A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin Daniel Ambort1, Daniel Stalder2, Daniel Lottaz1, Maya Huguenin1, Beatrice Oneda1, Manfred Heller2 and Erwin E Sterchi1 Institute of Biochemistry and Molecular Medicine, University of Berne, Switzerland Department of Clinical Research, University Hospital, Berne, Switzerland Keywords astacin family; image analysis; Madin–Darby canine kidney cells; meprin; protease proteomics Correspondence E E Sterchi, Institute of Biochemistry and Molecular Medicine, University of Berne, Buhlstrasse 28, CH-3012 Berne, Switzerland ¨ Fax: +41 31 631 3737 Tel: +41 31 631 4199 E-mail: erwin.sterchi@mci.unibe.ch (Received 14 April 2008, revised July 2008, accepted 10 July 2008) doi:10.1111/j.1742-4658.2008.06592.x In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin–Darby canine kidney epithelial cells A simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin– Darby canine kidney cells expressing meprin in zymogen or in active form The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS ⁄ MS analysis Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively The astacin-like zinc-dependent metalloendopeptidase human meprin (hmeprin) (EC 3.4.24.18) was first discovered in 1982 for its ability to hydrolyze N-benzoyl-l-tyrosyl-p-aminobenzoic acid, a chymotrypsin substrate used for assessing exocrine pancreas function [1] N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase (PPH) was subsequently purified and characterized from human small intestinal mucosa [2] At the same time, PPH orthologs, called meprin (metal endopeptidase from renal tissue) or endopeptidase-2, were found in mouse and rat kidney, respectively [3,4] Two similar subunits, termed meprina and meprinb, with molecular masses of 95 and 105 kDa, respectively, were identified Human meprin cDNA was expressed in Madin–Darby canine kidney (MDCK) cells, a well- established cell system for polarized epithelial cells To date, no such thoroughly characterized model system exists for human epithelial cells Hmeprina is secreted into the culture medium of MDCK cells as inactive homodimers, whereas hmeprinb is primarily membrane-bound [5] Hence, heterodimers of hmeprina ⁄ b allowed for localization of the a-subunit to the plasma membrane [6] Inactive zymogens of hmeprina and b are processed by limited proteolysis with trypsin into their active forms [5,6] Hmeprina, but not b, may alternatively be activated by plasmin [7,8] A first step towards the elucidation of the biological function of meprin was achieved by testing putatively cleavable polypeptide substrates A variety of protein and peptide substrates were processed in vitro; Abbreviations ADAM, a disintegrin and metalloprotease; BMP-1, bone morphogenetic protein 1; CID, collision-induced dissociation; ECM, extracellular matrix; hmeprin, human meprin (EC 3.4.24.18); ICAT, isotope-coded affinity tag; MDCK, Madin–Darby canine kidney; MMP, matrix metalloproteinase; PPH, N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase; TLD, tolloid 4490 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al biologically active peptides [2,9], as well as gastrointestinal peptides and extracellular matrix (ECM) components, such as collagen type IV, fibronectin and laminin-nidogen [10–12] These findings suggest that meprin may be involved in processes such as renal clearance of vasoactive peptides from blood plasma, regulation of cell movement, secretory activity and growth of intestinal tract, and tissue remodelling In addition, marked differences between a- and b-subunits in substrate and peptide bond specificity point to distinct functions for the two forms [10] Meprina selects for small (e.g serine, alanine and threonine) or hydrophobic (e.g phenylalanine) residues in the P1 and P1¢ sites and proline in the P2¢ position Meprinb prefers acidic amino acids in the P1 and P1¢ sites and selects against basic residues at P2¢ and P3¢ In conclusion, protease-substrate discovery executed by these in vitro cleavage assays was rather haphazard Thus, meprin and its substrate repertoire may be studied in a complex biological context