Organellar Protein Complexes of Caco-2 Human Cells Analyzed by

Organellar Protein Complexes of Caco-2 Human Cells Analyzed by Two-Dimensional Blue Native/SDS-PAGE and Mass Spectrometry Jean-Paul Lasserre,*,†,‡ Loı¨k Sylvius,† Raymonde Joubert-Caron,† Michel Caron,† and Julie Hardouin*,‡,§ Laboratoire de Biochimie des Proteínes et Proteómique, Universite´ Paris 13, UMR CNRS 7033, 74 rue Marcel Cachin F-93017 Bobigny cedex, France, Institut de Biochimie et Geńe´tique Cellulaires, Universite´ Bordeaux 2, UMR CNRS 5095, 1 rue Camille Saint-Sae¨ns F-33077 Bordeaux Cedex, France, and Laboratoire Polyme`res, Biopolyme`res, Surfaces, Equipe BRICS, Universite´ de Rouen, UMR CNRS 6270, Boulevard Maurice de Broglie F-76821 Mont-Saint-Aignan cedex, France Received April 28, 2010

The complexome is essential for a better understanding of protein functions. In order to study protein complexes, an approach allowing the extraction and the analysis in native conditions is needed. Twodimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) technology is thus an interesting and powerful approach for this purpose. This report deals with the analysis and the identification of the organellar protein complexes of Caco-2 human cells using 2D BN/SDS-PAGE and HPLC-chip-MS. We identified 58 protein complexes (26 heteromultimeric and 32 homomultimeric complexes) and 4 monomeric proteins. Among them, 32 protein complexes were pointed out, providing insights into the function of previously uncharacterized human proteins. Keywords: Two-dimensional blue native/SDS-PAGE • Complexome • colorectal cancer • native protein complex • organellar • heteromultimer • homomultimer

Introduction Proteomics is a powerful approach to study and to obtain information on proteins. Generally, proteins of an extract are separated by two-dimensional electrophoresis (2DE) and identified by mass spectrometry (MS), giving a complete and interesting two-dimensional map. Although this strategy allows the researchers to know the name of the proteins of a cell, only little information concerning the protein function can be obtained. An interesting way is to be able to point out the interactions between the proteins in order to have access to their functions and to understand the molecular mechanisms. Biomolecular interactions play a critical role in the vast majority of cellular processes. It has become evident over the past few years that many complex cellular processes are carried out by sophisticated multisubunit protein machines maintained by different kinds of interactions. Various methods based on distinct principles have been developed to identify protein-protein interactions like two-hybrid yeast system,1-5 protein microarrays,6,7 and affinity purification-mass spectrometry.8-15 The twohybrid yeast system has been the spread method but it essentially allows the detection of binary interactions. The TAPTag and the immunoprecipitation approaches are currently used to a greater extent to detect protein interactions. However, * To whom correspondence should be addressed. Dr. Jean-Paul Lasserre, phone, +33 (0)556991120; fax, +33 (0)556999059; e-mail, [email protected]. Dr, Julie Hardouin, phone, +33 (0)235146709; fax, +33 (0)235146704; e-mail, [email protected]. † Universite´ Paris 13. ‡ Universite´ Bordeaux 2. § Universite´ de Rouen. 10.1021/pr100381m

 2010 American Chemical Society

all these techniques know some drawbacks since they give a high number of false-positives and/or false-negatives. Analytical methods like MS can be used too, but a pure solution is often needed in order to be able to interpret the recorded data.16 Despite these drawbacks, the results obtained with all these techniques have enriched the databanks leading to the development of protein-protein interaction networks.17,18 Generally, protein complexes are maintained with weak bonds (van der Waals, hydrogen bonds). So, whatever the approach used, native conditions are imperative to prevent protein dissociation. Two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D BN/ SDS-PAGE) offers an attractive proteomic solution for the analysis of protein complexes keeping most of interactions between the partners. By 2D BN/SDS-PAGE, the protein complexes are separated in the first native dimension and the proteins belonging to the same protein complex are separated in the second denaturing dimension. The proteins are then digested by a specific enzyme, in general trypsin, and identified by MS. Initially, 2D BN/SDS-PAGE was described and used for the separation of protein complexes belonging to the respiratory chain of mitochondria19-27 and Paracoccus denitrificans cells.26,28 As this method shows reproducibility, the detection of protein complex deficiencies of membrane of mitochondria was achieved.29-34 In the same way, it has been successfully applied to study assembly photosynthetic complexes of membranes of cyanobacteria like Synechocystis,35,36 protein complexes of the mitochondria, and chloroplast membranes of plants.37-41 Moreover, an alternative method using agarose instead of acrylamide was achieved to study protein complexes Journal of Proteome Research 2010, 9, 5093–5107 5093 Published on Web 07/22/2010

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Lasserre et al. 42

with apparent molecular weights of more than 1200 kDa. As illustrated previously, many works used 2D BN/SDS-PAGE for the study of protein complexes belonging to the respiratory chain. But few studies have been done on whole cell extracts from various origins such as prokaryote43-45 or eukaryote46,47 and some improvements have to be achieved to use 2D BN/ SDS-PAGE in this context. For many years, our laboratory has worked on proteomic studies of colorectal cancer.48,49 Despite advances in diagnosis, cancer remains a major cause of mortality worldwide. No current specific and sensitive markers exist for early diagnosing disease development. The proteome is the global representative of all biological processes that take place during cancer development. Nowadays, the knowledge of the protein-protein interactions in colorectal cancer would be very important for understanding the biological mechanisms. Thus, we choose 2D BN/SDS-PAGE followed by MS, as a proteomic approach, for characterizing the main organellar protein complexes of colorectal cancer cells (Caco-2) used as model at the laboratory. In this report, we presented, in a first section, a discussion on the 2D BN/SDS-PAGE technique dealing with the optimizations of both the sample preparation and the separation conditions for the native electrophoresis, its advantages and its limits. In a second section, we presented the results of the approach. We pointed out 32 new protein complexes that have never been described in the human cells.

