The Medicago truncatula Small Protein Proteome and Peptidome

The Medicago truncatula Small Protein Proteome and Peptidome Kerong Zhang,† Carolyn McKinlay,‡ Charles H. Hocart,†,‡,§ and Michael A. Djordjevic*,†,§ Australian Research Council Centre of Excellence for Integrative Legume Research, Genomic Interactions Group, and Mass Spectrometry Facility, Research School of Biological Science, Australian National University, G.P.O. Box 475, Canberra, ACT 2601, Australia Received July 10, 2006

The small protein and native peptide component of plant tissues is a neglected area of proteomic studies. We have used fractionation techniques for denatured and nondenatured protein preparations combined with 2-D LC tandem mass spectrometry to examine the sequences of small proteins and peptides in four tissues of the model legume, Medicago truncatula: the root tip and root of germinating seedlings, nitrogen fixing nodules, and young leaves. The isolation and fractionation strategies successfully enriched the small protein and native peptide content of the samples. Eighty-one small M. truncatula proteins and native peptides were identified. Most samples were dominated by ribosomal and histone proteins, and leaf samples possessed photosynthesis-related proteins. Secreted proteins such as lipid transfer proteins were common to several tissues. Twenty-four hours after germination, the roots and root tip tissues possessed several “seed-specific” and late-embryogenesis proteins. We conclude that these proteins are present in cells prior to germination and that they are subsequently used as a nutritional source for the young tissues. Native UV absorbing peptides were detected in very low molecular weight fractions and sequenced. Each peptide shared C-terminal residues and showed homology to the seed storage protein legumin. The strategies used here would be suitable for combining bioassays and mass spectrometry to identify bioactive peptides in the M. truncatula peptidome. Keywords: model legume • native peptides • nodules • Sinorhizobium meliloti • secreted proteins • lipid transfer proteins • legumin

Introduction There is increasing interest in using proteomics to isolate and identify small proteins and native peptides in plants. One reason is that there is a need to understand how plant cells perceive endogenous and exogenous peptide signals, as a great knowledge gap exists between the large number of membranebound orphan receptors and the potential peptide ligands they interact with. For example, plant genomes encode very large gene families of receptor-like kinases (RLKs). A major subclass of these receptors possesses leucine-rich repeat extracellular domains (LRRs).1 Many of these LRR-RLKs interact with peptide ligands.2 One of the best examples is the interaction of the CLAVATA3 C-terminal peptide cleavage product with the CLAVATA1 and CLAVATA2 receptors.3-5 There is increasing evidence that other members of the LRR-RLK family interact with native peptide ligands to control, for example, stem cell differentiation and the innate immune system in plants,2,3,5-7 but until very recently, the existence of these peptides in plants had not been directly demonstrated using mass spectrometry.8,9 Another reason for the relative dearth of knowledge of bioactive * Author for correspondence. Phone, + 61 2 61253088; e-mail, [email protected]. † Australian Research Council Centre of Excellence for Integrative Legume Research, Australian National University. ‡ Mass Spectrometry Facility, Australian National University. § Genomic Interactions Group, Australian National University. 10.1021/pr060336t CCC: $33.50

 2006 American Chemical Society

peptides in plants is that small proteins of 25 kDa) contaminating the lower molecular weight fractions, suggesting that our protein isolation strategy and general handling procedure was effective at isolating intact small plant proteins. The Journal of Proteome Research • Vol. 5, No. 12, 2006 3357

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Table 1. Denatured M. truncatula Young Leaf Proteins Identified from 2 Week Old Plants

no.

protein

1 similar to photosystem II oxygen-evolving complex protein 3-like (Arabidopsis thaliana)

prep MW cell Mascot mass TC/GB acc (kDa)/aa fraction score observed charge TC106703

25.9/231 125-130

peptidea

coverage (%)

140

638.89

2

K.AWPYLQNDLR.L

30.3

2 2 2 3 2

R.FYLQPLTPTEAAAR.A K.LFQDIDNLDYAAK.V K.VGGPPAPSGGLPGTLNSDEAR.D R.YDLNTIISAKPK.D K.LPLFGATDASQVLK.E

15

2 similar to Ribulose bisphosphate carboxylase small chain chloroplast precursor

TC93920

20.2/180 125-130

97

790.07 763.70 975.87 455.19 729.91

3 similar to ribulose 1 5 -bisphosphate carboxylase small subunit

TC93917

20.2/180 125-130

239

782.43 840.49

2 2

K.KFETLSYLPPLTR.E K.ELDEVVAAYPTAFVR.I

23.3

528

719.12 783.23 731.19 527.38

2 2 2 2

K.FETLSYLPPLTR.E K.KFETLSYLPPLTR.E K.LPLFGATDASQVLK.E K.AAYPESFIR.I

41.8

115

1047.35 789.74 738.43 706.44 839.78 558.77

2 2 2 3 2 2

K.KFETLSYLPPLTEDQLAK.E R.KGWVPCLEFELEK.G K.LPLFGATDSSQVLK.E K.LPLFGATDSSQVLKELAEAK.A R.QVQCISFIAHTPATY.R.AYTVQFGTCK.F

19.3

451

734.83 503.76 619.95

2 2 2

K.FPENFTGCQDLAK.Q R.LATSGANFAR.A K.ISTSTNCATIRA.-

25.2

3 2 2

R.QAACNCLKSAAGAISGLNPNIAAGLPGK.C K.SAAGAISGLNPNIAAGLPGK.C K.FFETFAAPFTK.R

14.4

4 homologue to Ribulose bisphosphate TC106570 carboxylase small chain chloroplast precursor

5 homologue to Photosystem I reaction centre subunit N chloroplast precursor (PSI-N) (ARATH)

6 similar to nonspecific lipid -transfer protein precursor [Chickpea Garbanzo] (Cicer arietinum)

