Supporting Information for Proteomics DOI 10.1002/Pmic.200402059

Supporting Information for Proteomics DOI 10.1002/pmic.200402059

Cornelia Czupalla, Hannu Mansukoski, Theresia Pursche, Eberhard Krause and Bernard Hoflack

Comparative study of protein and mRNA expression during osteoclastogenesis

ª 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de Supplementary Table 1. Summary of proteins with increased abundance in osteoclasts

identified by MS

Spot NCBI Protein Mascot Sequence Predicted Change in

No. gene scorea) coverage expressionb)

identifier (%) Mr pI Protein mRNA

Lysosomal

1-04 20892559 vacuolar ATPase A subunit 189 32 68283 5.42 4.00 ↑

1-05 1184661 vacuolar ATPase B2 subunit 217 32 56549 5.57 5.46 ↑↑

1-08 1184661 vacuolar ATPase B2 subunit 253 36 56549 5.57 3.57 ↑↑

1-23 309202 cathepsin B 64 21 37340 5.46 2.00 -

1-27 29150255 vacuolar ATPase D2 subunit 173 41 40433 5.14 68.99 ↑↑

c) 1-28 29150255 vacuolar ATPase D2 subunit 500 22 (12 ) 40433 5.14 15.72 ↑↑

1-30 6753556 cathepsin D 62 19 44925 6.71 2.33 -

3-12 6754186 hexosaminidase B 80 14 61077 8.30 2.00 ↑

5-06 13097342 vacuolar ATPase E1/E2 128 60 26141 8.44 5.41 ↑↑

subunit

5-12 45504359 vacuolar ATPase E1 subunit 189 61 26141 8.44 3.03 ↑↑

5-25 31982433 cathepsin K 88 26 36865 8.61 3.97 ↑↑

Metabolism/Energy Generation

1-02 31980648 mitochondrial ATPase β 156 34 56265 5.19 2.81 -

subunit

1-06 10946574 creatin kinase 201 49 42686 5.40 6.44 ↑↑

1-07 38084969 succinate CoA ligase β 166 32 46811 6.58 2.69 -

subunit

1-09 46593021 ubiquinol cytochrome c 182 31 52818 5.81 3.35 ↑

reductase core protein 1

1-10 18250284 isocitrate dehydrogenase 3α 158 32 39613 6.27 3.27 ↑↑

1-11 10946574 creatin kinase 226 42 42686 5.40 3.45 ↑↑

1-12 38084969 succinate CoA ligase β 150 29 46811 6.58 2.23 - subunit

1-13 18152793 pyruvate dehydrogenase β 124 35 38912 6.41 2.37 ↑

1-15 20839603 NADH dehydrogenase Fe-S 96 31 30131 6.67 2.94 ↑

protein 3

1-16 20913657 mitochondrial ATPase δ 181 67 18738 5.52 2.92 ↑

subunit

1-17 22164792 mitochondrial ribosomal 116 39 21695 9.34 3.55 ↑

protein L12

1-19 38082750 NADH dehydrogenase flavo- 103 31 27139 7.63 3.14 ↑

protein 2

1-20 13124347 metaxin 2 283 17 (8c)) 29739 5.44 2.00 -

1-21 46195430 NADH dehydrogenase Fe-S 115 31 24023 5.89 3.38 -

protein 8

1-24 6680690 peroxiredoxin 3 106 24 28109 7.15 3.41 -

1-25 6680690 peroxiredoxin 3 72 17 28109 7.15 2.48 -

1-26 18266680 3-oxoacid CoA transferase 1 306 10 (11c)) 55953 8.73 3.99 -

1-32 2506545 78 kDa glucose-regulated 312 40 72377 5.07 2.45 ↓↓

protein

1-33 2506545 78 kDa glucose-regulated 208 31 72377 5.07 2.19 ↓↓

protein

1-35 3219998 Hsp60 112 18 60917 5.91 2.07 -

1-36 21704020 NADH dehydrogenase Fe-S 192 23 79698 5.51 2.57 ↑

protein 1

1-37 21704020 NADH dehydrogenase Fe-S 136 23 79698 5.51 2.92 ↑

protein 1

1-42 13879408 Hsp75 239 33 80027 6.25 2.25 ↑

1-43 29427692 mitofilin 668 31 (15c)) 83848 6.18 2.03 ↑

1-44 20071885 dihydrolipoamide S-acetyl- 446 16 (11c)) 67899 8.81 3.62 ↑

transferase

1-50 3123245 NADH-ubiquinone 71 90 (4c)) 1099 6.75 2.45 ↑

oxidoreductase 24 kDa subunit (fragment)

