Mitosis-Dependent Protein Expression in Neuroblastoma Cell Line N1E-115 Amedeo A. Azizi,†,‡ Sung-Ung Kang,‡ Angelika Freilinger,§ Mariella Gruber-Olipitz,†,‡ Wei-Qiang Chen,‡ Jae-Won Yang,‡ Markus Hengstschla¨ger,§ Irene Slavc,‡ and Gert Lubec*,‡ Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Wa¨hringer Gu ¨ rtel 18-20, A-1090 Vienna, Austria, and Department of Medical Genetics, Obstetrics and Gynecology, Medical University of Vienna, Wa¨hringer Gu ¨ rtel 18-20, A-1090 Vienna, Austria Received February 26, 2008
Systematic work on differential protein expression in mitosis is limited, and we therefore used neuroblastoma cells (N1E-115) incubated with either colcemid or nocodazole to arrest mitosis. Proteins were identified by MALDI-TOF/TOF and nano-LC-ESI-MS/MS with subsequent quantification of spot volumes with specific software. Immunoblotting was used for verification of selected proteins. Levels of 10 individual proteins were increased and levels of 6 proteins were decreased concordantly by both treatments. These proteins were constituents of heat shock and chaperone, cytoskeleton, proteasomal, heterochromatin, and DNA replication signaling as well as housekeeping and metabolic systems. Identification of mitosis-dependent proteins is of importance for the interpretation of previous work and for designing future experiments. Keywords: mitosis • protein expression • N1E-115 cell line • mouse
Introduction A legion of proteins is directly or indirectly involved in mechanisms leading to mitosis, but information on systematic protein profiling to identify corresponding protein cascades and pathways is limited. Identification of mitosis-dependent protein expression is mandatory and challenging because it is essential for the search for tumor markers and pharmacological targets.1,2 And indeed, these investigations are hampered and biased by the presence of a series of confounding factors. Protein profiling studies are mainly comparing tumor and nontumor cells, and differentially expressed proteins are considered tumor-related, tumorspecific, tumor-candidate marker proteins, or even tumor markers. In literature, it is hardly taken into account that differences in protein expression may well be due to different abundance of mitoses, that is, the mitotic rate in tumor tissues and cells. Likewise, proteins differentially expressed between tumorous and nontumorous samples may not be simply interpreted as potential pharmacological targets.1 These considerations along with the biological importance of the knowledge of mitosisassociated proteins per se formed the rationale for the current study. So far no reports on a systematic study to search for mitosisassociated proteins have been published to the best of our * To whom correspondence should be addressed. Prof. Dr. Gert Lubec, Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Wa¨hringer Gu ¨ rtel 18-20, A-1090 Vienna, Austria. Telephone: +43.1.40400.3215.Fax:+430.1.40400.6065.E-mail:
[email protected]. † These two authors contributed equally to this publication. ‡ Department of Pediatrics and Adolescent Medicine. § Department of Medical Genetics, Obstetrics and Gynecology.
3412 Journal of Proteome Research 2008, 7, 3412–3422 Published on Web 06/27/2008
knowledge, although there is data for differential gene expression at the nucleic acid level between resting and nocodazoletreated HeLa cells in mitotic arrest.3 Herein we aimed to identify mitosis-associated proteins by a proteomic approach trying to eliminate drug-related changes of protein levels by the use of two distinct microtubuledisrupting agents, colcemid and nocodazole. This attempt should reduce drug-related effects on protein levels because aberrant levels were only assigned to mitosis when concordant changes in colcemid- and nocodazole-treated cells were observed. This approach of quantifying protein spots followed by mass spectrometrical identification successfully and specifically identified 16 mitosis-associated proteins.