TC100474

TC93922

19.7/177 125-130

18.5/171 122-126

16.3/159 118-123

7 homologue to Photosystem I psaH precursor (Nicotiana sylvestris)

TC94297

15.6/146 118-123

128

890.44 890.43 653.46

8 similar to Photosystem I reaction center subunit IV B chloroplast precursor (Arabidopsis thaliana)

TC106666

15.5/147 118-123

74

609.69 636.32

2 2

-.IQIKTNILMK.L K.GTGSVVAVDQDPK.T

14.3

9 similar to Nonspecific lipidtransfer protein precursor [Chickpea Garbanzo] (Cicer arietinum)

TC94140

14.4/142 115-120

290

989.59 583.29

1 2

K.TRYPVVVR.F K.ISTSTNCATIR.A

21.8

TC93932

14.1/135 115-120

377

1768.96 478.72

2 2

K.QAAGAISGLNTAAASALPGK.C K.GVYQFVDK.Y

42.2

2 3 2 2 2

K.GVYQFVDKYGANVDGYSPIYEPK.D K.IKTDTPYGTGGGMALPDGK.D K.TDTPYGTGGGMALPDGK.D K.YGANVDGYSPIYEPK.D K.IQDKEGIPPDQQR.L

17.5

10 similar to Photosystem II 10 kDa polypeptide chloroplast precursor

11 homologue to ubiquitin fusion protein-fission yeast (Schizosaccharomyces pombe)

TC100230

14.1/126 115-120

78

1305.44 632.51 819.60 837.55 761.89

12 similar to cysteine proteinase inhibitor (Arabidopsis thaliana)

TC94890

12.9/116 112-117

70

532.78 963.01

2 2

R.TLADYNIQK.E K.NYEALVWEKPWLHLK.N

25.9

825.44 688.45

2 2

K.VIKGETQVVSGTNYR.L R.EGDILTLLESER.E

29.5

3 2 2 2

K.GPVREGDILTLLESER.E K.EDQFFETDPILK.K K.GGGFGGLFAK R.CESACPTDFLSVR.V

28.7

13 homologue to ribosomal protein S28-like (Arabidopsis thaliana)

TC107020

10.9/95

89-114

90

14 unknown protein

TC95304

10.3/95

89-114

100

15 homologue to photosystem I iron-sulfur protein psaC-rice

TC95569

8.9/80

81-95

52

595.81 742.00 455.76 713.81

16 similar to copper chaperone homologue CCH [imported] -soybean

TC107176

8.4/78

81-95

65

517.77 535.78

2 2

R.VYLGPETTR.S K.GNVEPDTVLK.T

17.9

17 similar to unknown protein (Arabidopsis thaliana)

TC103348

8.3/76

81-95

86

748.43 708.47

2 2

K.VVVKGNVEPDTVLK.T K.AETGGVSSINPDIR.K

34.2

623.14

2

K.VVDAVVLSEVSK.A

a

Underlined M ) oxidized methionine; underlined C ) carbamidomethyl cysteine.

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Medicago truncatula Small Protein Proteome and Peptidome Table 2. Denatured M. truncatula Proteins Identified from Newly Emerged Root Tips TC/GB acc

MW (kDa)/aa

prep cell fractions

Mascot score

mass observed

charge

similar to Legumin A precursor

TC100252

60.4/524

138-145

107

653.52

2

R.DFLEDALNVNR.H

3.1

homologue to Glyceraldehyde-3 -phosphate dehydrogenase

TC106518

36.6/339

134-140

76

956.83 581.35

2 2

R.DFLEDALNVNRHIVEK.L K.AGIALNDNFVK.L

9.7

137

417.33 852.36 981.577

2 2 2

K.IGINGFGR.I K.LVSWYDNELGYSTR.V K.LAVLQFYK.V

24.2

1 2 2 3 3

R.LIFAGK.Q K.IQDKEGIPPDQQR.L R.TLADYNIQK.E R.TLADYNIQKESTLHLVLR.L K.ETAQSGKDNSAGFLQQTGEK.V

18.9

no.

protein

1 2

3

homologue to ubiquitin/ribosomal protein S27 (white lupin)

TC93921

21.5/186

125-130

peptidea

coverage (%)

4

similar to Late embryogenesis abundant protein 1 (CapLEA-1)

TC94388

17.6/164

120-125

56

648.48 762.51 533.61 705.74 699.51

5

homologue to protein T19E23.13 [imported] (Arabidopsis thaliana)

TC107514

17.1/152

120-125

137

531.94 511.78

2 2

K.GMAQGATEAVK.N K.EGIPPVQQR.L

20.3

114

535.05 762.51 533.61 681.44

2 2 2 2

K.ESTLHLVLR.L K.IQDKEGIPPDQQR.L R.TLADYNIQK.E K.MELPEWTDIVK.T

24.6

2 2 2 2

R.RDLDQVAGR.I R.SGKMELPEWTDIVK.T R.TVQDVSSHEFVK.A R.AAQLDVVVTNK.L

10.6

6

similar to ribosomal protein S19 (Arabidopsis thaliana)

TC93964

16.0/142

118-123

7

homologue to ribosomal protein L32 (Arabidopsis thaliana)

TC106732

15.6/132

118-123

93

514.77 816.41 687.84 579.46

8

homologue to ubiquitin fusion protein (Nicotiana sylvestris)

TC107028

14.6/127

115-120

61

749.79 761.89

2 2

R.AAQLDVVVTNKLAR.L K.IQDKEGIPPDQQR.L

17.3

9

homologue to 60S ribosomal protein L37a

TC93929

13.3/120

112-117

118

532.78 776.50

2 2

R.TLADYNIQK.E K.AGGAYTLNTASAVTVR.S

22.5

10

histone H4 (H4C13) (Zea mays)

TC107116

11.4/102

95-115

65

620.44 581.27

2 2

K.GCRDMGMQGLR.R.DAVTYTEHAR.R

19.6

11

homologue to ribosomal protein small subunit 28 kDa (Helianthus annuus)