Experimental Procedures Cell Culture. Unless otherwise stated, all chemicals were purchased from Invitrogen (Invitrogen SARL, Cergy-Pontoise, France). Human colorectal cancer Caco-2 cells were grown to confluence in DMEM (D Minimum Essential Medium Modified) supplemented with 20% fetal calf serum (FCS), 1% nonessential amino acids, gentamycin (8 µg/mL), penicillin, and streptomycin (100 IU/mL). Extraction of Protein Complexes. The Caco-2 cells were washed with 0.9% NaCl and PBS, and scraped using a plastic cell scrapper. The cells were centrifuged at 800g for 10 min at 4 °C. The cell pellet was abundantly washed in ice-cold PBS. The final pellet was suspended in lysis buffer containing 50 mM Tris, 750 mM 6-amino-n-caproı¨c acid, 1 mM phenyl methyl sulfonyl fluoride (PMSF), and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Cells were broken using a Dounce glass homogenizer at 4 °C. The lysate was centrifuged at 15 000g for 15 min at 4 °C in order to obtain the organellar fraction in the pellet. The pellet was suspended in lysis buffer and washed three times by centrifugation. The organellar fraction was suspended in lysis buffer and broken again using Thomas Teflon pestle tissue homogenizer at 4 °C and centrifuged at 100 000g for 45 min at 4 °C. The supernatant corresponded to organellar protein complexes. After the spermine addition to a final concentration of 1 mM, organellar protein complexes were centrifuged at 4 °C at 100 000g for 45 min, and then filtered through miracloth paper (Calbiochem, San Diego, CA). These samples were used immediately or stored at -20 °C until further analyses. After protein extraction, the concentration of extract was 11 µg/µL. 2D BN/SDS-PAGE. The sample preparation and BN-PAGE were carried out as described by Scha¨gger et al.25 with the following modifications. Gel dimensions were 22 cm ×16.5 cm × 0.1 cm. Separating gels (first dimension) with linear 3-18% (w/v) acrylamide gradient gels were used. Anode and cathode buffers contained 50 mM Tris, 75 mM glycine. The cathode 5094

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buffer was supplemented with 0.002% (w/v) Coomassie Brillant Blue G-250 (Bio-Rad, Hercules, CA). Before loading, 1 µL of sample buffer (500 mM 6-amino-n-caproic acid, 5% (w/v) Coomassie Brillant Blue G-250) was added to 20 µL of sample. The gel was run at 4 °C with 1 W, 100 V, and 28 mA. Thyroglobulin (669 kDa) and BSA (66 kDa) (Sigma, Saint-Louis, MO) were used as molecular weight size standards for BNPAGE analysis. An individual lane from BN-PAGE was cut and equilibrated in 1% (w/v) SDS, 6 M urea, 0.125 M Tris/HCl, pH 6.8, for 5 min. This individual lane was dipped for 20 min into equilibrating buffer supplemented with 100 mM dithiothreitol (DTT), and then soaked for 20 min into equilibrating buffer supplemented with 55 mM iodoacetamide. An ultimate washing step was performed in the equilibrating buffer for 5 min. The lane was slipped between two glass plates at the top, above the stacking and separating gels previously poured. Then, stacking gel mixture was supplementary poured. Following the migration, proteins were visualized after staining of the gels using the PROTSIL2 Silver staining kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The number of 2D spots observed on the 2D gels is 105. Protein Digestion. Silver-stained proteins separated by 2D BN/SDS-PAGE were excised and destained using PROTSIL2 Silver staining kit (Sigma-Aldrich, Saint-Louis, MO) according to the manufacturer’s instructions. Spots were subsequently rinsed twice in ultrapure water for 5 min and shrunk in solution containing 50% ethanol and 50 mM NH4HCO3 for 30 min. This step was repeated three times. Spots were shrunk twice in acetonitrile (ACN). After ACN removal, gel pieces were dried at room temperature, covered with the trypsin solution (10 ng/ µL in 50 mM NH4HCO3), and rehydrated at 4 °C for 45 min. The excess was removed and the spots were finally incubated overnight at 37 °C in 50 mM NH4HCO3. The supernatant was collected, and a H2O/ACN/trifluoroacetic acid (TFA) (49.9:50: 0.1) extraction solution was added onto gel slices for 20 min. The supernatant was kept and ACN was added onto gel slices for 20 min. Supernatants were pooled and concentrated by drying at 37 °C for 2 h. HPLC-Chip-IT-MS/MS. The tryptic samples were then analyzed by nanoLC-MS/MS. All experiments were done on an Agilent 1100 LC/MSD Trap SL series system. The ionization system was the Chip Cube using HPLC-Chip-MS (Agilent Technologies, Santa Clara, CA). The chip was automatically loaded and positioned into the MS nanospray chamber. The chip contained a Zorbax 300SB-C18 (43 mm ×75 µm, 5 µm) column and a Zorbax 300SB-C18 (40 nL, 5 µm) enrichment column. The sample loading into the enrichment column was done at a flow rate set to 4 µL/min with the mix of the two following solutions at a ratio of 98/2: H2O/0.1% formic acid (FA) and ACN/0.1% FA. LC gradient (mobile phase A, H2O/ 0.1% FA; mobile phase B, ACN/10% H2O/0.1% FA) was delivered with flow rate set at 300 nL/min. Tryptic peptides were eluted from the reversed-phase column into the mass spectrometer using a linear gradient elution of 15-55% B over 30 min. The capillary voltage was set to 1800 V, the flow and the dry gas temperature were 4 L/min and 280 °C, respectively. The mass range was from m/z 400 to m/z 2200. The two most intense precursor ions were automatically selected for a MS/ MS study. The MS and MS/MS recorded data were analyzed by Data Analysis software version 3.3 (Agilent Technologies, Santa Clara, CA). The processing parameters used in the

Human Caco-2 Cell Complexomic creation of the peak list from the raw data were the smoothing (algorithm, Gauss; width, m/z 0.2; and cycle, 1), the baseline subtraction of mass spectra (flatness (0-1): 0.8), the retention time window for compound detection (0.5 min) and number of compounds (250). Protein Identifications. The MS data were analyzed using the Matrix Science program with Mascot Daemon client application version 2.0 (Matrix Science, London, U.K.). Proteins were identified using the MS/MS ion search and the SwissProt database. The search parameters were restricted to Homo sapiens taxonomy (206 586 sequences), allowed for one missed trypsin cleavage site, carbamidomethyl modification and variable oxidation of methionine. Using an ESI ion trap instrument, settings included a peptide tolerance of 0.4, MS/MS tolerance of 0.4, and a peptide charge of 2+ and 3+. An accepted result had a score of at least 26.