3-01 6680690 peroxiredoxin 3 104 19 28109 7.15 2.81 -

3-03 21704140 3-hydroxyisobutyrate 75 40 35440 8.40 3.68 ↑

dehydrogenase

3-05 21729786 hydroxyacyl glutathione 76 47 28883 6.50 3.86 ↑↑

hydrolase

3-07 33859640 transaldolase 1 220 47 37363 6.57 2.55 -

3-08 31982522 acyl-CoA dehydrogenase, 171 31 44861 8.68 4.03 ↑

short chain

3-10 27370092 EF-Tu 220 43 49477 7.23 2.85 ↑

3-11 6679651 enolase 3 β 214 22 46995 6.73 4.73 ↑↑

3-12 6680748 95 23 59716 8.29 2.00 mitochondrial ATPase α1 ↑

subunit

3-13 18202835 leucine aminopeptidase 64 22 52751 6.60 3.95 -

3-19 15030102 succinate dehydrogenase Fp 206 35 72539 7.11 2.83 ↑↑

subunit

3-20 15030102 succinate dehydrogenase Fp 200 31 72539 7.11 2.49 ↑↑

subunit

3-21 29427692 mitofilin 260 33 83848 6.18 2.31 ↑

3-22 29427692 mitofilin 92 14 83848 6.18 2.00 ↑

3-25 15030102 succinate dehydrogenase Fp 222 55 72539 7.11 2.00 ↑↑

subunit

3-29 33585932 dihydrolipoamide branched 147 20 53227 8.78 2.54 -

chain trans-acylase E2

3-30 23346461 NADH dehydrogenase Fe-S 123 21 52592 6.52 2.45 ↑

protein 2

3-32 29427692 mitofilin 167 39 83848 6.18 4.17 ↑

3-33 29427692 mitofilin 70 11 83848 6.18 2.21 ↑

5-01 31980762 superoxide dismutase 2 91 29 24588 8.80 3.14 ↑

5-02 34328230 adenylate kinase 2 131 43 25589 6.97 4.09 ↑↑

5-03 13385168 ubiquinol-cytochrome c 79 17 29349 8.91 3.05 ↑ reductase, Rieske iron-sulfur

polypeptide 1

5-07 26368552 NADH-ubiquinone 91 40 37995 8.70 2.13 ↑

dehydrogenase 1α

subcomplex assembly factor

5-08 33859640 transaldolase 1 240 48 37363 6.57 2.79 -

5-09 12805413 enoyl CoA hydratase, short 82 34 31237 8.76 2.46 -

chain 1

5-10 33859640 transaldolase 1 195 45 37363 6.57 2.13 -

5-11 21759114 electron transfer flavoprotein 199 62 27293 8.57 2.02 -

β subunit

5-14 26353108 64 21 30237 9.06 2.14 mitochondrial ATPase γ1 ↑

subunit

5-15 31982186 malate dehydrogenase 2 270 55 35589 8.93 2.02 ↑

5-16 31982186 malate dehydrogenase 2 262 55 35589 8.93 2.49 ↑

5-17 18700024 isocitrate dehydrogenase 3β 61 17 42168 8.76 2.80 ↑

5-18 13385942 citrate synthase 113 12 51703 8.72 2.05 ↑

5-19 33859554 fumarate hydratase 1 187 31 54336 9.12 2.65 ↑

5-20 6680618 acetyl-CoA dehydrogenase, 103 18 46452 8.60 2.67 ↑

medium chain

5-21 6680748 184 23 59716 9.22 2.52 - mitochondrial ATPase α1

subunit

5-22 21450129 acetyl-CoA acetyltransferase 159 19 44787 8.71 2.01 -

1 precursor

5-23 13385942 citrate synthase 78 12 51703 8.72 2.00 ↑

5-24 20810027 acetyl-CoA acyltransferase 2 71 16 41831 8.33 2.01 ↑↑

5-26 22267442 ubiquinol-cytochrome c 172 33 48205 9.26 4.74 ↑

reductase core protein 2

5-27 20810027 acetyl-CoA acyltransferase 2 207 39 41831 8.33 5.21 ↑↑ 5-28 21312298 serine hydroxymethyl 71 12 55724 8.73 2.33 ↓