TC107020

10.9/95

89-114

69

590.31 713.38

2 3

R.ISGLIYEETR.Q K.GPVREGDILTLLESEREAR.R

30.5

12

similar to embryonic abundant protein (soybean)

TC96799

10.7/100

89-114

95

547.82 804.74

2 2

K.HAVVVKVMGR.T E.RAEEEGIDIDESKFK.T

47

3 3 2 2

R.KEQLGTEGYQEMGR.K E.KSGGERAEEEGIDIDESKFK.T G.RSLEAQEHLAEGR.S K.KTGESIKETAANIGASAK.S

30

82

13

similar to seed maturation protein LEA 4 (Glycine tabacina)

TC94509

10.6/100

89-114

121

547 698.56 669.52 887.97

14

similar to acyl-CoA-binding protein (Gossypium hirsutum)

TC94665

10.0/89

89-114

111

671.84 302.28

2 3

R.VNQAELDKEAAR.E R.AKWDAWK.A

654.98 970.55 701.78 798.77 595.74

3 1 2 3 2

K.QATVGNVDTARPGMFNMR.D K.QLLEAAGVAV.K.SKEEAMSDYITK.V K.VKTLTESPSNEDLLILYGLYK.Q K.WDAWKAVEGK.

a

Underlined M ) oxidized methionine.

largest protein found in the denatured protein preparations was legumin, but this was present in higher fraction numbers (Table 2). The TFA-soluble M. truncatula proteins were also further fractionated using size exclusion (Superdex Peptide HR 10/30) chromatography under nondenaturing conditions. The fractions containing the lower molecular weight proteins from root

tip and nodule tissues are shown in Figure 2, and fractions 1315 in panel A and fractions 13 and 14 in panel B are particularly enriched for low molecular weight proteins. However, fractions 10-13 contained low molecular weight proteins as well as higher molecular weight species (20-70 kDa). There was an overlap in the sizes of the protein species present in adjacent fractions. Proteins detected in fractions 10-15 by 2-D LC-MS Journal of Proteome Research • Vol. 5, No. 12, 2006 3359

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identified after size fractionation procedures enriching for low molecular protein species were indeed small proteins ranging between 6.4 and 20 kDa as listed in Tables 1-5 for M. truncatula and 7.3-13 kDa for S. meliloti (Table 6). This was particularly so for the proteins identified after denaturing fractionation, where the proteins present were of a tight size range (Figure 1; Tables 1 and 2). Similarly, size fractionation of the nondenatured material generated proteins of a wider range of sizes (especially in fractions 10-12), and this was reflected by the wider sizes of the proteins identified by mass spectrometry (Figure 1; Tables 3-5). The congruency of these results was reassuring, especially when the same protein was identified in independent sample runs and in different fractions of the same sample (Tables 1-6).

Figure 2. 1-D PAGE showing the size range of TFA-soluble M. truncatula proteins after size-exclusion chromatography. Fractions 10-16 are shown after separation of nondenatured proteins using Superdex Peptide HR 10/30 chromatography. Panel A contains root tip proteins, and panel B contains root nodule proteins. Fractions 14 and 15 contain the smallest detectable proteins after 1-D PAGE separation.

of root tip, root radicle, and nodule tissues (Tables 3-5) were consistent with the size fractionation profile seen in Figure 2. Fractions 10-12 contained the majority of the higher molecular weight (>20 kDa) species including legumin, as well as low molecular weight proteins. Fractions 13 and 14 contained many small proteins ranging in size from 6.4 to 11 kDa (Table 3-5). Many more small proteins ranging from 2.8 to 13 kDa were identified than are listed in Tables 3-5, but these were identified from data from one peptide and, hence, were excluded from the results (see below). In total, 78 small M. truncatula proteins were identified in the tissues examined (Tables 1-5) representing 62 individual proteins, and most lie within the expected size range based on estimates of the sizes of the species in the fractions examined. To our knowledge, this represents the most extensive list of small M. truncatula proteins identified to date. In addition, 7 small S. meliloti proteins were identified in the nodule tissues of M. truncatula (Table 6), and only two have been described previously.21,22 Several proteins were found in multiple fractions as indicated in Tables 1-5. Special Considerations in Identifying Small Proteins by Mass Spectrometry. This study raises several issues with regard to the specific difficulties in identifying small proteins by current LC mass spectrometry methods. Most of the proteins 3360


However, 80 proteins that were identified were excluded from Tables 1-5, although they generated significant Mascot scores. This was because the protein identification was based upon one peptide which is generally regarded as unreliable for mass spectrometry identification.20 However, there are several parameters that suggest that these assignments are correct. First, all but two of these 80 excluded proteins were in the size range of 5-20 kDa. Second, about 43% of these excluded hits, the theoretical number of unmodified tryptic peptides possible (assuming no miscleavages) ranged between 1 and 4. Therefore, by identifying one peptide, between 25 and 100% of the possible peptides were found. Third, each of the peptides was found in a minimum of three independent experiments. Fourth, many of the hits were assigned to ribosomal and histone proteins that are known to be present and which are known to be highly modified (see below), thus, making it difficult to identify all of the peptides unless prior knowledge of the modification is known. Several examples of the excluded proteins are given in Table 7. Because of these parameters, we propose that a different set of rules govern the identification of small proteins. Clearly, identifying a single peptide from a large protein is unsatisfactory for protein identification, since sequence data from other peptides from that protein should be able to be gathered using mass spectrometry. However, this is not the case for small proteins, especially where the total number of possible peptides generated may be as few as four or less. Some weighting may need to be given for single peptide identifications if: (a) the material is pre-fractionated for size, (b) the peptide is found in independent experiments and in different fractions of the same sample, (c) a significant score is achieved in all repeats, and (d) where the total number of possible (unmodified) tryptic peptides is four or less. In some entries in Table 7, the Mascot score is greater than those for proteins where two or more peptides are identified (compare Table 7 with selective matches in Tables 1-5), and the percent coverage ranges from 8 to 22%. In addition, the current guidelines exclude a positive identification when 100% (i.e., one of one) of the possible peptides is identified. Common Small Proteins Identified in M. truncatula Tissues. A large proportion of the small proteins identified in the TFA-soluble fractions of the tissue samples of M. truncatula are highly basic (arginine- and lysine-rich) histone and ribosomal proteins ranging in size from about 6 kDa (56 amino acids), for the S29-like ribosomal protein, to over 20 kDa, for the S6 ribosomal proteins (Tables 1-5). These ribosomal and histone proteins are abundant in all plant tissues and would be expected to be found in all samples. A large number of singlepeptide identifications excluded from this study also matched to ribosomal and histone proteins (Table 7). A possible reason