Results and Discussion For the analysis of the organellar protein complexes in colorectal cancer human cells, we first presented the optimizations of the 2D BN/SDS-PAGE methodology concerning both the sample preparation and the protein complexes separation. After these optimizations, correct electrophoretic migrations were obtained allowing us to identify 58 protein complexes in the organellar fraction of Caco-2 human cells 1. Optimization of the 2D BN/SDS-PAGE Separation for the Analysis of Organellar Protein Complexes in Colorectal Cancer Human Cells. The 2D BN/SDS-PAGE methodology is a technique that can be divided in three major steps: (i) the preparation of the protein extract under native conditions, (ii) the complexes separation in the first dimension under native conditions, and (iii) the proteins separation in the second dimension under denaturing conditions. The last step is very well controlled. Conversely, the two first steps, and particularly the preparation of the sample, are very difficult because the use of native conditions is necessary to preserve the proteinprotein interactions. For the organellar protein complexes in colorectal cancer human cells, we optimized the 2D BN/SDSPAGE technique and we summarized here the optimization at the level of the sample preparation and at the level of the first dimension. 1.1. Optimization of the Sample Preparation. 2D BN/SDSPAGE is currently used to study the protein complexes belonging to the membrane of mitochondria or chloroplasts. However, the study of all protein complexes of eukaryotic or prokaryotic cells presented some problems due to the sample complexity. Camacho-Carvajal et al.46 explained that small molecules have to be removed by dialysis since they prevent a correct electrophoretic migration. Therefore, the sample preparation is an important step for the success of the separation in the first dimension of the protein complexes. The first important point was the elimination of DNA (negatively charged) before the electrophoresis separation since it obstructs the gel pores leading to an incorrect protein complex migration. Several methods could be used for the DNA removal. Here, we used the spermine and centrifugation to remove DNA but to keep the interactions. This step was performed at 4 °C in order to decrease the protein degradation by proteases that frequently occurs during the step of digestion by DNase carried out at 25 °C. The second essential point concerned the presence of lipids which generate some electrophoretic migration problems

research articles because lipids possess charges. The lipids were removed using a miracloth paper as described in details in the Experimental Procedures. These optimizations on the sample preparation of Caco-2 human cell extract enabled us to obtain a fraction corresponding to the organellar protein complexes coming from particularly mitochondria and nucleus free of DNA and lipids. 1.2. Optimization of the Protein Complex Separations. Protein complexes were separated according to their size, charge, and shape in the native polyacrylamide gradient gel (first dimension). We used a very slow migration using only 1 W constant in order to avoid not only heating, but also the protein complex dissociation. Because the protein complexes are essentially maintained by ionic interactions, a weak current helps to preserve the interactions. Indeed, a higher current could release subunits linked to the protein complex with a weak ionic interaction. Furthermore, although 1 W limits the heating, the electrophoresis needed to be carried out at 4 °C. To improve the organellar protein complexes separation in the first dimension, a 3-18% (w/v) acrylamide gradient was used for the BN/PAGE. Both the complex focalization and the separation in a given range size (60-1500 kDa) were improved. From these optimizations, resolute and reproducible gels for the Caco-2 extract were obtained enabling us to observe and to interpret many protein complexes. 1.3. Interpretation of the 2D BN/SDS-PAGE Data. Proteins belonging to the same protein complex co-migrated in the first native dimension but migrated independently in the second denaturing dimension. Consequently, proteins from the same complex presented aligned spots with a similar shape on the gel.43 When spots of different shapes were aligned, they were considered as proteins coming from different complexes. Considering these criteria, after the gel analysis, 58 distinct protein complexes and 4 monomeric proteins were determined for the organellar Caco-2 Human cells (Figures 1 and 2). These protein complexes could be classified in five groups: (i) the first group concerned 4 heteromultimeric protein complexes previously described in the scientific bibliography (Table 1 and Figure 1), (ii) the second group deals with 22 unknown heteromultimeric protein complexes in Human cells (Table 2 and Figure 1), (iii) the third group was formed of 22 homomultimeric protein complexes previously described in the scientific literature (Table 3 and Figure 2), (iv) the fourth group concerned 10 new homomultimeric protein complexes in human cells (Table 4 and Figure 2), and (v) the last group is composed by 4 monomeric proteins (Table 5 and Figure 2). We essentially focused on the new homomultimeric and heteromultimeric protein complexes (Tables 2 and 4) since they provided new information on protein complexes in Caco-2 human cells. 1.4. Validity of the Approach. Among the 26 heteromultimeric protein complexes, 4 protein complexes (complexes nos. 1-4) were previously partially or totally described (Table 1) and could validate both the technology and the analysis criteria. For example, the protein complex no. 1 (Table 1 and Figure 1), composed of ETFA (spots 75, 76 and 77) and ETFB proteins (spots 84, 85 and 182) was previously described as an heterodimer of alpha and beta subunits.50,51 Moreover, during the first dimension, this complex migrated at the correct molecular weight of 63 kDa. A second example concerned the protein complex no. 3 constituted by the SPTA2 (spot 156) and the SPTB2 proteins (spot 156) identified as a 1200 kDa complex in Journal of Proteome Research • Vol. 9, No. 10, 2010 5095

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Figure 1. 2D BN/SDS-PAGE gel of organellar heteromultimeric protein complexes from Caco2 Human cells. Unknown protein complexes are underlined. The acrylamide gradient gel used in the first dimension was 3-18% (w/v) following by a second dimension in denaturing conditions (SDS-PAGE) and silver staining.

Figure 2. 2D BN/SDS-PAGE gel of organellar homomultimeric protein complexes from Caco2 Human cells. Unknown protein complexes are underlined. The monomeric proteins are indicated with an asterisk. The acrylamide gradient gel used in the first dimension was 3-18% (w/v) following by a second dimension in denaturing conditions (SDS-PAGE) and silver staining. 5096


SF3A1

202

6

10, 9

16

SPTA2

HNRPC

24

3

SPTB2

57, 128

156

PSA6

87

2

8, 3, 7

6, 6, 5

peptide matchesa

Q15459

P07910

Q13813

Q01082

P60900

P25786

P38117

P13804

Swiss-Prot accession number

Electron transfer flavoprotein alpha-subunit, mitochondrial precursor. The electron transfer flavoprotein serves as a specific electron acceptor for several dehydrogenases. It transfers the electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF dehydrogenase). Electron transfer flavoprotein beta-subunit, mitochondrial precursor. The electron transfer flavoprotein serves as a specific electron acceptor for several dehydrogenases. It transfers the electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF dehydrogenase). Proteasome subunit alpha type 1. The proteasome is a multicatalytic proteinase complex which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. Proteasome subunit alpha type 1. The proteasome is a multicatalytic proteinase complex which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. Spectrin beta chain, brain 1. Fodrin, which seems to be involved in secretion, interacts with calmodulin in a calcium-dependent manner and is thus candidate for the calcium-dependent movement of the cytoskeleton at the membrane. Spectrin alpha chain, brain. Fodrin, which seems to be involved in secretion, interacts with calmodulin in a calcium-dependent manner and is thus candidate for the calcium-dependent movement of the cytoskeleton at the membrane. Heterogeneous nuclear ribonucleoproteins C1/C2. Binds pre-mRNA and nucleates the assembly of 40S hnRNP particles. May play a role in the early steps of spliceosome assembly and pre-mRNA splicing. Interacts with poly-U tracts in the 3′-UTR or 5′-UTR of mRNA and modulates the stability and the level of translation of bound mRNA molecules. Splicing factor 3 subunit 1. Subunit of the splicing factor SF3A required for ‘A’ complex assembly formed by the stable binding of U2 snRNP to the branchpoint sequence (BPS) in pre-mRNA. Sequence independent binding of SF3A/SF3B complex upstream of the branch site is essential, it may anchor U2 snRNP to the pre-mRNA. May also be involved in the assembly of the ‘E’ complex.

