[page 35↓]

4.  Results

The aim of this study was to establish a reference 2D-electrophoresis map of human mitochondrial proteins. Since this basic work should find its application in future diagnostic tests for patients with mitochondriopathies, I chose a material that can easily be obtained from patients. Immortalized cultured lymphoblastoid cells are easy to handle, can grow in non-adherent cell-cultures and were therefore the material of choice. The combined methods of 2D-electrophoresis and mass spectrometry were used to separate and to identify the proteins. All procedures and their respective results are summarized in Fig. 4-1. The whole project started with the establishment of an immortalized lymphoblastoid cell line. The electron microscopic photographs from the cells show that each lymphoblastoid cell contains about 15-20 mitochondria (Fig. 4-2). The mitochondria were isolated and enriched by homogenization followed by density gradient centrifugation. All three ensuing fractions were examined by electron microscopy (Fig. 3-2). Only the third fraction, which contained the mitochondria, was used in subsequent experiments. The mitochondrial proteins were separated by 2D-electrophoresis and stained with Coomassie brilliant blue. The protein identification was performed with MALDI-TOF mass spectrometry and subsequent database searching. The identified proteins were marked on the 2D-electrophoresis map to get the final reference map.

Fig. 4-2: Electron microscopic picture of a lymphoblastoid cell . Each cell contains about 15-20 mitochondria.

4.1. Mitochondrial isolation

In order to study the mitochondrial proteome specifically, I chose as raw material the mitochondrial fraction instead of whole lymphocyte preparations. In Fig. 4-3 the 2D-electrophoresis gels of the lymphocyte proteome (A) and the mitochondrial proteome (B) are compared to each other. On the mitochondrial gel there are much less spots than on the whole lymphocytes’ gel. Some spots, which are fairly weak or even barely visible on the lymphocytes’ gel, show up intensely on the mitochondrial gel (highlighted with red arrows in the mitochondrial gel). For isolation of mitochondria from lymphoblastoid cells the first aim was to gain enough mitochondria from as little material as possible. The second aim was to modify the current protocols of mitochondrial isolation to increase the purity of the mitochondrial fractions. In my experiments atotal of 108 cells was sufficient to obtain enough purified mitochondria from one patient to be able to perform two large gel 2D-electrophoresis runs, i.e. about 108 cells yielded 12-20 mg mitochondria. At the mitochondrial isolation procedure (see chapter 3.2.4), after ultracentrifugation I detected three distinct fractions in the hybrid discontinuous gradient. Floating material could be found between the surface of the gradient and the


[page 36↓]

Fig. 4-1: This summary describes the procedures and the results of this study step by step. The work started from the establishment of a lymphoblastoid cell line. The electron microscopic photographs showed that each lymphocyte contains about 15-20 mitochondria. The mitochondria were isolated after homogenisation of the lymphocytes followed by density gradient centrifugation. The three fractions were examined by electron microscopy. Only the third fraction was enriched of mitochondria. This fraction was used in subsequent experiments. The mitochondrial proteins were separated by 2D-electrophoresis and stained with Coomassie brilliant blue. The various protein-spots were cut out and were digested with trypsin. A peptide mass fingerprint or a peptide ladder were then generated with MALDI-TOF or MALDI-QTOF respectively. After database searching, the identified proteins were marked on the 2D-electrophoresis gel in order to obtain the final reference map.

interface between sample and 6% Percoll; a second ring was seen at the interface between 6% Percoll and 17% Metrizamide; and a third fraction lay at the interface between 17 % and 35 %


[page 37↓]

Fig. 4-3: Comparison of the 2D-electrophoresis maps of lymphoblastoid cells and of isolated mitochondria . Sub-cellular fractionation can intensify low abundant proteins and let them become visible. (A) depicts the map of lymphoblastoid cells, (B) depicts the map of the mitochondrial subfaction. The red arrows point to some intensified protein spots on the mitochondrial map. The green arrows point to the corresponding spots in the lymphoblastoid map.