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Medicago truncatula Small Protein Proteome and Peptidome

Table 3. M. truncatula Root Tip Proteins Identified after Nondenaturing Fractionation and Mass Spectrometry no. 1

2

3

protein similar to Legumin A precursor

homologue to Ribosomal protein S6

similar to ribosomal protein L6

acc. no.

MW (kDa)aa

TC100252

60.4/524

TC106523

TC94539

28.2/248

26.0/233

superdex fraction(s) 10, 11, 12

10, 11, 12

mascot score

mass observed

charge

161

654.11

2

R.DFLEDALNVNR.H

121

843.29 580.34 686.62 722.30

2 2 2 1

R.EQQENEGGNIFSGFK.R R.GGLSFVTPPER.Q K.VEGGLSIMSPPER.Q K.LFNLSK.D

12.1

70

521.60 818.92 549.22

2 2 2

R.LVTPLTLQR.K R.ISQEVLGDALGEEFK.G K.AGVKPHELVF.-

16.3

2 2 1 2 1 2

R.ASITPGTVLILLAGR.F K.QLPSGLLLVTGPFK.I R.ALHIVGPDK.K K.IATPANWKPGEPVVISPDVTNDQAK.D R.NMDEVLR.V R.AFLVEEQK.I

11, 12

peptidea

coverage (%) 9.5

4

1-cys peroxiredoxin

TC108877

24.4/217

10, 11

65

5

homologue to 60S ribosomal protein L34

TC101425

21.7/190

10, 11, 12, 13

81

742.24 736.28 950.82 1324.74 876.94 483.04

1 2 2

R.AYGGVLSGGAVR.E R.IQGIPHLRPTEYK.R K.FSQVVSNQLDMK.L

10.8

18.9

17.4

6

homologue to 40S ribosomal protein S18

TC94283

21.4/185

11, 12

64

1107.14 777.43 699.31

7

homologue to ribosomal protein L23

TC94128

19.4/171

11, 12

60

524.76 563.43

2 2

K.VPDWFLNR.K K.FPLTTESAMK.K

13.5

8


TC95376

18.4/162

10, 11

66

1434.80 521.72

1 2

R.LTPDYDALDVANK.I K.LTWTAMYR.K

20.4

2 1 2 2 2

R.SDSQVFLFVNSK.C R.SIVGATLEVIQK.K R.AGLQFPVGR.I R.HVLLAVR.N K.DPNTVFVFK.F

17.6

similar to Histone H2A

TC100976

15.8/147

10, 11, 12

51

10

homologue to ribosomal protein S19

TC106741

15.7/136

10, 11

52

686.63 1258.19 473.64 405.19 534.61

11

homologue to ribosomal protein L27-5

TC106617

15.6/134

12

52

801.89 428.77

2 2

K.STGFGLIYDSVENAK.K K.AVIVLQGR.Y

11.9

12

homologue to Histone H2B

TC97961

15.2/137

10, 11, 12

125

968.04 866.26

2 2

R.YTLDVDLK.E K.AMGIMNSFINDIFEK.L

17.5

146

940.32 393.96

1 2

R.LVLPGELAK.H K.AELALLR.V

34.4

2 2 2 2 2 2 2

R.LNIAQVLTVISQK.Q R.LSIAQVLTVISQK.Q K.YLPLDLRPK.K R.AEEEGIDIDESK.F R.KEQLGTEGYQEMGR.K K.SLEAQEHLAEGR.S K.LITPSVLSDR.L

34.9

19.5

9

13


TC100995

14.2/122

11, 12

14

homologue to Em protein

TC96799

10.7/100

11, 12, 13

113

15

homologue to 40S ribosomal protein S25-1

TC100588

10.7/106

10, 11, 12

147

714.63 701.03 558.76 668.62 813.98 671.06 551.38

139

746.08 831.26 885.26

2 2 2

R.LVSAHSSQQIYTR.A K.VNNMVLFDQGTYDK.L K.QAAGAISGLNTAAASALPGK.C

886.07

3

R.QAACNCLKQAAGAISGLNTAAASALPGK.C

16

a

similar to UP|NLTP_CICAR (O23758) Nonspecific lipid-transfer protein precursor (LTP)

TC94140

14.3/143

11, 12, 13

10.9

38


for this is that ribosomal proteins are known to be extensively post-translationally modified (e.g., reduction of glutamic acid, pseudo-uridinylation, phosphorylation, acetylation, and methylation).13,23 These post-translational modifications would normally hamper identification unless specific knowledge of the modification and its position in the protein was available prior to database searches. Also, a high number of trypsin cleavage sites (due to the high abundance of Lys and Arg residues in the basic proteins) can also generate peptides too small to be identified by mass spectrometry. The identification of ribosomal proteins is further complicated by the presence of multiple genes encoding ribosomal proteins with regions of identical amino acid sequence. This can make it difficult to distinguish one homologue from another (for example, ribosomal proteins S19 and S19-3; Table 4). In Arabidopsis, each

ribosomal protein is encoded, on average, by four genes, generating a range of isoforms,13 and different isoforms of the same Arabidopsis ribosomal protein are encoded by the nuclear, mitochondrial, and chloroplast genomes.13 A comparison of Root Tip Proteins Separated by Denaturing or Nondenaturing Fractionation. The results show that after the selection for solubility in 1% TFA, the subsequent use of different protein solubilization and fractionation techniques leads to different proteins being identified in the same sample by mass spectrometry. We conclude this because (a) there was high intra-sample reproducibility (>80% on independent runs) and (b) there was little overlap in the proteins identified by mass spectrometry of root tip proteins fractionated using denaturing gel electrophoresis (Table 2) or size-exclusion nondenaturing chromatography (Table 3). Of a combined total Journal of Proteome Research • Vol. 5, No. 12, 2006 3361