protein functions

169

537, 431

779

1048

149

61

351, 130, 283

299, 326, 280

scoreb

7

28, 25

8

13

15

7

32, 11, 31

19, 25, 21

% covc

88888

33725

285150

275259

27838

29822

28054

35400

MW (Da)d

5.15

4.95

5.22

5.41

6.34

6.15

8.24

8.62

pIe

N

N

C, ck

mb

C, N

C, N

mt

mt

locationf

ref

Swiss-Prot

80-82

55, 56

50, 51

a Number of peptide masses matching the top hit. b Mascot protein score obtained by HPLC-Chip-MS spectra. c Sequence coverage refers to the percentage of the protein sequence that corresponds to matched peptides. d Predicted molecular weight (MW) in dalton (Da) according to protein sequence. e Predicted isoelectric point (pI) of the protein according to sequence. f Location of the protein according to Swiss-Prot: C, cytoplasm; N, nucleus; Mt, mitochondria; Mb, membrane; Ck, cytoskeleton.

4

3

PSA1

78

ETFB

85, 84, 182

2

ETFA

75, 76, 77

1

gene names

number spot

no.

Table 1. Heteromultimeric Protein Complexes Known from Organellar Fractions

Human Caco-2 Cell Complexomic

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5098

209

5


OAT CISY PRDX3

GSTP1

152, 207 153 91, 169

91

9

183 168 184 216

12

13

218

11

10

8

1

SUCB2

7 5 6 5 2

2

SUCB1

PRDX1 PRDX2 P5CR1 SEPINH1 EIF4A3

3

1

11, 5 9 6, 5

8 7 11 8

KCRB

LDHB LDHA G3P DLDH

15, 15

ACTN4

65 70 63 179

6, 6

ACTN1

7

4, 18

ENPL

3, 9

6

1

1433G

1433E

1

peptides matchesa

1433Z

gene names

6

79

number spot

no.

Q06830 P32119 P32322 P50454 P38919

Q96I99

Q9P2R7

P12277

P09211

P04181 O75390 P30048

P07195 P00338 P04406 P09622

O43707

P12814

P14625

P62258

P61981

P63104


14-3-3 protein zeta/delta. Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. 14-3-3 protein gamma. Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. 14-3-3 protein epsilon. Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. Endoplasmin precursor. Molecular chaperone that functions in the processing and transport of secreted proteins. Alpha-actinin-1. F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. Alpha-actinin-4. F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. L-lactate dehydrogenase B chain. L-lactate dehydrogenase A chain. Glyceraldehyde-3-phosphate dehydrogenase. Dihydrolipoyl dehydrogenase, mitochondrial precursor. Lipoamide dehydrogenase is a component of the glycine cleavage system as well as of the alpha-ketoacid dehydrogenase complexes. Ornithine aminotransferase, mitochondrial precursor. Citrate synthase, mitochondrial precursor. Thioredoxin-dependent peroxide reductase, mitochondrial precursor. Involved in redox regulation of the cell. Protects radical-sensitive enzymes from oxidative damage by a radical-generating system. Glutathione S-transferase P. Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. Creatine kinase B-type. Reversibly catalyzes the transfer of phosphate between ATP and various phosphogens (e.g., creatine phosphate). Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa. Succinyl-CoA ligase [ADP-forming] beta-chain, mitochondrial precursor. Succinyl-CoA ligase [GDP-forming] beta-chain, mitochondrial precursor. Peroxiredoxin-1. Peroxiredoxin-2. Pyrroline-5-carboxylate reductase. Collagen-binding protein 2 precursor (Colligin 2). Probable ATP-dependent RNA helicase DDX48.

protein functions

Table 2. Heteromultimeric Protein Complexes Unknown from Organellar Fractions

218 171 310 229 68

44

46

133

60

445, 202 351 296, 259

288 275 413 410

756, 770

324, 308

206, 716,

36 23 26 14 5

2

4

7

7

27, 13 21 28, 22

27 22 36 17

20, 21

8, 8

6, 20

25

5

46

302

5

% covc

67

scoreb

22324 21918 33568 46525 47126

46824

50641

42902

23438

48846 51908 28017

36769 36819 36070 54686

105245

103563

92696

29326

28325

27899

MW (Da)d

8.27 5.67 7.18 8.75 6.3

6.15

7.05

5.34

5.44

6.57 8.45 7.67

5.72 8.46 8.58 7.59

5.27

5.25

4.76

4.63

4.8

4.73

pIe

C C ? ERL N

mt

mt

C

?

mt mt mt

C C C mt

C, N

C

ER

C

C

C

locationf

ref

58

58

59

59-63

research articles Lasserre et al.

151

30, 31 30 171

16

17

215

222

19

20

18

15

60 74 150

number spot

14

no.