Experimental Section Cell Culture. The cell line used for this study was the mouse neuroblastoma cell line N1E-115 (ATCC no. CRL-2263) purchased from American type Culture Collection (ATCC, LGC Promochem, Middlesex, UK). For mitotic arrest, N1E-115 cells were treated for 16 h with 20 ng/mL colcemid or with 40 ng/mL nocodazole. For cytofluorometric analyses of DNA distribution, cells were harvested by trypsinization and fixed by rapid submersion in ice-cold 85% ethanol. After overnight fixation at -20 °C, cells were pelleted and DNA was stained in 1 mL of staining solution (0.25 mg/ mL propidium iodide, 0.05 mg/mL RNase, 0.1% Triton X-100 in citrate buffer, pH 7.8). Cells were analyzed on FACScan (Beckton Dickinson, San Jose, CA). Sample Preparation. After harvesting, cells were washed three times with 10 mL PBS (phosphate buffered saline) (Gibco, 10.1021/pr800149p CCC: $40.75
2008 American Chemical Society
Mitosis-Dependent Protein Expression Gaithersburg, MD) and centrifuged for 10 min at 800 g at 20 °C. The supernatant was discarded, and the pellet was suspended in 1.0 mL of sample buffer consisting of 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO), 4% CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]1-propanesulfonate) (Sigma, St. Louis, MO), 65 mM DTT (1,4dithioerythritol) (Merck, Germany), 1 mM EDTA (ethylenediaminetraacetic acid) (Merck, Germany), protease inhibitors complete (Roche, Basel, Switzerland), and 1 mM phenylmethylsulfonyl chloride. Sonification of the samples was carried out for approximately 15 s. After homogenization, samples were left at 22 ( 1 °C for one hour, and then centrifuged at 14.000g for one hour. For desalting and protein concentration, the supernatant was transferred into Ultrafree-4 centrifugal filter unit (Millipore, Bedford, MA). Protein content of the supernatant was quantified by Bradford protein assay system,4 bovine serum albumin was used to generate the standard curve. The absorbance was measured at 595 nm. Two-Dimensional Gel Electrophoresis (2-DE). 2-DE was performed as described elsewhere.5 Five 2-D gels for each group (control, colcemid, and nocodazole) were produced. Isoelectric Focusing (IEF): 700 µg protein were applied on immobilized pH 3-10 nonlinear gradient strips at their basic and acidic ends. Focusing was started at 200 V, and the voltage was gradually increased to 8000 V over 31 h and then kept constant for a further 3 h (approximately 150 000 Vh in total). After the first dimension, strips (18 cm) were equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS, and 2% DTT and then for 15 min in the same buffer containing 2.5% iodo-acetamide instead of DTT. Second Dimension: After equilibration, strips were loaded on 9-16% gradient sodium dodecylsulfate polyacrylamide gels for second-dimensional separation. Gels (180 × 200 × 1.5 mm) were run at 40 mA per gel. Immediately after the second dimension run, gels were fixed for 18 h in 50% methanol, containing 10% acetic acid; the gels were then stained with Colloidal Coomassie Blue (Novex, San Diego, CA) for 12 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Biorad Laboratories, Hercules, CA) covering the range 10-250 kDa. pI values 3-10 were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess of dye was washed out from gels with distilled water and gels were scanned with ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Photoshop 7.0 (Adobe, San Jose, CA). Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. Spots were excised with a spot picker (PROTEINEER sp, Bruker Daltonics, Bremen, Germany) and placed into 96well microtiter plates. In-gel digestion and sample preparation for MALDI analysis were also performed by an automated procedure (PROTEINEER dp, Bruker Daltonics).6,7 Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/µL trypsin (Promega, Madison,WI) in enzyme buffer (consisting of 5 mM Octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 h at 30 °C. Peptide extraction was performed with 10 µL of 1% trifluoroacetic acid (TFA) in 5 mM OGP. Extracted peptides were directly applied onto a target (AnchorChip, Bruker Daltonics), which was loaded