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Table 4. M. truncatula Root Proteins Identified after Nondenaturing Fractionation and Mass Spectrometry no. 1

2

3

4 5

6

7

8

protein similar to legumin A precursor

weakly similar to 51 kDa seed maturation protein precursor

similar to Maturation polypeptide

similar to peroxidase 1B precursor homologue to ribosomal protein S6

weakly similar to maturation protein pPM32

similar to 60S ribosomal protein L6

1-cys peroxiredoxin

TC/GB acc

MW (kDa)aa

TC100252

60.4/524

TC100258

TC95538

TC106484 TC106523

TC101811

TC100531

TC108877

55.4/513

41.2/372

38.3/353 28.2/247

26.4/244

25.9/233

24.4/217

superdex fraction(s) 10

10

10, 11

12, 13 10, 11, 12

10, 11, 12

10, 11, 12

10, 11, 12

Mascot score

mass observed

charge

109

654.21

2

R.DFLEDALNVNR.H

86

949.46 1523.57 695.18 1321.84

2 3 2 1

R.GREQQENEGGNIFSGFK.R R.RPSYSNAPQEIFIQQGSGYFGMVFPGCPETFEEPQESEQR.E K.VEGGLSIMSPPER.Q K.EAAETWTEWAK.E

107

865.14 617.25 886.54 892.20

1 2 2 2

K.ITEGLGFK.D R.NEDEEKGAIVK.V K.NKIQDVASGTGEHSAEK.A K.EQHDQEKPGVIGSVMK.A

77

1340.24 668.58 495.98

2 2 2

R.LKAADQMTGQTFNDVGPLDDEGVTR.V K.MGEYKDYTAEK.A K.MGNIGVLTGK.K

112

938.44 327.21

2 2

K.TSTIASEQDAGPNINSLR.R R.LLLHR.G

16.6

124

819.21 744.59 590.49 834.83

2 3 3 2

R.ISQEVNGDALGEEFK.G R.ISQEVNGDALGEEFKGYVFK.I K.KGENDLPGLTDVEKPR.M R.ARETMSEGVDSEDIK.Q

25.4

102

725.78 887.94 805.19 1233.92 958.04

2 1 2 1 1

K.DRTNEAAGSVWDK.A R.DYAYEVK.D R.KPSTNWAYDHSSSK.T K.TGEVAGSATEALK.S K.AIDSVPDLK.T

28.3

120

741.96 841.82 736.42 1280.91 1077.93

2 2 2 1 1

R.ASITPGTVLILLAGR.F R.HDPVAKPAAPVEKPPK.F K.QLPSGLLLVTGPFK.I R.VNQAYVIGTSTK.V R.ALHIVGPDKK.I

38.2

1 2 1 2 1 2 2

K.DMFPQGFK.T K.IATPANWKPGEPVVISPDVTNDQAK.D K.LSFLYPAQTGR.N K.MAQYASEFNKR.G R.NMDEVLR.V K.VNYPIISDPKR.E R.AYGGVLSGGAVR.E

10.5

peptidea

coverage (%) 15.4

9.2

14

7.9

9


TC101425

21.7/190

10, 11

51

969.82 1324.98 1253.05 602.19 876.90 652.31 554.42

10


TC94128

19.4/171

10

64

483.07 563.78

2 2

R.AFLVEEQK.I K.FPLTTESAMK.K

28.6

200

1434.80 637.10 869.20 544.58 1394.76

1 2 1 2 1

R.LTPDYDALDVANK.I R.NKLDHYQILK.F K.MYDIQAK.K R.TTVTFHRPK.T K.DNSAGFLQQTGEK.V

30.6

52

698.7 540.55 832.80 646.28 699.24

2 2 2 2 2

K.ETAQSGKDNSAGFLQQTGEK.V K.GMAQGATEAVK.N K.SNQMMGNIGDKAQAAK.E K.VKGMAQGATEAVK.N K.FSQVVSNQLDMK.L

17.1

58

416.69 524.53 655.28

2 2 3

R.HYWGLR.V K.VPDWFLNR.K R.FKELAPYDPDWYYVR.A

19

107

689.26 843.78

2 2

R.TVQDVSSHEFVK.A R.IYDVKDPNTVFVFK.F

30.1

112

705.91 801.87 1157.96

2 2 1

R.KQFVIDVLHPGR.A K.STGFGLIYDSVENAK.K R.AAQLDVVVTNK.L

42.4

699.51 791.21 733.78

2 2 2

K.FVVHNVQDLELLMMHNR.T K.GVTLMPNIGYGSDKK.T R.TYCAEIAHNVSTR.K

11

12

similar to late embryogenesis abundant protein 1 (CapLEA-1)

40S ribosomal protein S18

TC94389

TC94282

17.7 /163

17.6/152

10, 11, 12

11

13

similar to 40S ribosomal protein S19-3

TC93964

16.0/142

11

14


TC106741

15.7/136

10, 12

15

3362


TC106733

15.6/132

10, 11, 12


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Medicago truncatula Small Protein Proteome and Peptidome Table 4 (Continued) TC/GB acc

MW (kDa)aa

similar to late embryogenesis abundant protein (Fragment)

BG454018

15.45/138

10, 11


TC100995

14.2/122

13

no.