2

ROAA

2

HNRPL

8

2

DHSA

THIL

13

8

LPPRC

GRP75

7, 14 7 13

6

EF1A1

ALDH2 SSDH OXRP

2

7

DHE3

ENOA

7 4 21

peptides matchesa

LDHB 3HIDH PDIA3

gene names

Table 2. Continued

Q99729

P24752

P14866

P31040

P38646

P42704

P05091 P51649 Q9Y4L1

P68104

P06733

P00367

P07195 P31937 P30101


L-lactate dehydrogenase B chain. 3-hydroxyisobutyrate dehydrogenase, mitochondrial precursor. Protein disulfide-isomerase A3 precursor. Catalyzes the rearrangement of -S-S- bonds in proteins. Glutamate dehydrogenase 1, mitochondrial precursor. May be involved in learning and memory reactions by increasing the turnover of the excitatory neurotransmitter glutamate (By similarity). Alpha-enolase. Multifunctional enzyme that, as well as its role in glycolysis, plays a part in various processes such as growth control, hypoxia tolerance and allergic responses. May also function in the intravascular and pericellular fibrinolytic system due to its ability to serve as a receptor and activator of plasminogen on the cell surface of several cell-types such as leukocytes and neurons: Elongation factor 1-alpha 1. This protein promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis Aldehyde dehydrogenase, mitochondrial precursor. Succinate semialdehyde dehydrogenase, mitochondrial precursor. 150 kDa oxygen-regulated protein precursor. Has a pivotal role in cytoprotective cellular mechanisms triggered by oxygen deprivation. May play a role as a molecular chaperone and participate in protein folding (By similarity). 130 kDa leucine-rich protein. May play a role in RNA metabolism in both nuclei and mitochondria. In the nucleus binds to HNRPA1-associated poly(A) mRNAs and is part of nmRNP complexes at late stages of mRNA maturation which are possibly associated with nuclear mRNA export. May bind mature mRNA in the nucleus outer membrane. In mitochondria binds to poly(A) mRNA. May play a role in translation or stability of mitochondrially encoded cytochrome c oxidase (COX) subunits. May be involved in transcription regulation. Cooperates with PPARGC1A to regulate certain mitochondrially encoded genes and gluconeogenic genes and may regulate docking of PPARGC1A to transcription factors. Seems to be involved in the transcription regulation of the multidrug-related genes MDR1 and MVP. Stress-70 protein, mitochondrial precursor (75 kDa glucose regulated protein). Implicated in the control of cell proliferation and cellular aging. May also act as a chaperone. Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial precursor. Heterogeneous nuclear ribonucleoprotein L (hnRNP L).This protein is a component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes which provide the substrate for the processing events that pre-mRNAs undergo before becoming functional, translatable mRNAs in the cytoplasm. L is associated with most nascent transcripts including those of the landmark giant loops of amphibian lampbrush chromosomes. Acetyl-CoA acetyltransferase, mitochondrial precursor). Plays a major role in ketone body metabolism. Heterogeneous nuclear ribonucleoprotein A/B. Binds single-stranded RNA. Has a high affinity for G-rich and U-rich regions of hnRNA. Also binds to APOB mRNA transcripts around the RNA editing site.

protein functions

100

332

35

44

565

448

352, 691 358 527

265

98

279

273 243 987

scoreb

6

18

4

3

23

7

17, 34 13 15

13

6

15

25 19 42

% covc

36704

45456

60719

73672

73920

146306

56859 58034 111494

50451

47350

61701

36769 35705 57146

MW (Da)d

9.04

8.98

6.65

7.06

5.87

5.5

6.63 8.62 5.16

9.1

6.99

7.66

5.72 8.38 5.98

pIe

N

mt

N

mb, mt

mt

mt, N

mt mt ERL

C

C, N, Mb

mt

C mt ER

locationf

ref


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5100

GRP75

HSP7C

130


225

8 5

16, 24

3

9, 12, 11 2 4 6

Q9UKM9 Q96C36

P14625

P23246

P40926 P04406 P04406 P05388

P11142

P38646

Q14103

P00558


Phosphoglycerate kinase 1. In addition to its role as a glycolytic enzyme, it seems that PGK-1 acts as a polymerase alpha cofactor protein (primer recognition protein). Heterogeneous nuclear ribonucleoprotein D0. Binds with high affinity to RNA molecules that contain AU-rich elements (AREs) found within the 3′-UTR of many proto-oncogenes and cytokine mRNAs. Also binds to double- and single-stranded DNA sequences in a specific manner and functions a transcription factor. Each of the RNA-binding domains specifically can bind solely to a single-stranded nonmonotonous 5′-UUAG-3′ sequence and also weaker to the single-stranded 5′-TTAGGG-3′ telomeric DNA repeat. Binding of RRM1 to DNA inhibits the formation of DNA quadruplex structure which may play a role in telomere elongation. May be involved in translationally coupled mRNA turnover. Implicated with other RNA-binding proteins in the cytoplasmic deadenylation/translational and decay interplay of the FOS mRNA mediated by the major coding-region determinant of instability (mCRD) domain. Stress-70 protein, mitochondrial precursor. Implicated in the control of cell proliferation and cellular aging. May also act as a chaperone. Heat shock cognate 71 kDa protein. Chaperone. Isoform 2 may function as an endogenous inhibitory regulator of HSC70 by competing the cochaperones. Malate dehydrogenase, mitochondrial precursor. Glyceraldehyde-3-phosphate dehydrogenase. Glyceraldehyde-3-phosphate dehydrogenase. 60S acidic ribosomal protein P0. Ribosomal protein P0 is the functional equivalent of E. coli protein L10 Splicing factor, proline- and glutamine-rich. DNA- and RNA binding protein, involved in several nuclear processes. Essential pre-mRNA splicing factor required early in spliceosome formation and for splicing catalytic step II, probably as an heteromer with NONO. Binds to pre-mRNA in spliceosome C complex, and specifically binds to intronic polypyrimidine tracts. Interacts with U5 snRNA, probably by binding to a purine-rich sequence located on the 3′ side of U5 snRNA stem 1b. May be involved in a pre-mRNA coupled splicing and polyadenylation process as component of a snRNP-free complex with SNRPA/U1A. Endoplasmin precursor. Molecular chaperone that functions in the processing and transport of secreted proteins. RNA-binding protein Raly. Pyrroline-5-carboxylase reductase 2 (EC 1.5.1.2).

protein functions

395 226

727, 990

86

369, 556, 533 34 179 260

339

474

109

110

scoreb

25 20

6, 26

4

32, 42, 38 10 17 23

13

20

6

10

% covc

32501 33958

92696

76216

35965 36070 36070 34423

71082

73920

38581

44854

MW (Da)d

9.2 7.66

4.76

9.45

8.92 8.58 8.58 5.71

5.37

5.87

7.62

8.3

pIe

N ?

ER

N

mt C C ?

C, N

mt

N

C

locationf

ref

a Number of peptide masses matching the top hit. b Mascot protein score obtained by HPLC-Chip-MS spectra. c Sequence coverage refers to the percentage of the protein sequence that corresponds to matched peptides. d Predicted molecular weight (MW) in dalton (Da) according to protein sequence. e Predicted isoelectric point (pI) of the protein according to sequence. f Location of the protein according to Swiss-Prot: ?, represents absence of data; C, cytoplasm; N, nucleus; Mt, mitochondria; Mb, membrane; ER, endoplasmic reticulum; ERL, endoplasmic reticulum lumen; Ck, cytoskeleton.

26

RALY P5CR2

ENPL

8, 11

127 126

SFPQ

8

25

24

MDHM G3P G3P RLA0

144, 146, 147 147 59 59

23

7

10

2

HNRPD

22

4

peptides matchesa

PGK1

223

21

gene names

number spot

no.