research articles with R-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics) matrix thinlayer. The mass spectrometer used in this work was an Ultraflex TOF/TOF (Bruker Daltonics) operated in the reflector mode for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode using the FlexControl software. An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M + H]+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1-17 and clip 18-39). Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analyzed by PMF from MALDI-TOF were additionally analyzed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analyzed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd., London, UK). Database searches, through Mascot, using combined PMF and MS/MS data sets were performed via BioTools 2.2 software (Bruker Daltonics, Bremen, Germany). A mass tolerance of 25 ppm and no missing cleavage site for PMF and MS/MS tolerance of 5 ppm and 1 missing cleavage site for MS/ MS search were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was basically used as criterion for correct identification. Furthermore, database searches were also performed using MSFit and ProFound. The algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere.8 In-Gel Digestion of Proteins and Protein Identification with Nano-HPLC-ESI-Q-TOF Mass Spectrometry. Gel pieces were cut into small pieces to increase surface and put into a 0.6 mL tube. They were washed with 50 mM ammonium bicarbonate to remove dust and then washed two times with 50% 50 mM ammonium bicarbonate/50% acetonitrile for 30 min with occasional vortexing. The washing solution was discarded at the end of each wash. One-hundred microliters of 100% acetonitrile was added to each tube covering the gel piece completely and incubated for 5 min. Gel pieces were dried completely in a Speedvac (Eppendorf, Germany). Cysteines were reduced with a 10 mM dithiothreitol solution in 0.1 M ammonium bicarbonate for 60 min at 56 °C. The same volume of a 55 mM solution of iodoacetamide in 0.1 M ammonium bicarbonate buffer was added and incubated in the dark for 45 min at 25 °C to alkylate cysteine residuces. The reduction/alkylation solutions were removed by washing with 50 mM ammonium bicarbonate buffer for 10 min. Buffer was removed, and the dried gel pieces were reswollen with 12.5 ng/ mL trypsin solution buffered in 25 mM ammonium bicarbonate and incubated for 16 h at 37 °C. Supernatants were transferred to new 0.6 mL tubes, and gel pieces were extracted subsequently with 50 µL of 0.5% formic acid/20% acetonitrile for 15 min in a sonication bath. The volume was reduced to 10 and 20 µL HPLC-water added. Nano-ESI-LC-MS/MS analyses were carried out with the UltiMate 3000 system interfaced to the QSTAR Pulsar mass spectrometer. The gradient was (A ) Journal of Proteome Research • Vol. 7, No. 8, 2008 3413
research articles
Azizi et al.
Table 1. Primary Antibodies Used for Western Blotting protein
T-complex protein 1, alpha subunit B (chaperone) Stress-70 protein, mitochondrial (precursor) (syn: Mortalin) Osmotic stress protein 94 (syn:Heat shock 70-related protein APG-1) Proliferation-associated protein 2G4 (syn: p38-2G4)
UniProtKBAcc#
P11983 P38647 P48722 P50580
Heat shock cognate 71 kDa protein Proteasome activator complex subunit 1 DNA replication licensing factor MCM7 Selenide, water dikinase 1Syn: Selenophosphate synthetase 1