protein

16

17

superdex fraction(s)

Mascot score

mass observed

charge

120

681.08

2

K.MNQAELDKLAAR.E

21

77

830.26 786.84

2 1

R.VGGNPNATGYTTGGTYK. K.AELALLR.V

23.8

105

714.39 558.59 473.35

2 2 2

R.LNIAQVLTVISQK.Q K.YLPLDLRPK.K R.AGLQFPVGR.I

28.6

peptidea

coverage (%)

18

homologue to histone H2A.vD

TC100310

14.1/133

12

19

similar to nonspecific lipid-transfer protein precursor (LTP)

TC94140

11.4/116

12, 14

84

1448.75 1166.99

2 1

R.VGATAAVYSAAILEYLTAEVLELAGNASK.D K.ISTSTNCATIR.A

33.6

20

similar to Nonspecific lipid-transfer protein precursor (LTP)

TC94138

11.3/113

12, 13

108

886.49 884.70

3 2

R.QAACNCLKQAAGAISGLNTAAASALPGK.C K.QAAGAISGLNTAAASALPGK.C

24.8

21

homologue to ribosomal protein small subunit 28

TC107020

10.9/96

12, 13, 14

54

886.17 688.76

3 2

R.QAACNCLKQAAGAISGLNTAAASALPGK.C R.EGDILTLLESER.E

19.8

22

similar to seed maturation protein LEA 4

TC94509

10.5/101

11, 12, 13, 14

116

454.93 1419.07

2 2

K.FLDDQNR.H R.EHNAAASAGHQLGVGGHHTTGTGGAAQNRA.

67.3

50

517.76 521.60 825.10 673.28 602.33

2 2 2 2 2

K.ETAANIGASAK.S K.MTAHDPLQK.E K.TGESIKETAANIGASAK.S R.VNQAELDKEAAR.E R.INQGYAAALPGK.C

38.9

49

585.64 366.41 680.87 767.16 707.28

2 2 2 1 1

K.ISASFNCASIR R.NIVSAAR.T R.RINQGYAAALPGK.C R.VYLPFK.I K.EIGFIK.Y

28.6

529.19

2

R.VCGNSHGLIR.K

23

24

a

lipid transfer protein-like protein

homologue to ribosomal S29-like protein

TC110477

TC93973

10.0/95

6.4/56

11, 12, 13, 14

12, 13, 14


of 31 root tip proteins identified (Tables 2 and 3), only two proteins were common to both samples (TC96799 and TC100252), but a further 2 others were ribosomal proteins of the same identity but with different TC assignments (e.g., L27 and S19). Given that intra-sample protein identification reproducibility was high (∼80%), the differential solubilization and handling strategies used for the denatured and nondenatured samples are a likely cause of the lack of congruency between the root tip proteins identified in Tables 2 and 3. In the case of the denatured proteins (Table 3), proteins were solubilized in sample buffer and separated using reagents containing detergent before purification (which can result in sample loss) and analysis by mass spectrometry. In contrast, the concentrated TFA-soluble proteins in the root tip extract were solubilized and chromatographed in 30% acetonitrile before concentration, digestion, and mass spectrometer analysis. The use of SDS followed by cleanup procedures compared to 30% acetonitrile may have led to a subset of proteins being present in the fractions introduced into the mass spectrometer. Finally, although efforts were made to remove SDS from the denatured samples, trace SDS in samples remained, and we would not recommend this as a viable approach, since detergents are very undesirable for LC-MS experiments. Seed Storage Proteins Are Present in Root and Root Tip Samples of Young Germinated Seedlings. Several “seed-

specific” seed storage proteins and late-embryogenesis proteins were found in the root radicle and root tip samples of germinated seedlings of M. truncatula (Tables 2-4), but not in older 21 day nodule or 14 day leaf samples (Tables 1 and 5). These proteins included legumin A, several seed maturation proteins, LEA type proteins, and the EM protein. The expression of seed storage protein genes is thought to be seed-specific,24 but some proteins such as the late-embryogenesis-abundant (LEA) proteins accumulate in vegetative organs during prolonged periods of stress.25 Recently, Boudet et al.25 identified both seed storage proteins and dehydration stress-induced embryogenesis proteins in young M. truncatula root radicles that were subjected to osmotic stress. They showed that prolonged osmotic stress can re-establish desiccation tolerance and the induction of these “seed-specific” genes in newly emerged root radicles of 2.8 mm but not ones of 5 mm. The storage and stress proteins identified in our study occur in unstressed root radicles (length of 15-20 mm) that are more mature than those used by Boudet et al.25 Therefore, the most likely explanation for the presence of “seed-specific” storage-type proteins in these tissues is that vestiges of the storage proteins are present in the root apical meristem cells prior to germination, root radicle emergence, and development, and that these are carried for a time in root apical meristem and daughter cells of the root radicle providing a temporary nutrient source. Most of these storage and lateJournal of Proteome Research • Vol. 5, No. 12, 2006 3363

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Zhang et al.

Table 5. M. truncatula Nodule Proteins Identified after Nondenaturing Fractionation and Mass Spectrometry no.

protein

TC/GB acc

MW (kDa)/aa


Mascot score

mass observed

charge

peptidea

coverage (%)

1

homologue to protein disulfide-isomerase precursor (PDI)