to identify physiologically relevant substrates The introduction of protease proteomics enabled identification of protease and protease-substrate repertoires on an organism-wide scale by means of proteomic techniques [13] Using different cell-based systems [14– 16] a variety of hitherto unkown substrates were found in conditioned media for the metzincin metalloendopeptidases, a disintegrin and metalloprotease (ADAM)-17 and matrix metalloproteinase (MMP)-14 Human plasma was also used to identify substrates for recombinant MMP-14 in a cell-free system [17] Two methodological platforms were successfully applied for protein separation: LC-MS ⁄ MS and 2D IEF ⁄ SDS ⁄ PAGE [14–17] These standard techniques were used in combination with lectin-affinity pre-fractionation and quantitative tags such as isotope-coded affinity tags (ICAT) or cyanine dyes for differential in-gel electrophoresis From these protease proteomic studies, it became obvious that metalloendopeptidases are key modulators of diverse signalling pathways and not merely ECM degrading entities [18] For example, the major role of the MMP family is the control of cellular responses critical to homeostatic regulation of the extracellular environment and the immune response [19,20] We decided to apply protease proteomics to identify novel physiologic substrates for meprin, aiming to elucidate its key functions at the cellular level For the above described techniques, some conceptual problems may arise: first, ICAT-based approaches compare pairs of peptides, and therefore it is not possible to discover cleaved protein fragments with shortened (non-trypsin-generated) N- or C-termini; second, nonglycosylated proteins and fragments escape from lectin-affinity purification We thus designed a simple Meprin protease proteomics 2D IEF ⁄ SDS ⁄ PAGE-based protease proteomic approach that remedied these limitations and circumvented complicated quantitative and statistical evaluation Hmeprina ⁄ b was transfected into MDCK cells and activated in situ by limited trypsin treatment at confluent cell stage Conditioned media of meprin activated and non-activated cells were concentrated with ultrafiltration and then separated by 2D IEF ⁄ SDS ⁄ PAGE A simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure allowed for detection of protein spots unique to 2D gels produced from conditioned media of meprin activated cells LC-MS ⁄ MS analysis of candidate substrates confirmed the validity of this protease proteomic approach for the discovery of shortened (nontrypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments Results Design and application of a simple 2D IEF/SDS/ PAGE-based protease proteomic approach in substrate finding Traditionally, 2D IEF ⁄ SDS ⁄ PAGE-based image analysis is performed on two sets of gels and protein spots are matched to the same reference gel within one single analysis Statistical tools are then applied to quantitatively assess subtle but significant changes in peak volumes to find up- or down-regulated protein spots Unfortunately, error-prone matching to wrong reference spots is often underestimated, making quantitative statistical information useless Hence, annotations of interesting candidate spots to wrong spots in the reference gel leads to misinterpretation of the data set and protein spots unique to only one specific condition are then not properly displayed in the corresponding reference gel A remedy to false-positive data interpretation is the stepwise reduction in complexity of such an analysis Therefore, we designed a simple image analysis procedure in which digitized 2D gels were cut into four parts or quadrant sections This procedure enabled the performance of four independent image analyses in which the gel parts of each corresponding quadrant were used to construct four independent reference gels instead of one The corresponding quadrant sections were grouped into sets of gels termed level match-sets for each condition (activated meprin versus non-activated meprin) and then into supersets of level match-sets (higher-level match-sets) (Fig 1) The four level match-sets are the