P63017
Thimet oligopeptidase 1
Q8K2D4
Beta-centractin Protein disulfide isomerase associated 3 Heat shock protein 75 kDa, mitochondrial (precursor)(syn: TNFR-associated protein 1) Ubiquitin carboxyl-terminal hydrolase isozyme L1 Proteasome subunit beta type 3
Q8R5C5 Q99LF6
P97371 Q61881 Q8BH69
Q9CQN1 Q9R0P9 Q9R1P1
antibody
host species
Stressgen, Hines Drive, MI
Rat monoclonal (clone #91a)
Abcam Ltd., Cambridge, UK
Mouse monoclonal
Santa Cruz Biotechnology, Inc., Santa Cruz, CA
Rabbit polyclonal
Anti-PA2G4 (Human), Affinity-Purified Polyclonal IgY Antibodies (A22060F) Hsc70 antibody [13D3] (ab2788) Proteasome Activator 11S REG alpha antibody (ab3333) MCM7 antibody [47DC141] (ab2360) SEPHS1 monoclonal antibody (M01), clone 4D3-6A2 (H00022929-M01) Mouse monoclonal antibody to Thimet Oligopeptidase [4D6 ] (NB400-146) ACTR1B antibody (ab13802) ERp57 antibody [MaP.Erp57] (ab13506) TRAP1 antibody [TRAP1-6] (ab2721)
GenWay Biotech, Inc., San Diego, CA
IgY, Chicken Polyclonal Antibody
Abcam Ltd., Cambridge, UK
Mouse monoclonal
Abcam Ltd., Cambridge, UK
Rabbit polyclonal
Abcam Ltd., Cambridge, UK
Mouse monoclonal
Abnova, Taipei City, Taiwan
Mouse monoclonal
Novus Biologicals, Littleton, CO
Mouse monoclonal
Abcam Ltd., Cambridge, UK Abcam Ltd., Cambridge, UK
Goat polyclonal Mouse monoclonal
Abcam Ltd., Cambridge, UK
Mouse monoclonal
PGP9.5 antibody - Neuronal Abcam Ltd., Cambridge, UK Marker (ab10404) Proteasome 20S beta 3 Abcam Ltd., Cambridge, UK antibody [MCP102] (ab22670)
0.05% TFA in water, B ) 80% ACN/0.04% TFA in water) from 0 to 50% B in 30 min, 90% B in 5 min, 0% B in 25 min. Peptide spectra were recorded over the mass range of m/z 400-1600, and MS/MS spectra were recorded in information-dependent data acquisition over the mass range of m/z 50-1600. One peptide spectrum was recorded followed by three MS/MS spectra on the QSTAR Pulsar instrument; the accumulation time was 1 s for peptide spectra and 2 s for MS/MS spectra. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. Doubly or triply charged peptides were chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS spectra were interpreted by the MASCOT software (Matrix Science). Quantification. Protein quantification was performed using Proteomweaver 3.0 (Biorad Laboratories, Hercules, CA) a program designed for 2-D proteome analysis. Images of five gels per group (control, colcemid and nocodazole) were analyzed. Spots were automatically recognized by Proteomweaver and matched between gels. Relative protein amounts were deduced by the program from spot volume using a specific algorithm. In order to optimize spot matching all pairs were reviewed and obvious mismatches were corrected manually. Only a few spots were edited manually if they were slightly merged with one another or if their borders were not properly detected. Statistics. The geometric mean of corresponding spot volumes was calculated and a Students t test was performed for intergroup comparison. A spot was considered up-regulated when the regulation factor was higher than 1.2 (i.e., spot volume of a group was higher than 120% compared to controls) and p-value (Students t test) was 1.2], p ) 0.05)a.
Figure 2. 2-D gel electrophoresis. Protein (700 µg) was applied on 18 cm nonlinear gradient strips (pH 3-10) (first dimension) and loaded on 9-16% gradient sodium dodecylsulfate polyacrylamide gels for second-dimensional separation (180 × 200 × 1.5 mm3). All protein spots exhibiting increased (black) or decreased (white, italics) levels in at least one of the treated groups as compared to untreated cells are depicted in a sample gel and identified by their UniProtKB accession number (see text).