Metrizamide. I investigated the pellet of each of the three fractions by electron microscopy (Fig. 3-2). The material of the first fraction contained mainly membranes from both cell and [page 38↓]subcellular organelles. In the second fraction I found a few lysosomes and some other material which was difficult to identify. Only the third fraction was highly enriched in mitochondria. I only used the pellet from the third fraction. The high purity of the mitochondrial fraction was confirmed by the subsequent experiments. Protein analysis by MALDI-TOF mass spectrometry identified only few non-mitochondrial proteins.

4.2. Preparation of protein samples

The protein samples of the mitochondria were prepared according to a protocol by Klose (1999a) (see chapter 3.3). The sample was treated with high concentrations of urea and with a detergent (CHAPS) to solubilize the membrane proteins. The protein concentration of the final sample was measured by using the BCA protein assay method (see chapter 3.4).Fig. 4-4 gives an example of the result of the protein concentration measurement. The concentration of the protein samples of each experiment was around 8-10 mg/ml.

Fig. 4-4: Analysis of the protein concentration with the BCA protein assay. The result has to be multiplied by ten (highlighted with red), since the sample had been diluted 10 times before the measurement.

4.3. 2D-electrophoresis of mitochondrial proteins

4.3.1. The pH-gradient of the IEF-gel

The pH-gradient of the IEF-gel was measured as described above (Section 3.5.4.3). The results are shown in Fig. 4-5A. The measured spots tend to lie on a straight regression line (y [pH]= -0.1088x [length of the gel] + 8.8965, starting from the basic end, with a dispersion of R2=0.939 (p<0.0001)). The gradient is linear between the pH-values of 5-9. The effect of the protein sample on the pH-gradient was also investigated (Fig. 4-5B). Despite the fact that the pH-gradient of the protein containing gel fluctuated more than that of the empty gel, the linear regression lines were nearly similar (y = -0.098x + 8.85, R2=0.922 (p<0.0001)). The regression lines, the best fit and the variance were caluclated with the statistic software package “StatView version 4.5.”

4.3.2. Two-dimensional electrophoresis

A total of about 500 µg protein sample was loaded on the anodic end of the isoelectric focussing gel for a thick (1.5 mm) Coomassie stained large gel. In contrast, only one tenth of the protein sample was required for a thin (0.9 mm) silver stained gel. A broad pH-range am[page 39↓]pholine mixture of pH 2-11 was used for isoelectric focussing in order to get a “panorama view” of the mitochondrial proteins. The isoelectric point of the most acidic spot detectable was at pH 5.00 and that of the most basic one at pH 9.75.

Fig. 4-5A-B : pH-gradient of 40 cm IEF gels . The pH-measurement was performed as described in section 3.5.4.3. The results of the empty control gel are depicted in (A). The measured spots tend to lie on a regression line (y = -0.1088x+8.8965, R2 = 0.939 (p<0.0001)). The gradient is linear between the pH-values of 5-9. The effect of the protein sample on the pH-gradient is shown in (B). Although the pH-gradient of the protein containing gel fluctuated more than that of the empty gel, the linear regression lines are similar (y= -0.098x+8.85, R2 = 0.922 (p<0.0001)).

The second dimension gel (SDS-PAGE) was run with a molecular weight standard. The molecular weight range of the standard marker lay between 17.5 and 76 kDa. The reproducibility of the 2D-electrophoresis runs was demonstrated by analyzing an identical sample on four different gels. Most spots were reproduced on each gel except in the case if the extreme ends of the first dimension gels were accidentally lost in the process of expulsion from the glass tube. In Fig. 4-6 a comparison of the same region of the four different gels is shown.