Table 2. Continued


SODM

GNPI ODO2

TERA CLPP

ACTA LDHA ENPL

ALDOA HTRA2 ACDSB

5

4 6

14 3

2 9 11

6 2 5

P04179

P46926 P36957

P55072 Q16740

P62736 P00338 P14625

P04075 O43464 P45954

P23141 P34897 P00367 P07954 P60709 P26440 P24752 P16219

P40925 Q8TCS8

P11310

Acyl-CoA dehydrogenase, medium-chain specific, mitochondrial precursor. Malate dehydrogenase, cytoplasmic. Polyribonucleotide nucleotidyltransferase 1, mitochondrial precursor. Liver carboxylesterase 1 precursor. Serine hydroxymethyltransferase, mitochondrial precursor. Glutamate dehydrogenase 1, mitochondrial precursor. Fumarate hydratase, mitochondrial precursor. Actin, cytoplasmic 1 (Beta-actin) Isovaleryl-CoA dehydrogenase, mitochondrial precursor. Acetyl-CoA acetyltransferase, mitochondrial precursor. Acyl-CoA dehydrogenase, short-chain specific, mitochondrial precursor. Fructose-bisphosphate aldolase A. Serine protease HTRA2, mitochondrial precursor. Acyl-CoA dehydrogenase, short/branched chain specific, mitochondrial precursor. Actin, aortic smooth muscle (Alpha-actin-2) L-lactate dehydrogenase A chain. Endoplasmin precursor. Molecular chaperone that functions in the processing and transport of secreted proteins. Transitional endoplasmic reticulum ATPase. Putative ATP-dependent Clp protease proteolytic subunit, mitochondrial precursor. Glucosamine-6-phosphate isomerase. Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase. The 2-oxoglutarate dehydrogenase complex catalyzes the overall conversion of 2-oxoglutarate to succinyl-CoA and CO(2). It contains multiple copies of 3 enzymatic components: 2-oxoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3). Destroys radicals which are normally produced within the cells and which are toxic to biological system

protein functions

205

175 276

590 175

27 297 471

243 98 221

24

17 13

21 15

5 25 15

20 5 15

296, 381 13, 18 392 21 258 16 221, 269 14 228, 341 14, 28 337 20 233, 305 16, 14 193 11

24878

32819 48952

89819 30446

42381 36819 92696

39720 48868 47797

62766 56414 61701 54773 42052 46803 45456 44611

36500 86510

47015

% covc MW (Da)d

138, 125 15, 10 190, 264 7, 10

381

scoreb

8.35

6.42 9.01

5.14 8.26

5.23 8.46 4.76

8.39 10.1 6.53

6.15 8.76 7.66 8.85 5.29 8.45 8.98 8.13

6.89 7.87

pIe

mt

C mt

C, N mt

C, ck C ER

mt mt

ER mt mt mt, C C, ck mt mt mt

mt mt

mt

4

6 multi

12 7

multi 4 2

4 3 4

3 and 6 4 6 4 multi 4 4 4

2 3

4

ref

67 swissprot

6, 65, 66

4

6 multi

12 7

2 4 2

4 3 4

6, 85-88

6, 84 swissprot

82 83

swissprot 80 swissprot

77 78, 79 swissprot

3 or 6 6, 68, 69 4 70 6 71 4 72 1 and 2 6, 73 4 6, 74 3 75 4 76

2 3

4

subunit subunit locationf describedg approxh

a Number of peptide masses matching the top hit. b Mascot protein score obtained by HPLC-Chip-MS spectra. c Sequence coverage refers to the percentage of the protein sequence that corresponds to matched peptides. d Predicted molecular weight (MW) in dalton (Da) according to protein sequence. e Predicted isoelectric point (pI) of the protein according to sequence. f Location of the protein according to Swiss-Prot. ?, represents absence of data; C, cytoplasm; Mt, mitochondria; Mb, membrane; ER, endoplasmic reticulum; Ck, cytoskeleton. g Represents a number of subunits described in scientific bibliography. h Represents an estimation of number of subunits.

141

48

12 83

44 45

137 157

220 69 10

41 42 43

46 47

132 158 131

38 39 40

6, 8 10 8 5, 6 5, 9 8 6, 5 4

8

30 25, 180 EST1 31 33 GLYM 32 35 DHE3 33 38, 136 FUMH 34 42, 47 ACTB 35 50 IVD 36 52, 53 THIL 37 56 ACADS

ACADM

peptides Swiss-Prot matchesa accession number

4, 3 6, 7

49

27

gene names

28 68, 208 MDHC 29 13, 210 PNPT1

number spot

no.

Table 3. Homomultimeric Protein Complexes Known from Organellar Fractions


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5102


176,177, 178, 181, 212, 213 138 133

6 15

55 140 201

154 142

49

50 51

52 53

54 55 56

57 58

CH10 D3D2

SET NPS3A AATM

HSP74 GRP75

TPIS BCAT2

CH60

9 4

3 4 8

8 9

9, 23, 22, 6, 14, 16 5 5

peptides matchesa

P61604 P42126

Q01105 Q9UFN0 P00505

P34932 P38646

P60174 O15382

P10809

Swiss-Prot accession number 60 kDa heat shock protein, mitochondrial precursor. Triosephosphate isomerase. Branched-chain-amino-acid aminotransferase, mitochondrial precursor. Heat shock 70 kDa protein 4. Stress-70 protein, mitochondrial precursor. SET protein. Protein NipSnap3A. Aspartate aminotransferase, mitochondrial precursor. 10 kDa heat shock protein, mitochondrial. 3,2-trans-enoyl-CoA isomerase, mitochondrial precursor.

protein functions

369 206

150 137 352

396 446

479, 1097, 967, 266, 537, 611 317 135

scoreb

67 15

11 17 19

12 18

20, 44, 43, 13, 28, 26 19 14

% covc

10794 33080

33469 28563 47844

95096 73920

26807 44658

61187

MW (Da)d

8.91 8.8

4.23 9.21 9.14

5.18 5.87

6.51 8.88

5.7

pIe

mt mt

C, ER, N C mt, mb

C mt

? mt (isoforme A)

mt

locationf

6 2

? ? 2

? ?