Azizi et al.
spot nr./method
2085 14 LC-MS/MS
6.75 28701 284 MS
6.27 39639 13 LC-MS/MS
6.19 48355 96 MS/MS
5.13
5.91 73528 1 LC-MS-MS(379 MS)
6.67 57873 Not ident.
pI
16
5
19
5
71(26)
162 MS
spot nr./ method
78
268
173
262
32 MS
3 LC-MS/MS
235 MS
4 LC-MS/MS
3017(110) 102 MS
Mascot score
colcemid
24
5
19
3
35
13
161
97
91
114
292
66
t test p)
spot nr./ method
0.5491 0.0021 327 MS
0.7329 0.0322 8 LC-MS/MS
0.7902 0.0284 154 MS
0.6489 0.0302 9 LC-MS/MS
0.6772 0.0346 178 MS
0.4055 0.0286 123 MS
peptide Mascot regul. matches score factor
nocodazole
20
2
20
5
35
16
117
41
112
267
212
74
t test p)
0.6843 0.0169
0.7255 0.0208
0.7409 0.0160
0.5594 0.0049
0.6910 0.0426
0.6421 0.0478
peptide Mascot regul. matches score factor
This table furthermore shows the method of protein identification, number of peptide matches, and the Mascot score as well as the UniProtKB accession number, pI, and MW.
T-complex protein 1, zeta subunit Stress-70 protein, mitochondrial (precursor) (syn: Mortalin) Chromobox protein homologue 3 (syn: HP1 gamma, heterochromatin protein 1 homologue gamma) Ornithine aminotransferase, mitochondrial (precursor) Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial precursor Phosphoglycerate mutase 1
protein name
peptide matches
control
F-Actin capping protein beta subunit (Syn.: CapZ beta) Osmotic stress protein 94(syn: Heat shock 70-related protein APG-1) Proteasome subunit alpha type 2 Heat shock cognate 71 kDa protein Heat shock cognate 71 kDa protein 3-phosphoglycerate dehydrogenase Heat shock protein 75 kDa, mitochondrial (precursor)(syn: TNFR-associated protein 1) Proteasome subunit beta type 3
protein name
MW (kDa) spot nr./method
6.15 22965 5 LC-MS/MS(236 MS/MS)
6.25 80209 31 MS
6.12 56586 13
5.37 70871 140 MS
5.37 70871 265 MS
8.42 25794 250 MS/MS
5.54 94382 Not identified
5.47 31214 196 MS
pI
18(14)
30
15
18
11
13
19
peptide matches
111 MS
130 MS
319 MS
231 MS
58 MS
330 MS
363 MS
17
46
23
23
25
12
22
22
84
323
141
107
134
72
70
121
t test p)
spot nr./ method
1.3482 0.0420 319 MS/MS
1.6601 0.0140 21 MS
1.4926 0.0054 97 MS/MS
2.0204 0.0315 252 MS
2.3728 0.0141 333 MS
1.4061 0.0119 375 MS
1.3847 0.0198 Not identified
1.2296 0.0372 299 MS
spot nr./ peptide Mascot Regul. method matches score factor
681(101) 27 MS
117
91
65
63
109
114
Mascot score
colcemid
43
19
24
24
12
19
243
203
108
116
71
106
t test p)
1.2860 0.0723
1.3718 0.2032
1.2663 0.0651
1.5765 0.1609
1.9155 0.0416
1.1897 0.1780
1.1845 0.5042
1.1888 0.0792
peptide Mascot Regul. matches score factor
nocodazole
a This table furthermore shows the method of protein identification, number of peptide matches, and the Mascot score as well as the UniProtKB accession number, pI and MW. P63017 was found in two separate up-regulated spots.
Q9R1P1
Q9CQN1
Q75SV9
P63017
P63017
P49722
P48722
P47757
UniProtKB Acc#
control
Table 5. Protein Spots Found by in silico Quantification (Proteomweaver 3.0) to be Up-Regulated in the Group Treated with Colcemid Compared to Controls (>120% [) regulation factor >1.2], p ) 0.05), but Not in the Nocodazole Treated Groupa
a
Q9DBJ1
Q9D6R2
P29758
P23198
P38647
P80317
UniProtKB Acc#
MW (kDa)
Table 4. Protein Spots Found by in silico Quantification (Proteomweaver 3.0) to be Down-Regulated in Both Mitosis Arrest Groups (Treated with Colcemid or Nocodazole) Compared to Controls (
