412, 9–711) HTU-5Ribonuclease UK114 (5–307, 4–235) ML-1Ribosome-binding protein 1 (31–1349, 16–353) CGTH W-1S-formylglutathione hydrolase (6–495, 12–710) FTC-133Serine-threonine kinase receptor-associated protein (13–708, 19–1062) CGTH W-1Septin-11 (8–285, 10–367) CGTH W-1, HTU-5Sialic acid synthase (13–785) HTU-5Sideroflexin-1 (5–179, 6–229) ML-1Single-stranded DNA-binding protein, mitoch. (7–444, 8–434) FTC-133S-methyl-50-thioadenosine phosphorylase (12–702, 7–508) FTC-133SUMO-activating enzyme subunit 1 (21–2133, 16–1482) CGTH W-1Transgelin-2 (11–548, 14–784) FTC-133, CGTH W-1Tryptophanyl-tRNA synthetase, cytoplasmic (9–329, 19–888) ML-1Tubulin folding cofactor B (8–555, 8–544) FTC-133Tubulin-specific chaperone A (10–711, 11–907) CGTH W-1UMP-CMP kinase (7–716, 6–536) FTC-133Vacuolar ATP synthase catalytic subunit A (20–1012,16–802) ML-1, FTC-133Xaa-Pro dipeptidase (7–273, 15–1059) FTC-133

These proteins could not be found in the literature described as proteins of human thyroid cells. The highest numbers of peptidesobserved and of MASCOT scores are indicated within brackets.



gel (data not shown). These proteins have various functions

and it may not be possible in a first approach to draw

conclusions regarding entire signaling pathways. However, it

will be of interest whether thyroid cells enhance plastin-3 and

dihydropyrimidinase-related protein-2 under s-mg, while they

form three-dimensional aggregates and modify their cytoske-

leton simultaneously [10]. These proteins regulate the shape

of nerve cells by controlling axon formation [48, 49].

200

250

300

Rel

ativ

e d

ensi

tom

etri

c u

nit

s # #

100

150# #

150

200#

249 11245 690

50

100

150

Alpha-enolase Phosphoglyceratekinase 1

A

112 12087 780

50

Annexin 1 Annexin 2

B

Rel

ativ

e d

ensi

tom

etri

c u

nit

s

Glutathione S-transferase

49 1560

50

100

C

Rel

ativ

e d

ensi

tom

etri

c u

nit

s

1 g s-µg

Figure 4. Western blot analysis of FTC-133 cell lysates cultivated for 3 days either under 1 g or s-mg. The protein contents of Alpha-enolase

and Phosphoglycerate kinase 1 which were found in the gel pieces 40 and 40�(A), of Annexin 1 and Annexin 2 which were found in the gel

pieces 41 and 41� (B), and Glutathione S-transferase P which was found in the gel pieces 30 and 30� (C) were determined. The densi-

tometric units of each protein were normalized to the protein content of glyceraldehyde 3-phosphate dehydrogenase, respectively. The

numbers at the base of each column states the mean value of the densitometric analysis. The rhombus (]) indicates a significant

difference (p 5 0.004).

Table 2. MS analysis of comparable pieces of gels

1g emPAI s-mg emPAI

Gel band 40 of Fig. 3 Gel band 40� of Fig. 3

a-Enolase 58.70 a-Enolase 34.14Phosphoglycerate kinase 1 18.13 Phosphoglycerate kinase 1 12.90Pyruvate kinase isozymes M1/M2 4.63 Pyruvate kinase isozymes M1/M2 3.97Fumarate hydratase, mitochondrial 2.73 Fumarate hydratase, mitochondrial 2.06Putative elongation factor 1-a-like 3 0.9 Putative elongation factor 1-a-like 3 0.76


Glyceraldehyde-3-phosphate dehydrogenase 30.86 Glyceraldehyde-3-phosphate dehydrogenase 27.86Annexin A2 26.70 Annexin A2 14.92Aldose reductase 5.51 Aldose reductase 4.35Annexin A1 4.74 Fructose-bisphosphate aldolase 4.05Fructose-bisphosphate aldolase 3.22 Annexin A1 2.97Electron transfer flavoprotein subunit a 2.36 Electron transfer flavoprotein subunit a 4.03Alcohol dehydrogenase [NADP1] 1.39 Alcohol dehydrogenase [NADP1] 1.17


Translationally controlled tumor protein 15.96 Translationally controlled tumor protein 28.35Ras-related protein Rab-1B 8.39 Ras-related protein Rab-1B 8.16Ferritin heavy chain 6.67 Glutathione S-transferase P 5.07Glutathione S-transferase P 4.63 Ferritin heavy chain 2.16Sorcin 3.77 Ras-related protein Rab-18 1.89GTPase Nras 1.76 Ras-related protein Rab-7a 1.44Ras-related protein Rab-7a 1.52 Programmed cell death protein 6 1.34Programmed cell death protein 6 1.23 GTPase NRas 1.27Proteasome subunit b type-9 1.23 Proteasome subunit b type-9 1.13Ras-related protein Rab-18 0.96 Sorcin 0.9

The pieces of gels are shown in Fig. 3, where their positions and the type of proteins applied are indicated. 1 g and s-mg indicate the cultureconditions of the originating cells.

Proteomics 2010, 10, 904–913 911


4 Concluding remarks

Applying FF-IEF followed by SDS-gel electrophoresis and

MS, we were able to identify 235 different proteins in lysates

of four different human thyroid cell lines. The reproduci-

bility of the FF-IEF allowed us to compare pairs of gel pieces

containing proteins from cells cultured under 1 g or s-mg.

Significant differences in the emPAI values of equal

proteins in comparable gel pieces may indicate different

expression of these proteins under different gravitational

conditions.

The authors have declared no conflict of interest.

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Application of free-flow IEF to identify protein candidates changing under microgravity conditions