transferase 2

5-29 6680748 116 17 59716 9.22 2.00 - mitochondrial ATPase α1

subunit

5-30 6680748 121 17 59716 9.22 2.44 - mitochondrial ATPase α1

subunit

5-32 13435984 serine hydroxymethyl 133 22 55726 8.72 2.58 ↓

transferase 2

6680748 90 12 59716 9.22 mitochondrial ATPase α1 -

subunit

5-34 23956084 acyl-CoA dehydrogenase, 197 28 70831 8.91 2.00 ↑

very long chain

5-35 34328415 aldehyde dehydrogenase 65 21 61772 8.58 2.01 ↑

5-36 18079339 aconitase 2 128 23 85410 8.08 3.76 ↑↑

5-37 18079339 aconitase 2 86 17 85410 8.08 5.24 ↑↑

5-38 18079339 aconitase 2 126 23 85410 8.08 4.20 ↑↑

5-39 18079339 aconitase 2 121 23 85410 8.08 4.95 ↑↑

5-40 18079339 aconitase 2 97 17 85410 8.08 4.57 ↑↑

5-41 18079339 aconitase 2 122 23 85410 8.08 4.64 ↑↑

5-42 18079339 aconitase 2 64 12 85410 8.08 4.67 ↑↑

5-43 18079339 aconitase 2 64 12 85410 8.08 4.77 ↑↑

5-44 18079339 aconitase 2 67 12 85410 8.08 3.82 ↑↑

5-45 18079339 aconitase 2 64 12 85410 8.08 3.60 ↑↑

5-48 18266680 3-oxoacid CoA transferase 1 107 17 55953 8.73 2.00 -

5-50 18266680 3-oxoacid CoA transferase 1 81 22 55953 8.73 2.12 -

5-51 18266680 3-oxoacid CoA transferase 1 64 12 55953 8.73 2.08 -

5-54 31982856 dihydrolipoamide 132 22 54238 7.99 2.11 -

dehydrogenase

5-55 31982522 acyl-CoA dehydrogenase, 184 40 44861 8.68 2.08 ↑

short chain 5-56 33859554 fumarate hydratase 1 149 19 54336 9.12 2.47 ↑

5-57 33859554 fumarate hydratase 1 69 22 54336 9.12 2.37 ↑

5-58 6680748 141 23 59716 9.22 2.47 - mitochondrial ATPase α1

subunit

5-59 13385942 citrate synthase 81 17 51703 8.72 2.00 ↑

5-60 18700024 isocitrate dehydrogenase 3β 86 17 42168 8.76 2.00 ↑

Cytoskeleton

1-01 49868 β-actin/γ-actin 212 44 39161 5.78 2.00 -

809561 207 44 40992 5.56

1-12 809561 γ-actin 79 24 40992 5.56 2.23 -

1-18 12847411 actin related protein 2/3 169 52 16278 5.47 2.36 -

complex subunit 5

1-29 49868 β-actin 241 15 (6c)) 39161 5.78 2.12 -

1-31 202368 vimentin 227 50 53641 5.06 2.04 -

1-34 202368 vimentin 158 30 53641 5.06 6.90 -

1-38 49868 β-actin/γ-actin 184 39 39161 5.78 2.09 -

809561 181 40 40992 5.56

1-40 90508 gelsolin 337 6 (7c)) 80827 5.53 3.30 ↑

1-41 90508 gelsolin 609 15 (13c)) 80827 5.53 4.09 ↑

1-45 21704120 T-plastin 567 17 (13c)) 70697 5.42 2.64 -

1-46 21704120 T-plastin 297 9 (5c)) 70697 5.42 2.07 -

3-09 29126784 actin related protein 2 124 31 44601 6.31 2.08 -

5-04 23621467 actin related protein 2/3 87 21 344336 6.84 3.22 -

complex subunit 2

Nuclear

1-03 6679108 nucleophosmin 1 116 28 32540 4.62 9.14 -

1-39 128843 nucleolin C-23 666 17 (16c)) 76677 4.69 3.34 -

1-51 9845253 heterogeneous nuclear 192 10 (4c)) 49248 5.89 2.82 -

ribonucleoprotein H2 3-14 91032 lamin A/C 173 28 74140 6.05 2.44 ↑d)