TC106707

57.0/513

10

69

817.67

2

K.AASILSTHEPPVVLAK.V

6

2

similar to peroxidase 1B precursor

TC106484

38.3/354

10

92

867.52 495.98

2 2

K.FSGEEYDNFIALAEK.L K.MGNIGVLTGK.K

10.5

2 2 2

R.RLDVINQIK.T K.TSTIASEQDAGPNINSLR.R R.ISQEVNGDALGEEFK.G

9.3

9.2

3


TC106523

28.2/247

10

76

550.81 938.44 819.00

4

homologue to ribosomal protein

TC106330

19.7/173

10

48

415.04 431.87

2 2

K.QGVLTPGR.V K.FILDLLK.N

5


TC100705

18.5/163

10

43

545.25 874.11

2 1

R.YLEDVLAHK.Q K.DIAQEAVK.K

12.3

5

homologue to basic blue protein

TC93931

13.6/125

11, 12, 13, 14

477

629.92 818.64

2 2

R.SIVGATLEVIQK.K K.MHNVVLVDQTGYDK.C

31.2

79

1628.80 546.19 582.48

1 2 2

K.SFVAGDVLDFGYNPK.M R.TGSDQIELVK.G R.DAVTYTEHAR.R

43.7

664.32 591.55 734.74

2 2 2

R.DNIQGITKPAIR.R R.ISGLIYEETR.G K.TVTAMDVVYALKR.Q

7

histone H4-tomato (Lycopersicon esculentum)

a

TC106934

11.4/103

10, 11

Underlined M ) oxidized methionine.

Table 6. S. meliloti Small Proteins Identified from Root Nodules no.

protein

Sm number

MW (kDa)/aa


Mascot score

mass observed

charge

peptide

coverage (%)

1

signal peptide hypothetical/global homology

SMc01418

13.3/124

10, 11, 12

84

1256.10

1

K.DGMPLYFWVK.D

22.6

2

50S ribosomal protein L7/L12 (LB)

SMc01318

12.8/126

12

49

945.64 828.86

2 1

K.MGDVTGDGVKGEWDVARP.K.DLVEGAPK.A

20.6

3

GroES2 chaperonin

SMa0745

10.5/98

13

40

4

30S ribosomal protein S15

SMc00323

10.1/89

11

36

5

50S ribosomal protein L27

SMc03772

9.4/89

12

34

6

histone-like protein

SMc01906

9.3/90

10, 11, 12, 13, 14, 15

84

959.46 323.94 404.43 536.70 504.90 786.42 922.92 770.96

2 3 2 2 2 2 1 2

K.IVEDLSSLTVLEAAELSK.L K.GGVIIPDTAK.E K.WSGTEVK.I R.RGLLALVSSR.R R.RSLLDYLK.K K.KFGGEAVIPGNIIVR.Q R.VYVSVMPK.A K.GRNPSTGAEVDIPAR.N

54

1182.94 702.83 664.36 559.89

1 2 2 2

R.LVGFGNFSVSR.R -.MNKNELVAAVADK.A R.NPSTGAEVDIPAR.N R.AGLSTLKDGQK.V

626.73 782.84

2 1

K.VSFELTQDRR.S K.WFNSTK.G

7

transcription regulator cold shock protein

SMc01428

7.4/69

11, 12

embryogenesis proteins occurred in the fractions possessing proteins of their corresponding weight. However, proteolytic breakdown of seed storage proteins is known to be part of a natural degradation process providing sustenance for root radicle cells.26 Indeed, Boudet et al.25 found evidence for breakdown products legumin in their study.26 Lipid Transfer Proteins and Other Secreted Proteins. Four different lipid transfer proteins (LTPs) were identified in the tissues examined (TCs 93922, 94140, 110477, and 94138). TC94140 was common to root, root tip, and leaf tissue but was not detected in nodules. Leaf (TC93922) and root (TC94138 and TC110477) may possess tissue-specific LTPs, but this requires independent verification. LTPs are ∼10 kDa, basic, and invari3364


17.3 20.2 25.8 43.3

39.1

ably secreted. LTPs consist of a large gene family of 71 members in Arabidopsis.27 These proteins are implicated in plant defense as antimicrobials. Specific LTPs also compete with elicitins for high affinity binding sites on plant membranes.28 Their 3-D shape forms a hydrophobic cleft that can bind to lipophilic molecules in vitro including plant hormones such as jasmonic acid29 and are implicated in long-distance, systemic-acquired resistance signalling.30 Further work is required to determine their precise biological functions, but mass spectrometry is clearly a useful tool to examine tissue-specific accumulation of LTPs. Apart from the seven identified secreted LTPs, five other secreted proteins were present in the samples including

research articles


Table 7. Selected Small Proteins of M. truncatula Determined by Identifying One Peptide in Multiple Samples

no. 1

2 3 4 5 6 7

possible protein identity similar to Nonspecific lipid-transfer protein precursor [Chickpea Garbanzo] (Cicer arietinum) homologue to 40S ribosomal protein S30 (Arabidopsis thaliana) homologue to ribosomal protein S27 (Arabidopsis thaliana) homologue to ribosomal protein S23-2 (Arabidopsis thaliana) similar to At4g31830 (Arabidopsis thaliana) similar to Nucleolar RNA-binding Nop10p-like protein homologue to ribosomal protein S30 (Arabidopsis thaliana)

TC/GB acc

MW (kDa)/aa

prep cell fraction

TC93943

11.9/116

95-115

TC101459

6.9/61

TC100715

6.9/59

TC100637

15.8/141

coverage (%)

peptides observed/ predicted

R.LNNNQAAALPGK.C

10.3

1 of 4

2

R.FVTAVVGFGK.K

16.4

1 of 1

755.88

2

R.LVQSPNSFFMDVK.C

22

1 of 2

566.84

2

K.VSGVSLLALFK.E

mascot score

mass observed

charge

leaf

70

605.32

2

81-95

Leaf

73

512.29

81-95

Leaf

77

118-123

Root tip

147

tissue

peptide

TC109865

11.3/107

95-115

Root tip

83

782.39

2

K.VTAGVANPEDTHPKK.-

14.0

1 of 3

7.4/64

82-94

Root tip

65

735.35

2

R.FGLLPTQHPAPKY.-

18.9

1 of 3

TC101459

6.9/61

81-95

Root tip

91

512.29

2

R.FVTAVVGFGK.K

16.4

1 of 1

MW superdex (kDa)/aa fraction(s)

acc. no.