reference gels of the respective quadrants from the 2D gel sections of each condition and the four higher-level match-sets are the reference gels of the two different conditions (activated FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4491 Meprin protease proteomics D Ambort et al Fig Simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure The procedure is based on qualitative differences among reference gels (level match-sets) of each group of five gel replicates (three pooled biological gel replicates and two more technical gel replicates) Gel replicates of each group (activated meprin versus non-activated meprin) were cut virtually into four equally spaced quadrants for four independent image analyses Reference gels of each group were then clustered into a new set for higher-level image analysis The spot matching features of PDQUEST (version 7.3.1) allowed for detection of unique protein spots The combined higherlevel match-set is the final fusion of all annotated unique spots into one big 2D reference map meprin versus non-activated meprin) This procedure allowed for subsequent matching of protein spots first to reference gels of the same condition and thereafter to reference gels common to both conditions The stepwise annotation of protein spots to two independent levels of reference gels allowed for detection of unique spots in the final higher-level match-sets (Fig 2) These differential spots were unique to one specific condition and absent in the other or vice versa Applying the above procedure to conditioned media of MDCKa ⁄ b cells revealed that, among 817 protein spots displayed, 35 were unique to media of cells expressing activated meprina ⁄ b and 40 to media of cells with non-activated meprina ⁄ b (Table 1) These unique protein spots were therefore absent in the corresponding other condition Thus, unique spots were indicative of proteins released into or proteolytically cleaved in the extracellular milieu by hmeprina ⁄ b We then hypothesized that, upon LC-MS ⁄ MS analysis of candidate substrates, it may be feasible to find shortened (non-trypsin-generated) N- and C-termini in peptide fragments Such potential N- or C-terminally truncated cleavage products can be identified in protein spots unique to conditioned media of trypsin activated MDCKa ⁄ b cells, as shown below Fig Application of a simple 2D IEF ⁄ SDS ⁄ PAGE-based protease proteomic approach in substrate finding A representative image analysis of the first quadrant is shown Two hundred and fifty micrograms of conditioned medium protein from trypsin activated and non-activated MDCKa ⁄ b cells was separated by IEF in a 24 cm long IPG pH 3–10 NL strip Vertical separation was according to mass in a 12.5% SDS gel Optimized Ruthenium staining: for each condition (activated meprin versus non-activated meprin), three pooled biological gel replicates (from 18 dishes per pooled sample) and two more technical gel replicates (of one pooled sample) were produced for subsequent image analysis Unique protein spots are labelled in level and higher-level match-sets with SSP assigned by the image analysis software 4492 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al Meprin protease proteomics Table Protein spot matching statistics 2D IEF ⁄ SDS ⁄ PAGE-based image analysis was performed with PDQUEST (version 7.3.1) on five gel replicates (three biological replicates, two technical replicates) of conditioned media from trypsin activated and non-activated MDCKa ⁄ b cells Qualitative spot matching differences among reference gels (level match-sets) are expressed as unique spots (% of each corresponding quadrant section) Gel replicates Reference gel Condition Quadrant Replicate Replicate Replicate Replicate Replicate Level match-set Activated meprin Non-activated meprin In total Activated meprin Non-activated meprin In total Activated meprin Non-activated meprin In total Activated meprin Non-activated meprin In total Activated meprin Non-activated meprin In total 1 2 3 4 All All All 315 334 313 333 318 334 315 332 316 332 318 334 221 217 219 212 221 218 215 216 218 217 222 218 106 107 103 110 116 