4.3.3. Gel staining

The difference between silver stain and Coomassie stain is that the silver stain is more sensitive but the Coomassie stain has less influence on the proteins. The silver stained gel may show more spots, but for the purpose of protein identification, the Coomassie stain is more favorite since it does not modify the proteins covalently. The silver stain requires only one tenth the amount of protein sample compared to the Coomassie stain. Fig. 4-7 shows the comparison of two gels stained by these two methods. A total of 420 spots could be separated and detected on a silver stained gel from only 50 µg protein sample. In comparison, less than 10 spots were detected on the same gel stained with Coomassie. However, the subsequent protein analysis by MALDI-TOF mass spectrometry showed the drawback of the silver stain. For protein identification a total of 184 spots were excised from two gels stained with colloidal Coomassie brilliant blue. I could identify 115 spots by peptide mass fingerprinting via MALDI-TOF mass spectrometry. The rest of the Coomassie gel was then destained and subsequently restained with silver. The spots now appeared to be much darker than in the preceding Coomassie stain. Additional 300 spots that had not been excised before from the Coomassie-gels were cut out. After reduction of the silver with iodine salt and DTT and subsequent trypsin digestion these spots were also subjected to MALDI-TOF mass spectrometry. In contrast to the spots from the Coomassie gels I could not identify a single spot from the silver stained gel.


[page 40↓]

Fig.4-6: These four corresponding gel-sections demonstrate the reproducibility of 2D-electrophoresis . All four gels were stained with silver.

Fig. 4-7: These two gel-sections demonstrate the difference in sensitivity between the silver (A) and the Coomassie G-250-stain (B). Both gels have been run under the same conditions. A total of 525 µg mitochondrial protein were loaded on gel B. In contrast, only 47 µg protein were loaded on gel A. Moreover, it achieved a higher resolution than gel B.


[page 41↓]

4.3.4.  Influence of repeated freezing-thawing cycle on sample quanlity

In order to test the influence of repeated freeze-thaw cycles on sample quality, the same sample was run after the first thawing (gel A) and additionaly after refreezing and rethawing (gel B). Although the amount of sample loaded on gel B was 2 µl more than that of gel A many spots on gel B are much fainter than the corresponding ones on gel A. This experiment de­mon­strates the loss of proteins during the procedure of repeated freeze-thaw cycles.

Fig. 4-8: The effect of repeated freeze-thaw cycles on sample quality : Comparison of the two gels that have been prepared from frozen stocks. Gel A used the sample that was run on the 2D-gel after the first time thawing. Gel B used the sample that was run on the 2D-gel after an additional round of freezing and thawing. Although the amount of loaded sample of gel B was 2 µl more than that of gel A, many spots on gel B are much fainter than the corresponding ones on gel A.

4.4. Protein identification

4.4.1. MALDI-TOF and MALDI-QTOF tandem mass spectrometry

The proteins were identified by MALDI-TOF mass spectrometry on the basis of their peptide mass fingerprints, and by MALDI-QTOF tandem mass spectrometry on the basis of their peptide fragment ladders. A total of 184 spots was excised from two large Coomassie-stained 2D-electrophoresis gels. Following in-gel digestion with trypsin, the peptide mixture of each protein was analyzed by MALDI-TOF mass spectrometry (see sections 3.7-3.10).


[page 42↓]

Fig. 4-9: The mass spectrum of spot 3. A total of 60 peptides were detected by MALDI-TOF analysis. Using the Mascot search engine, 28 peptides labelled with a green dot ( ) could be matched to the theoretical trypsin digest of the protein (ATP-synthese beta-chain, mitochondrial precursor ). The parameters for this search were one missed cleavage, possible methionine oxidation and 0.1 Da mass tolerance. All methionine containing peptides and their oxidized derivatives are grouped with the red M (M ). The presence of double-peaks separated by 16 Da confirms the presence of methionine residues in the respective peptide fragment. One fragment resulting form trypsin self-digestion is marked with pink color (trypsin ). When less stringent criteria were used, some more peptides could be matched. If the mass tolerance is increased to 0.5 Da, additional fragments can be matched ( ). If the number of possible missed cleavages is increased to four, an additional large fragment can be matched ( ).