346883 190 30 65367 6.41

3-15 91032 lamin A/C 110 21 74140 6.05 3.31 ↑d)

346883 125 25 65367 6.41

3-16 91032 lamin A/C 172 30 74140 6.05 3.52 ↑d)

346883 160 19 65367 6.41

3-17 91032 lamin A/C 267 38 74140 6.05 4.55 ↑d)

346883 202 24 65367 6.41

3-18 91032 lamin A/C 250 41 74140 6.05 3.83 ↑d)

346883 272 31 65367 6.41

3-23 91032 lamin A/C 177 38 74140 6.05 3.49 ↑d)

346883 179 27 65367 6.41

3-24 91032 lamin A/C 127 25 74140 6.05 4.79 ↑d)

346883 138 19 65367 6.41

3-26 91032 lamin A/C 254 38 74140 6.05 2.61 ↑d)

346883 272 24 65367 6.41

3-27 91032 lamin A/C 149 24 74140 6.05 2.87 ↑d)

346883 160 20 65367 6.41

3-28 91032 lamin A/C 154 31 74140 6.05 3.21 ↑d)

346883 164 19 65367 6.41

5-46 91032 lamin A/C 78 18 74140 6.05 3.30 ↑d)

346883 89 21 65367 6.41

5-47 91032 lamin A/C 209 33 74140 6.05 3.04 ↑d)

346883 208 30 65367 6.41

5-49 91032 lamin A/C 233 34 74140 6.05 2.01 ↑d)

346883 249 27 65367 6.41

5-52 91032 lamin A/C 199 32 74140 6.05 2.01 ↑d)

346883 216 27 65367 6.41

5-53 91032 lamin A/C 253 41 74140 6.05 2.18 ↑d)

346883 275 33 65367 6.41

Signal Transduction 1-47 7305075 Ras-GAP SH3-domain 163 7 (5c)) 51797 5.41 3.97 ↑

binding protein

1-48 6755238 non-receptor protein tyrosine 406 20 (11c)) 49561 5.78 2.33 ↑↑

phosphatase 1

3-31 6755238 non-receptor protein tyrosine 65 37 49561 5.78 3.03 ↑↑

phosphatase 1

Other

1-14 7304963 chloride intracellular channel 207 60 28711 5.44 2.28 ↑↑

1-22 31981086 EF hand domain containing 191 52 26784 5.07 3.73 -

2/swiprosin 1

1-49 20833776 RIKEN cDNA 2610034 B18 408 32 (9c)) 25178 5.09 3.65 -

3-02 19526463 endoplasmic reticulum 104 33 28805 5.90 2.00 -

protein 29

3-04 27923993 60S acidic ribosomal protein 140 66 34165 5.91 2.04 -

3-06 6755965 voltage-dependent anion 78 46 31713 7.44 2.50 -

channel 2

5-05 6755965 voltage-dependent anion 136 25 31713 7.44 2.14 -

channel 2

5-13 6755963 voltage-dependent anion 78 20 30737 8.62 2.09 -

channel 1

5-31 33416534 interferon-activated gene 64 17 50377 8.30 8.40 ↑↑

202B

5-33 6755004 programmed cell death 8 121 20 66724 9.23 2.32 ↑

a) MASCOT was searched with a mass accuracy of 50 ppm b) Average fold change ratio in protein expression in Raw 264.7 cells vs. osteoclasts. Changes in mRNA expression in Raw 264.7 cells vs. osteoclasts are indicated by ↑/↓ (slight increase/decrease) and ↑↑ /↓↓ (strong increase/decrease, > twofold). (-) Indicates no change in mRNA expression; p values were < 0.05 c) Number of peptides sequenced by LC-MS/MS d) Only lamin A is present in DNA microarray Supplementary Table 2. Summary of proteins with decreased abundance in osteoclasts

identified by MS

Spot NCBI Protein Mascot Sequence Predicted Change in

No. gene scorea) coverage expressionb)