2 2

?

subunit describedg

6, 90, 91 6, 92

>8 >6

swissprot swissprot swissprot

3 >8 >10 >10 4

2 3

89, 92

ref

14

subunit approxh

gene names

4 2 1 6, 4

peptides matchesa

P02768 P07237 P42765 P27797


scoreb

% covc

Serum albumin precursor 66 5 Protein disulfide-isomerase precursor. 49 3 3-ketoacyl-CoA thiolase, mitochondrial. 79 3 Calreticulin precursor. Molecular calcium binding chaperone 262, 187 16, 11 promoting folding, oligomeric assembly and quality control in the ER via the calreticulin/calnexin cycle. This lectin interacts transiently with almost all of the monoglucosylated glycoproteins that are synthesized in the ER. Interacts with the DNA-binding domain of NR3C1 and mediates its nuclear export.

protein functions

71317 57480 42354 48283

MW (Da)d

locationf

subunit describedg

5,92 S ? 4,76 ERL 1, 2 and 4 8,32 mt 4 4,29 ERL, C, S, mb 1

pIe

1 1 1 1

subunit approxh

swissprot swissprot 81

ref

a Number of peptide masses matching the top hit. b Mascot protein score obtained by HPLC-Chip-MS spectra. c Sequence coverage refers to the percentage of the protein sequence that corresponds to matched peptides. d Predicted molecular weight (MW) in dalton (Da) according to protein sequence. e Predicted isoelectric point (pI) of the protein according to sequence. f Location of the protein according to swissprot. ?, represents absence of data; C, cytoplasm; Mt, mitochondria; Mb, membrane; ERL, reticulum endoplasmic lumen; S, secreted. g Represents a number of subunits described in scientific bibliography. h Represents an estimation of number of subunits.

59 19 ALBU 60 26 PDIA1 61 48 THIM 62 161, 162 CRTC

no.

number spot

Table 5. Monomeric Proteins from Organellar Fractions

a Number of peptide masses matching the top hit. b Mascot protein score obtained by HPLC-Chip-MS spectra. c Sequence coverage refers to the percentage of the protein sequence that corresponds to matched peptides. d Predicted molecular weight (MW) in dalton (Da) according to protein sequence. e Predicted isoelectric point (pI) of the protein according to sequence. f Location of the protein according to swissprot. ?, represents absence of data; C, cytoplasm; N, nucleus; Mt, mitochondria; Mb, membrane; ER, endoplasmic reticulum. g Represents a number of subunits described in scientific bibliography. h Represents an estimation of number of subunits.

number spot

no.

gene names

Table 4. Homomultimeric Protein Complexes Unknown from Organellar Fractions


Human Caco-2 Cell Complexomic the first dimension (Table 1 and Figure 1). It was previously described as a tetramer formed by two alpha-beta dimers.52-54 1.5. Limits of 2D BN/SDS-PAGE and MS. 2D BN/SDS-PAGE and MS are techniques that present some limits as (i) the spot resolution, (ii) the protein complex stability during the sample preparation and the separations, (iii) the subunit stoichiometry into a protein complex, (iv) the solubilization of the different subunits of a protein complex, (v) the gel penetration of large protein complexes, (vi) the detection threshold, or (vii) the absence of identification after MS analysis. For all these reasons, some protein complexes could be therefore partially observed on the gel as for the protein complexes nos. 2 and 4. Indeed, the protein complex no. 4 (spliceosome protein complex) is known to be constituted by several proteins (http://www.expasy.org/ uniprot/P07910). Here, only the heterogeneous nuclear ribonucleoproteins C1/C2 (HNRPC, spots 57 and 128) and the splicing factor 3 subunit 1 (SF3A1, spot 202) proteins were found (Table 1 and Figure 1). This protein complex migrated at 400 kDa; thus, we assumed that the other proteins could be present but not identified in this protein complex. A second example concerned the protein complex no. 2 (Table 1) known as the proteasome protein complex and composed of at least 15 individual subunits which form a highly ordered ring shape structure. In the first dimension, we noticed that the protein complex migrated as a 900 kDa complex. However, after the second dimension, only two proteins were identified: the proteasome subunit alpha type 1 (PSA1, spot 78) and the proteasome subunit alpha type 655,56 (PSA6, spot 87) (Table 1 and Figure 1). Other spots, aligned with these two proteins, were observed but no identification was obtained by MS. These two examples showed that this approach combining 2D BN/SDS-PAGE and MS does not always allow the identification of all the proteins involved in a protein complex. However, new and complementary insights can be collected to understand protein-protein interactions. 1.6. Advantages and Disadvantages of Protein-Protein Interaction Technologies. Protein-protein interactions studies can be carried out by different technologies: yeast two-hybrid system,1-5 affinity purification-mass spectrometry,8-15 2-D BN/SDS-PAGE.19-46,57 The two-hybrid system is a molecular genetic tool which facilitates the study of protein-protein interactions. This method has been the most used to detect interactions because it is fairly simple, rapid, and inexpensive. Unfortunately, the two-hybrid system has a tendency to produce frequently false positives caused by bait proteins, acting as transcriptional activators, or by proteins leading to nonspecific interactions for largely unknown reasons. This method also produces false negatives. For example, physiological protein-protein interactions are not detected by two-hybrid assays involving a sterical hindrance of the two fusion proteins so that transcriptional activation is prevented, or instability of proteins or failure of nuclear localization. Furthermore, two-hybrid systems permit only to determine essentially binary interactions. Affinity purification-mass spectrometry methods are currently more used to detect protein-protein interactions. These technologies give also false-negatives, and following the specificity of the antibody used, immunoprecipitation leads to falsepositives anytime. Several other methods are available to detect protein-protein interactions, such as X-ray crystallography, nuclear magnetic resonance, protein complex pull-down, and protein interaction studies performed on proteins.