Theoretically, all the measured peptide masses should match the corresponding “in silico” digested fragments of a certain protein in the database. However, in the experimental setting the highest percentage of matched peptide masses in my study was 76%. The average percentage of matched peptide masses was 44% and that of the covered sequence was 33%. In order to confirm these primary results, verification of each spot was carried out “by hand”. Fig. 4-9 shows an example using the mass spectrum information of spot 3. Each measured peptide mass was compared to the “in silico” digested ones. The matched peptides were highlighted and marked with a green dot. The methionine containing peptides and their corresponding oxidized derivatives (oxidation of every methionine leads to a mass increase of 16 Da), which strongly confirmed the identity of the peptide, were marked as a group. The peptide masses of the self-digested trypsin fragments were marked as well. Sometimes, in order to match more peptides, less stringent criteria were used. These peptides (e.g. with one or two missed cleavages) were also highlighted. If the mass spectrum was not good enough to secure a protein identity, the amino acid sequence of a single fragment was determined by MALDI-QTOF tandem mass spectrometry. The sequence information could then be used as sequence tag to find the protein in the database.


[page 43↓]

Fig. 4-10A: The mass spectrum of spot 30 measured with MALDI-TOF mass spectrometry. Only 6 peptides were detected including 3 peptides of the self-digested trypsin.

Fig. 4-10B: The mass spectrum of spot 30 after desalting with nano-scale reversed-phase chromatography. A total of 22 peptides was now detected by MALDI-TOF mass spectrometry. One isolated peptide was selected out for further MALDI-QTOF measurement (highlighted with a red arrow). Peptide ladder sequencing had to be performed because this spectrum was still not good enough to identify the protein.

In Fig. 4-10A-D an example of this “manual” process is shown using the mass spectrum information of spot 30. The mass spectrum of spot 30 was not satisfactory when analyzed by MALDI-TOF mass spectrometry. Only six peptides were detected including three peptides generated by trypsin self-digestion. Sometimes salts from the buffer interfere with the MALDI-TOF mass spectrum. Therefore the peptides were first desalted by reversed-phase chromatography (see section 2.4.2). After desalting, a total of 22 peptides of spot 30 could then be detected with MALDI-TOF mass spectrometry. However, since the spectrum


[page 44↓]

Fig. 4-10C : The mass spectrum of the 1168.67 Da peptide of spot 30 using MALDI-QTOF tandem mass spectrometry. These fragments are used to generate the protein sequence tag by GPMAW32-software on the internet (see following figure).

Fig. 4-10D: The sequence result of the isolated 1168.67 Da peptide of spot 30. The peptide fragment ladders were obtained by the MALDI-QTOF mass spectrometry. This sequence tag was later used for database searching in order to identify the protein.

of 22 peptide fragments was still not good enough to identify the protein, MALDI-QTOF analysis had to be carried out on one isolated peptide. The sequence result of the isolated polypeptide of spot 30 led to the identification of the 13 kDa subunit of complex-I. I identified a total of 115 protein spots that corresponded to 95 different proteins. Most of them were identified by MALDI-TOF mass spectrometry, only five spots needed to be analyzed with MALDI-QTOF tandem mass spectrometry. In Fig. 4-11 the strategy of protein identification is illustrated.


[page 45↓]

Fig. 4-11 : Strategy of protein identification.

4.4.2. Database search for protein identification

I used several search engines on the internet including Mascot, ProFound, and MS-Fit. These programs match the peptide masses from a protein spot with the “in silico” digested peptide masses of all known human proteins in the National Centre for Biotechnology Information non-redundant (NCBInr) protein database. At least five or more matching peptides were re[page 46↓]quired for a secure identity assignment. With the Mascot search engine, most samples could be identified satisfactorily with a significant probability score (p<0.05). However, several spectra had to be handled with other search engines like ProFound and MS-Fit. A total of eight spots could be identified additionally that way. As mentioned above there were still five spots that could only be identified by their peptide sequence information gained from MALDI-QTOF tandem mass spectrometry. Finally I identifie 115 spots, which are listed in Tab. 7-1.The theoretical and the experimental molecular weights (MW) and isoelectric points (pI) of the identified proteins, their corresponding SWISS-PROT accession numbers are also listed, along with the data from the mass spectrometric analysis; i.e. the numbers of matching peptides, the sequence coverage (in percent) and the probability of assignment of a random identity.