identifier (%) Mr pI Protein mRNA

Lysosomal

2-04 309202 cathepsin B 63 22 37340 5.46 -2.01 -

Metabolism/Energy Generation

2-05 6678131 spermidine synthase 104 26 33973 5.31 -2.02 -

2-07 6755212 proteasome 28 subunit a 97 28 28655 5.73 -2.00 ↓

2-09 6840949 PKCq-interacting protein 144 32 37758 5.42 -2.25 -

PICOT

2-10 33859640 transaldolase 1 234 48 37363 6.57 -2.58 -

2-11 16740761 thioredoxin domain 99 21 36092 5.18 -2.57 ↓↓

containing 5

4-01 33859640 transaldolase 1 154 36 37363 6.57 -2.71 -

4-02 31982541 biliverdin reductase A 92 26 33477 6.53 -2.07 ↓

4-03 20178035 phosphoglycerate mutase 71 20 28814 6.67 -2.00 -

4-04 12963491 α-enolase 233 41 47095 6.37 -2.00 -

4-05 6996917 glucose-6-phosphate 226 37 59225 6.06 -2.96 ↓

dehydrogenase X-linked

4-06 41529270 3-phosphoglycerate 133 23 56549 6.12 -3.11 ↓↓

dehydrogenase

4-07 31560737 adenylosuccinate 63 13 49990 5.98 -2.00 -

synthetase 2

6-01 6678413 triosephosphate 165 55 26696 6.90 -2.04 ↓

isomerase

6-02 6754976 peroxiredoxin 1 101 31 22162 8.26 -2.18 ↓

6-03 6754524 lactate dehydrogenase 1 88 20 36475 7.62 -2.08 - 6-05 6679937 similar to 159 37 35787 8.44 -2.00 -

glyceraldehyde-3-

phosphate dehydrogenase

6-06 20886743 phosphoserine 93 20 40447 8.15 -2.62 ↓

aminotransferase 1

6-07 6680067 glucose phosphate 121 17 62728 7.75 -2.54 -

isomerase 1

6-08 31981562 pyruvate kinase 3 184 23 57808 7.18 -3.46 -

6-09 31981562 pyruvate kinase 3 216 26 57808 7.18 -2.77 -

6-10 11066098 transketolase 212 25 60545 6.54 -2.29 -

6-12 31981562 pyruvate kinase 3 203 29 57808 7.18 -2.95 -

6-13 11066098 transketolase 205 30 60545 6.54 -2.00 -

6-14 6680067 glucose phosphate 94 14 62728 7.75 -2.30 -

isomerase 1

6-15 6679291 phosphoglycerate kinase 78 16 44508 7.53 -2.05 ↓

6-16 31981382 inosine 5'-phosphate 92 17 55780 6.84 -2.29 ↓

dehydrogenase 2

6-17 31981562 pyruvate kinase 3 392 63 57808 7.18 -2.85 -

6-18 31981562 pyruvate kinase 3 209 33 57808 7.18 -2.90 -

6-19 31981562 pyruvate kinase 3 82 17 57808 7.18 -2.45 -

6-20 31981562 pyruvate kinase 3 133 22 57808 7.18 -2.47 -

6-21 31981562 pyruvate kinase 3 99 17 57808 7.18 -2.46 -

6-22 31981382 inosine 5'-phosphate 72 14 55780 6.84 -2.19 ↓

dehydrogenase 2

6-23 14250422 phosphogluconate 271 48 53227 6.81 -2.00 -

dehydrogenase

6-24 13385434 phosphoribosyl- 235 34 46976 6.94 -2.00 -

aminoimidazole

carboxylase

6-25 6754278 isocitrate dehydrogenase 115 24 46630 6.48 -2.92 ↓

1 6-26 6754278 isocitrate dehydrogenase 106 20 46630 6.48 -2.03 ↓

Cytoskeleton

2-01 33563236 Rho-GDIβ 184 43 22836 4.97 -2.31 ↓

2-06 9789995 stathmin 1 192 67 17264 5.76 -5.81 ↓

2-08 7304887 annexin A3 236 52 36348 5.33 -2.43 ↓↓

2-12 7106439 tubulin β5 110 23 49639 4.78 -3.28 -

2-13 7106439 tubulin β5 173 34 49639 4.78 -3.85 -

2-14 7106439 tubulin β5 96 18 49639 4.78 -3.41 -

6-04 6680924 cofilin 1 86 31 18548 8.22 -2.00 -

Nuclear

2-02 6679108 nucleophosmin 1 74 21 32540 4.62 -4.64 -

2-03 6679108 nucleophosmin 1 88 21 32540 4.62 -4.12 -

2-15 7242171 proliferating cell 83 25 28766 4.66 -2.68 ↓↓

nuclear antigen

6-11 30172544 structural 63 35 134190 8.54 -2.52 ↓↓

maintenance of

chromosomes 2-like