research articles Another method of choice is the 2-D BN/SDS-PAGE that can theoretically give both false-positives and false-negatives. Indeed, the false-negatives are due to some protein interactions suppressed during the protein extract preparation or protein separation. Concerning the false-positives, the use of different acrylamide gradient gels in the first dimension could eliminate them. In summary, each technology possesses weaknesses and advantages. For these reasons, we consider that these different technologies are complementary and lead to the validation of the identified protein complexes. 2. Characterization of Organellar Protein Complexes in Colorectal Cancer Human Cells. 2.1. Identification of New Protein Complexes Using 2D BN/SDS-PAGE and MS. Using 2D BN/SDS-PAGE and HPLC-chip-MS, 58 protein complexes and 4 monomeric proteins were pointed out. Among them, 32 new protein complexes were described for the first time in Caco-2 human cells (Tables 2 and 4, Figure 2). All these new protein-protein interactions could not be explained with current knowledge. However, some of them were previously described in organisms other than H. sapiens or were in agreement with functional interactions. Among these 32 new protein complexes, 3 protein complexes (protein complexes no. 9-11, Table 2, Figures 1 and 3) were examples of metabolon. A metabolon is a protein association into the same complex, improving catalytic efficiency by channelling an intermediate from one active site of an enzyme to another active site of the next enzyme. For the protein complex no. 9, the citrate synthase protein (CISY, spot 153) participates in the Krebs cycle. Its catalytic activity yields citrate and CoA from oxaloacetate and acetyl-CoA. The second partner, ornithine aminotransferase (OAT, spot 152 and 207), transforms oxaloacetate and L-ornithine in L-amino acid and L-glutamate-5-semialdehyde (Figure 3). In this study, the protein complexes nos. 5-8, and 12 have never been described in H. sapiens but have been observed in other organisms. For examples, the interactions between alphaactinin-1 (ACTN1, spots 3 and 9) and alpha-actinin-4 (ACTN4, spots 3 and 9) (complex no. 6), between L-lactate dehydrogenase A chain (LDHA, spot 65) and L-lactate dehydrogenase B chain (LDHB, spot 70) (complex no. 7), or between peroxiredoxin-1 (PRDX1, 183) and peroxiredoxin-2 (PRDX2, spot 168) (complex no. 12) have been previously described in mouse cells,58,59 whereas the complex no. 8 has been observed in Escherichia coli.9 The heteromultimeric protein complex no. 5 (Table 2 and Figure 1) was determined to be composed by 3 proteins: 14-3-3 protein zeta/delta (1433Z, spot 209), 14-3-3 protein gamma (1433G, spot 209) and 14-3-3 protein epsilon (1433E, spot 79). Only the binary interactions between 14-3-3 protein epsilon and 14-3-3 protein zeta/delta, and 14-3-3 protein epsilon and 14-3-3 protein gamma, have been described in the human cells.60-63 The interaction between 143-3 protein gamma and 14-3-3 protein zeta/delta has been seen in the mouse cells.59 Here, for the first time, the interaction between the three subunits was pointed out using 2D BN/SDSPAGE and MS. Other protein complexes (protein complexes nos. 14, 17 and 22) could be explained by functional connections (Table 2 and Figure 1). In the heteromultimeric protein complex no. 22, the Stress-70 protein (GRP75, spot 130) and the heat shock cognate 71 kDa protein (HSP7C, spot 225) were found associated. These proteins are involved in the stress and can also act as chaperones. A second example concerned the heteromultimeric protein complex no. 17 in which each protein catalyzes a Journal of Proteome Research • Vol. 9, No. 10, 2010 5103

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Lasserre et al.

Figure 3. Representation of three metabolons. A metabolon is the association of proteins into the same protein complex improving catalytic efficiency by channeling an intermediate that is formed at one active site of an enzyme to the active site of the next enzyme.

Figure 4. Isomerism. The structure of the two molecules is very similar allowing the enzymes to use both compounds during the enzymatic reaction.

similar reaction. Indeed, the aldehyde dehydrogenase protein (ALDH2, spot 30 and 31) and the succinate semialdehyde dehydrogenase protein (SSDH, spot 30) belong to the aldehyde dehydrogenase family and act on aldehyde substrates using NADH as cofactor. A particular third example was the protein complex no. 14 composed by L-lactate dehydrogenase B chain (LDHB, spot 60) and 3-hydroxyisobutyrate dehydrogenase (3HIDH, spot 74). As seen in Figure 4, the structure of the molecules, transformed by these two enzymes, are very similar. Furthermore, the L-lactate dehydrogenase could catalyze the oxidation of hydroxybutyrate. 5104


2.2. Proteins Identified with Only One Peptide. Generally, proteins identified with only one peptide sequence were not included in the results. However, for these peptides, when a correct amino acid sequence (above 5 residues) was deduced from the MS/MS spectrum, and when there was logical biological interpretation with the others proteins of the complexes, these single ions were kept. The mass spectra were meticulously studied and the b- and y-type ions were carefully checked. In addition, the mass measurement error of these daughter ions was also considered. Five proteins were thus identified with only one peptide after the nanoLC-MS/MS

research articles

Human Caco-2 Cell Complexomic analysis, implied in protein complexes nos. 5, 10, 11, and 36 (spot 209, 91, 218, and 48) with correct mass spectra interpretation (data not shown). 2.3. Several Protein Locations during the First and the Second Dimensions. Many interactions in cellular signaling networks are subjected to dynamic temporal and spatial regulations. That implies transitory interactions that are typically difficult to detect. Protein complex formations are dependent on signal-induced or biological contexts like posttranslational modifications (PTM) such as phosphorylation or ubiquitination. Because of these modifications, the same protein complex or protein can migrate at different locations. The same protein could be found in different locations after the first native dimension migration. This could be explained by several reasons: (i) a protein could interact with different proteins; (ii) a protein complex could be in different oligomerization state; (iii) a protein complex could be formed by the same partners but in different state of phosphorylation. For example, the heteromultimeric protein complex no. 1 migrated in three places that could be explained by either different phosphorylation states, sequence variations, or different oligomerization states (Table 1 and Figure 1). The protein LDHB was also identified in two distinct spots, that means that this protein was implied in two different complexes: the heteromultimeric protein complex no. 7 (spots 65) and the heteromultimeric protein complex no. 14 (spot 60) (Table 2 and Figure 1). In the case of homomultimeric complexes, the example of the protein complex no. 49 is interesting. This complex named 60 kDa heat shock protein (CH60) was found as a 800 kDa complex and a 400 kDa complex in the first dimension corresponding, respectively, to a multimer of 14 subunits composed of two stacked rings of 7 subunits and one ring unknown in human cells but previously described in E. coli64,89 (Table 4 and Figure 2). The same phenomenon could occur during the migration in the second denaturing dimension and numerous other examples were observed. These results could be explained by either the physiological states of these proteins (like PTM) or the incomplete solubilization during the reduction step. For example, in the protein complexes nos. 4 and 10 named, respectively, heterogeneous nuclear ribonucleoproteins C1/C2 (HNRPC, spots 57 and 128) (Table 1 and Figure 1) and thioredoxin-dependent peroxide reductase (PRDX3, spots 91and 163) (Table 2 and Figure 1) illustrated very well this point since these proteins migrated at two locations in the second denature dimension.

Concluding Remarks In conclusion, the 2D BN/SDS-PAGE technique described in this work shows its capability and its utility for the complexome study. Numerous protein complexes were separated and proteins implied in these complexes were observed in one gel. From this study, 58 protein complexes and 4 monomeric proteins were pointed out. We described, for the first time, 32 new protein complexes in Human cells. From these data, we could complete the complexomic network by adding the protein-protein interactions observed here. Moreover, the determination of the protein interaction would be very interesting in order to understand some biological pathways in colorectal cancer pathology. These results confirmed that the association of 2D BN/SDSPAGE and MS allowed the identification of new proteins

complexes in the human cells. This approach can be considered as a complementary tool in the complexomic and proteomic areas. Abbreviations: 2D BN/SDS-PAGE, two-dimensional blue native/SDS-PAGE; FA, formic acid.

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