4.5. Mitochondrial proteome reference map

4.5.1. Mitochondrial proteome reference map

Fig.4-12shows a representative mitochondrial proteome map from human lymphoblastoid cells. All of the identified spots are highlighted in red with the corresponding spot number near them. The pI and MW are shown as well. The detailed information of the proteins is listed in Tab. 7-1 and also labelled directly in the 12 sectors of the reference map (see supplementary meterial: Fig. 4-12#1 to 4-12#12). A total of 184 spots from two Coomassie stained gels (corresponding to 141 different spots) resulted in the identification of 115 spots (corresponding to 95 different proteins). Out of the 400 visible protein spots on the silver gel I thus could identify 20%. Among the 95 identifed proteins, 77% (n=74) were annotated according to NCBI and SWISS-PROT databases, as mitochondrial proteins. Although our mitochondrial fraction was highly purified, it still contained some proteins from other subcellular organelles. In our sample, 15 spots corresponding to 11 different gene-products belong to subcellular components other than the mitochondria. The location of them is indicated below. There are ten proteins remaining whose functions and/or localization are unknown. For four of them (spots 46, 73, 129, 131) were only found as ESTs in the databases. All of the identified spots are grouped in Tab. 4-1 according to their location and function. The 25 spots that did not give a result on MALDI-TOF mass spectrometry were very weak spots that contained too little protein to produce a satisfactory spectrum.

4.5.2. Locations of the identified proteins

Based on the annotation in the NCBI and SWISS-PROT databases, a total of 74 identified proteins out of 90 spots were annotated as “mitochondria-associated” proteins. 27 of these proteins are located in the mitochondrial matrix, 16 in the mitochondrial inner membrane, 2 in the mitochondrial outer membrane, and 2 in the mitochondrial inter-membrane space. The remaining 27 proteins are surely mitochondrial proteins, however, their exact subcellular location is not clear. A total of eleven proteins out of 15 spots are located elsewhere. Seven of them are located in the cytoplasm, three at the endoplasmic reticulum, and one is known as B-cell-associated protein. The subcellular location of ten proteins is unknown. These data are listed in detail in Fig. 4-13 and in Tab. 4-2.

4.5.3. Functions of the identified mitochondrial proteins

According to the annotation in SWISS-PROT and to the classification system of MITOP, I sorted the identified mitochondrial proteins. The sorting is based on the protein function and is summarized in Tab. 4-1. Most of these proteins (59 proteins) are part of central metabolic [page 47↓]pathways, including the citric acid cycle, the pyruvate dehydrogenase complex, the respiratory chain, the β-oxidation, protein assembly or catabolism (urea cycle). Another group of proteins are transport proteins (seven proteins). Proteins that have a role in cell protection or apoptosis or heme-biosynthesis or cell maintenance, are grouped in “other functions” (eight proteins). The function of only one protein, the ES1 protein homologue mitochondrial percursor, is not yet characterized. However, this protein is assumed to play an important role, since a homologous protein has been identified in the zebra fish (Danio rerio) and in Escherichia coli. One of the most important functions of the mitochondrion is the oxidative phosphorylation at the respiratory chain. 16 of the identified proteins are part of its five protein complexes. Seven proteins belong to complex I, three proteins belong to complex III, two to complex IV, and four proteins are subunits of complex V. I detected no protein subunit of complex II.

4.5.4. Identified membrane proteins

In order to identify putative transmembrane proteins, I analyzed all of the identified proteins with a transmembrane prediction software (SOSUI), which predicts the transmembrane helices by calculating the hydrophobicity, the amino acid charges and the sequence length of a candidate peptide. Although about one third of the identified proteins are membrane-associated proteins located either at the inner membrane (27 proteins) or at the outer membrane (two proteins) of the mitochondrion, only four of them are really transmembrane proteins with one or two transmembrane helices. These are spot 13 (GTP-specific succinyl-CoA synthetase beta-subunit), spot 75 (NADH-ubiquinone oxidoreductase (complex I) B16.6 kDa subunit), spot 90 (isocitrate dehydrogenase, gamma-subunit), and spot 134 (sideroflexin 1). The location of these spots is highlighted in Fig. 4-14.