8

similar to Late embryogenesis abundant protein (Fragment) homologue to FK506-binding protein 2 precursor homologue to superoxide dismutase (Cu-Zn) homologue to 40S ribosomal protein S30-like weakly similar to lipid transfer protein precursor homologue to ribosomal protein L34e similar to lipid transfer protein

TC96465

18.1/176

11, 12

Root tip

105

830.03

2

R.VGGNPNATGYTTGGTYK.-

TC107232

16.4/150

10, 11

Root tip

87

801.92

2

R.NNPIDFELGGGQVIK.G

TC106823

15.2/151

10, 11

Root tip

73

668.86

2

R.AVVVHADPDDLGK.G

11 12 13 14

tissue

Mascot mass score observed charge

protein

10

1 of 3

TC96705

no.

9

7.8

peptide

peptide coverage observed/ (%) predicted 9.7 10 8.6

1 of 4 1 of 3 1 of 3

TC101898

6.9/61

11, 12, 13

Root tip

63

513.49

2

R.FVTAVVGFGK.K

16.4

1 of 1

TC108688

12.0/114

13

Root

54

761.77

2

R.IPGVNPDTVAALPEK.C

13.2

1 of 4

TC106476

13.7/119

11

Nodule

82

554.58

2

R.AYGGVLSGGAVR.E

10.1

1 of 3

TC102826

11.5/114

12

Nodule

55

654.81

2

K.ALASAVSTSEDKK.A

11.4

1 of 2

legumin (TC100252), seed maturation protein precursor (TC100258), a cysteine protease inhibitor (TC94890), peroxidase 1B precursor (TC 106484), and basic blue protein (TC93931). Several of these are known to be targeted to the xylem sap of plants.31,32 Identification of Novel Peptides from M. truncatula. Fraction 15 of the nondenatured proteins contains the smallest proteins detectable by 1-D gel electrophoresis (i.e., ∼4-5 kDa; Figure 2). Fractions 16 and 17 of the root and root tip samples have no detectable proteins, and these fractions were examined by HPLC analysis prior to 2-D LC mass spectrometry to determine if they contained novel peptides that could not be stained using Coomassie blue. Each fraction generated UV absorbing peaks (either at 280, 254 nm, or both; data not presented), suggesting the presence of discrete peptide species. Some peaks with the same retention time were present in both fractions 16 and 17. This is consistent with the overlap in proteins species present in fractions containing higher molecular weight species (Tables 1-5; Figures 1 and 2). Samples of fractions 16 and 17 were digested with trypsin and examined using 2-D LC-MS. The output was examined using Mascot software, and no significant peptide identities were determined from the M. truncatula database. Key MS/MS spectra were manually sequenced (Figure 3), and three glutamate-rich, putative tryptic peptides were identified that shared several C-terminal residues (underlined): -TFEEQPEESQER- (monoisotopic mass, 1509 Da), -SHVEFPETQER- (monoisotopic mass, 1533 Da), and -PEVEVEFPETQER- (monoisotopic mass, 1588 Da). Each of these peptides contained an aromatic amino acid which was consistent with absorption in the UV range, detected using the HPLC. A nine amino acid C-terminal sequence

(underlined) is shared by two of the peptides, and the Cterminal residues -QER- were shared by all three peptides. These sequences were used to interrogate the plant and M. truncatula databases. All three peptides showed similarity to legumin, a protein that was found in fractions 10-12 and 14 of the root tip sample and fraction 10 of the root sample (Tables 3 and 4); however, the mass of the peptides does not correspond to predicted masses of tryptic products of any Medicago or known plant legumins. It is possible that these represent natural breakdown products of legumin, and this is consistent with the detection of larger legumin fragments in the lower molecular weight fractions (e.g., Table 3). Small S. meliloti Proteins in Nodule Samples. Root nodules contain S. meliloti bacteria. To identify the small bacterial proteins in nodule samples, the mass spectrometer data were also used to interrogate the S. meliloti database. Consistent with the plant protein data (Tables 1-5), S. meliloti proteins of the expected size range (7-13 kDa) were identified with a bias toward ribosomal proteins and histones (Table 6). Only two of the 7 proteins identified had been previously identified in S. meliloti (SMc01418 and Smc01318), and these were found in nodule bacteria.12,21,22 The lack of any hits to larger proteins such as nitrogenase subunits or various transporter subunits or periplasmic binding proteins that are known to be abundant in nodule bacteria21,22 also suggests no apparent proteolytic activity during sample preparation, and that size fractionation was effective.

Conclusions We have used effective size fractionation strategies to isolate, enrich, and identify small proteins and novel peptides from Journal of Proteome Research • Vol. 5, No. 12, 2006 3365

research articles

Zhang et al.

several tissues of M. truncatula including root nodules containing S. meliloti. In the most comprehensive proteomic study of M. truncatula so far,15-17 the smallest proteins identified are greater than 11 kDa. About 80% of the proteins identified in this study have not been previously identified using proteomic analysis. This is most likely to be because previous studies15-17 relied upon 2-D gel separation of proteins. This study emphasizes the limitations of 2-D gel electrophoresis-based approaches in separating and resolving small proteins. In addition, the fractions containing nondenatured proteins and peptides identified using our approach would be suitable for subsequent bioassay.

Acknowledgment. This research is supported by a grant from the Australian Research Council Centre of Excellence Program (CE0348212). Peter Milburn of the Australian Cancer Research Foundation Biomoleular Resource Facility is acknowledged for separation of nondenatured proteins using Superdex Peptide HR 10/30 chromatography. Peta Holmes is acknowledged for isolating nodule tissue. References

Figure 3. Spectra showing protein sequence of novel peptides from M. truncatula. The de novo sequence of three peptides is shown. Each peptide generated extensive and complementary y- and b-series ions, and the determined sequence is shown. 3366


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PR060336T

Journal of Proteome Research • Vol. 5, No. 12, 2006 3367

The Medicago truncatula Small Protein Proteome and Peptidome

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