4.5.5. Multiple spot proteins

A total of 17 proteins on the reference map could be detected in more than one spot (Fig. 4-15). The majority of the multiple spot proteins are most probably isoforms, such as spots 9 and 10 (HSP 60 kDa), spots 11 and 12 (actin-beta), spots 34 and 118 (succinyl CoA: 3-oxoacid CoA transferase), spots 36 and 47 (ATP synthase, H+ transporting F1), spots 37, 123 and 124 (medium-chain acyl CoA isomerase), spots 38 and 39 (similar to delta 3,5-delta 2,4-deinoyl-CoA isomerase), spots 40 and 128 (electron-transfer-flavoprotein, alpha subunit), spots 48 and 49 (glutamate dehydrogenase 1), spots 50 and 51 (Tu translation elongation factor), spots 52 and 67 (acetoacetyl-CoA thiolase), spots 53 and 69 (voltage-dependent anion channel 1), spots 63 and 66 (complex III subunit II), spots 81 and 82 (isocitrate dehydrogenase), spots 85 and 87 (malate dehydrogenase 2), and spots 88 and 89 (glyceraldehyde-3-phosphate dehydrogenase). Spots 5 and 31 (complex IV, subunit Va) correspond to same gene product but have markedly different pI and MW. Similar to that, spot 114, which was identified by sequence information, corresponds to the same gene product as spots 11 and 12 (actin-beta). Spot 26 was at first identified as an unkown protein for (MGC:9832). After sequence alignment of the amino acids with the help of the protein-BLAST program (see list of internet sites), it was finally verified to be a short isoform of actin-beta.

4.5.6. Comparison of theoretical and the experimental pI and MW

The theoretical pI and MW of proteins shown in my protein list were calculated with the “Compute pI/MW” tool of the SWISS-PROT database. This tool calculates the pI of a protein [page 48↓]by calculating the mean of the pK values of its amino acids as described by Bjellqvist et al. (1993). The MW of a protein was calculated as the sum of the average isotopic masses of

Tab. 4-1 : Sorting of the identified proteins according to their subcellular locations and functions. Proteins with transmembrane domains predicted by the SOSUI-algorithm are listed and highlighted separately.

function

spot #

number of spots

number of proteins

mitochondrial

 

90

74

respiratory chain

 

19

16

complex I

[6] [29] [30] [75] [126] [137] [138]

7

7

complex II

no

  

complex III

[25] [55] [63]/[66]

4

3

complex IV

[5]/[31] [56]

3

2

complex V

[3] [18] [36]/[47] [106]

5

4

fat metabolism

 

19

13

ß-oxidation

[28] [37]/[123]/[124] [38]/[39] [40]/[128] [52]/[67] [54] [65] [79] [92] [133]

15

10

other proteins for fat metabolism

[34]/[118] [45] [64]

4

3

nucleotide metabolism

 

5

5

 

[61] [74] [93] [135] [130]

5

5

protein metabolism

 

14

12

 

[20] [35] [43] [48]/[49] [50]/[51] [84] [91] [119] [122] [125] [127] [140]

14

12

carbohydrate metabolism

   
 

[13] [15] [24] [27] [32] [60] [62] [80] [81]/[82] [83] [85]/[87] [90]

14

12

transport proteins

 

10

7

HSP

[7] [9]/[10] [33] [58]

5

4

VDAC

[53]/[69]

2

1

TOM

[67/2]

1

1

TIM

[21]/[22]

2

1

other functions

 

9

9

cell protection

[41] [72] [121]

3

3

heme biosynthesis

[68]

1

1

apoptosis

[44]

1

1

sulfide oxidation

[132]

1

1

iron transport

[134]

1

1

maintenance and cell growth

[70]

1

1

unknown

[71]

1

1

    

other compartments

 

15

11

cytoplasmic

[11]/[12]/[26]/[114] [16] [86] [88]/[89] [96] [101] [120]

11

7

endoplasmic reticulum

[1] [23] [42]

3

3

B-cell-specific

[103]

1

1

    

location unknown

 

10

10

function unknown

[2] [46] [73] [105] [129] [131] [136]

7

7

nucleotide metabolism

[117]

1

1

HSP

[8] [14]

2

2

    

Total

 

115

95

membrane proteins

[13] [75 ] [90] [134 ]

4

4


[page 49↓]

Fig. 4-13 : Classification of the identified proteins according to their location. The mitochondrial group (M) includes those proteins that are definitively mitochondrial but for which no further information exists on their exact location.

Tab. 4-2: Classification of the identified proteins according to their location. The mitochondrial group (M) includes those proteins that are definitively mitochondrial but for which no further information exists on their exact location.

location

spot number

protein number

percent

mitochondrial total

90

74

77%

M: mitochondria

31

27

28%

MM: mitochondrial matrix

36

27

28%

MIM: mitochondrial inner membrane

18

16

17%

MOM: mitochondrial outer membrane

3

2

2%

MIMS: mitochondrial intermembrane space

2

2

2%

    

other compartments

15

11

12%

C: cytoplasmic

11

7

8%

ERL: endoplasmic reticulum lumen

3

3

3%

cell: cell associated proteins

1

1

1%

    

location unknown

10

10

11%

    

Total

115

95

100%

amino acids in the protein and the average isotopic mass of one water molecule.


[page 50↓]

Fig. 4-12: The reference map of the mitochondrial proteome from human lymphoblastoid cells . All the measured spots are highlighted (red : mitochondrial proteins; green : other cytoplasmic proteins; yellow : proteins which locations remain unknown). The corresponding spot numbers are shown at the spots. The MW and pI ranges of the whole gel are shown at the left and above the gel.


[page 51↓]

Fig. 4-14: Identified mitochondrial membrane proteins . Among the 74 identified mitochondrial proteins, only four carry transmembrane domains. One spot (#75) belongs to complex I of the respiratory chain, other two spots (#13 and #90) function in the citrate acid cycle and the fourth spot (#134) is an iron transport protein.


[page 52↓]

Fig. 4-15: multiple spots proteins. A total of 17 proteins on the reference map could be detected in more than one spot. Most of the multiple spot proteins are probably isoforms of the same proteins. However, spots 5 and 31 (complex IV, subunit Va) correspond to the same gene but show a marked difference in pI and MW. Similar to that, spot 114 that was identified by sequence information, corresponds to the same gene as spots 11 and 12 (actin-beta). Spot 26, which originally corresponded to a unknown protein, was finally identified to be a short isoform of actin-beta.


[page 53↓]

The experimental pI of each protein was calculated by using the regression formula that I had obtained from the measurement of the pH gradient (see section 4.3.1). Similar to that, the experimental MW was calculated with the regression formula derived from a series of MW standard markers. The correlation between the experimental and the theoretical pI and MW values was analyzed with the ANOVA statistics software (Fig. 4-16). The correlation between the coupled pI or MW values was tested by the coupled t-test. The correlation coefficient (R) of the coupled pI values is equal to 0.873 (p<0.0001) and that of the MW values is equal to 0.941 (p<0.0001). The absolute differences between theoretical and experimental pI values are between 0.01 and 1.81 pH values. (median = 0.69 pH values, interquartile distance = 0.35 pH values). Similar to that, the absolute differences between theoretical and experimental MW values are between 0.08 to 43.09 kDa (median = 3.48 kDa, interquartile distance = 3.76 kDa).

Fig. 4-16 : Comparison of the theoretical and experimental pI and MW of the identified proteins. The left regression diagram depicts the relation of the pI values between the measured values (m) and the theoretical calculations (S). Similar to that, the right regression diagram depicts the relation between the two different MW values. The linear regression formulas and the lateral dispersion (R2) are depicted below the diagrams.


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