113 115 103 117 104 122 119 105 110 107 108 115 109 108 102 105 104 Higher-level match-set 115 111 Unique spots (%) (0.6) 18 (5.4) 336 10 (4.4) (2.6) 228 12 (9.2) (6.9) 131 Protein identification by means of LC-MS/MS, PHENYX-based and BLASTP-based protein database searching By visual inspection, the 35 protein spots unique to media of trypsin activated MDCKa ⁄ b cells could be reduced to 33 putative candidates The redundancy of two spots present in more than one quadrant from each set of 2D gels analysed prompted correction (Fig 2; see Fig S1) On colloidal Coomassie stained preparative 2D gels, 24 protein spots of interest were detectable These spots could be rematched to putative candidates found in fluorescence stained analytical gels (data not shown) Gel plugs were then prepared, in-gel digested with trypsin and peptides thereof separated ⁄ fragmented by LC-MS ⁄ MS Collisioninduced dissociation (CID) spectra interpretation with phenyx (version 2.1) against the uniprot-SwissProt protein database (release 48.8) led to 22 (17 nonredundant) protein identifications (Fig and Table 2) The taxonomic search space was restricted to Mammalia (40 084 sequence entries) To double-check significant hits, the same spectra were interpreted with the webbased search engine mascot (version 2.1) against the same database and parameter settings (data not shown) [21] The identification of nucleophosmin (protein spot SSP 2102; Table 2) was accepted because the peptide VDNDENEHQLSR and its in-source produced fragment DNDENEHQLSLR were unambiguously identified with good scores by phenyx and 11 (9.0) (5.7) 122 777 782 35 (4.3) 40 (4.9) 817 mascot In addition, the whole tryptic peptide MSVQPTVSLGGFEITPPVVLR was identified by phenyx and mascot as first ranking identification, but with scores below the chosen acceptance criteria (Table and data not shown) Beside six positive hits for dog, other species (e.g rat, human, rabbit and mouse) were predominantly represented The current release (51.3) of the uniprot-SwissProt protein database lists 664 sequence entries for dog and thus may explain the poor representation in this species Recently, the dog genome was sequenced to completion [22] Peptide sequence tags deciphered from our previous analysis permitted search with blastp (version 2.2.16) against the 33 527 dog RefSeq protein sequence entries of the NCBI [21] All top scoring significant hits corresponded to predicted dog protein sequence entries Finally, all equivocal uniprot-SwissProt protein database searches were successfully matched to predicted dog protein orthologs (Table 3) Discovery of shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments phenyx offers the remarkable feature to search for non-tryptic peptides (i.e half-cleaved peptides) In-gel tryptic digestion of proteins contained within gel plugs produces peptide fragments terminating C-terminally with a lysine or arginine residue Trypsin cleavage specificity is then fixed to the N- or C-terminus FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4493 Meprin protease proteomics D Ambort et al A B C Fig Two-dimensional reference maps on protein identifications Representative 2D gel images of conditioned medium protein from MDCKa ⁄ b cells (A) 2D gel of trypsin activated meprin (B) 2D gel of non-activated meprin Unique protein spots were labelled with SSP defined by image analysis software (C) Close-up view of one representative protein spot, namely, SSP 7006 LC-MS ⁄ MS analysis of candidate substrates confirmed the validity of this protease proteomic approach for the discovery of shortened (non-trypsingenerated) N- and C-terminally truncated cleavage products in peptide fragments (Table 2) In silico digestion of the theoretical full-length protein product with trypsin enables the determination of all tryptic peptides terminating with a lysine or arginine 4494 residue Hence, peptide fragments not featuring a lysine or arginine residue in the C-terminal ends or truncated in the N-termini by some amino acids relative to the preceding in silico-generated tryptic fragments are candidates for proteolytically processed (non-trypsin-derived) cleavage products In a protease proteomic approach, this option facilitates the discovery of shortened (non-trypsin-generated) N- or C-terminally truncated cleavage products defined by meprin protease activity To determine new peptide ends other than lysine or arginine, peptides must not be identified either C- or N-terminal to the truncated peptide We applied this strategy to all protein database searches performed with phenyx Several shortened half-cleaved peptides (not full-length tryptic peptides) were detected (Table 2) Half-cleaved peptides may also originate from in-source fragmentation of intact tryptic peptides during the ionization process Accordingly, the following half-cleaved peptides co-eluted with corresponding intact tryptic peptides after chromatographic separation: TDGNSEHLKR and DGNSEHLKR from protein spots SSP 602 ⁄ 9602 and SSP 1602, respectively; PGPVFGSK from protein spot SSP 1602; and DNDENEHQLSLR from protein spot SSP 2102 The halfcleaved peptides derived from the sequence stretching over amino acids 159–182 of clusterin (IDSLLENDRQQTHALDVMWDSFNR) found in protein spots SSP 502 and SSP 1502 were chromatographically separated and thus may not refer to in-source fragmentation products Those half-cleaved products are most probably related to in-gel digestion artefacts because cleavage within this protein sequence stretch by meprin must be excluded due to an overall amino acid sequence coverage of this protein that exceeded amino acid 182 In addition, the two half-cleaved peptides DQAVSDTELQEMSTEGSK (residues 23–40) and DTELQEMSTEGSK (residues 28–40) in SSP 502 and 1502 were chromatographically separated and were not in-source fragmentation products generated during the ionization process The former peptide represented the mature N-terminus of clusterin (aspartate at position 23) and hence was not generated by meprin activity The latter peptide was presumably produced by meprinb with acidic amino acids preferred in the P1¢ position and selecting against basic amino acids in the P2¢ and P3¢ positions [10] The leguminous lectin-like VIP36 was present in two different protein spots (SSP 1602 and SSP 602 ⁄ 9602) and also met our criteria for shortened (non-trypsin-generated) C-terminally truncated cleavage products in peptide fragments In both spots, the truncated peptide LFQLMVEH (residues 273–280) was identified with no further peptides towards the C-terminal end (not ending with a lysine FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS Protein identificationb ALDOA_HUMAN ANXA1_RABIT CAPG_HUMAN CLUS_CANFA CLUS_CANFA CLUS_CANFA SSPa 8111 1703 2208 502 1405 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 1502 P25473 P25473 P25473 P40121 P51662 P04075 SwissProt accession number 12 11 2 Number of unique peptidesc Experimental m ⁄ z (Th) 522.771 696.365 758.492 708.385 905.045 694.331 631.73 676.259 793.287 985.952 735.824 616.842 680.894 616.363 616.421 552.277 732.427 537.749 593.764 714.356 616.738 552.23 714.245 985.976 735.838 616.818 680.9 552.847 733.334 537.774 877.014 593.776 903.468 714.365 764.85 Sequenced,e (60)R ⁄ QLLLTADDR(69) (69)R ⁄ VNPC^IGGVILFHETLYQK(87) (111)K ⁄ GVVPLAGTNGETTTQ#GLDGLSER(134) (153)K ⁄ IGEHTPSALAIM*ENANVLAR(173) (289)K ⁄ C^PLLKPWALTFSYGR(304) (58)K ⁄ GVDEATIIDILTK(71) (113)K ⁄ TPAQFDADELR(124) (115)K ⁄ YQEGGVESAFHK(127) (321)Q ⁄ YAPNTQVEILPQGR(335i) (22)G ⁄ DQAVSDTELQEM*STEGSK(40j) (27)S ⁄ DTELQ#EM*STEGSK(40k) (57)K ⁄ TLIEQTNEER(67) (57)K ⁄ TLIEQTNEERK(68) (67)R ⁄ KSLLSNLEEAK(78) (68)K ⁄ SLLSNLEEAKK(79) (68)K ⁄ SLLSNLEEAK(78) (81)K ⁄ EDALNDTKDSETK(94) (158)R ⁄ IDSLLENDR(167) (167)R ⁄ QQTHALDVM*Q(177) ⁄ Dl (182)R ⁄ ASSIM*DELFQDR(194) (57)K ⁄ TLIEQTNEER(67) (68)K ⁄ SLLSNLEEAK(78) (182)R ⁄ ASSIM*DELFQDR(194) (22)G ⁄ DQAVSDTELQEM*STEGSK(40j) (27)S ⁄ DTELQ#EM*STEGSK(40k) (57)K ⁄ TLIEQTNEER(67) (57)K ⁄ TLIEQTNEERK(68) (68)K ⁄ SLLSNLEEAK(78) (81)K ⁄ EDALNDTKDSETK(94) (158)R ⁄ IDSLLENDR(167) (158)R ⁄ IDSLLENDRQQTHAL(173) ⁄ Dl (167)R ⁄ QQTHALDVM*Q(177) ⁄ Dl (167)R ⁄ Q#QTHALDVM*QDSFNR(182) (182)R ⁄ ASSIM*DELFQDR(194) (335)K ⁄ LYDELLQSYQEK(347) 1043.561 2087.087 2273.102 2122.084 1807.944 1386.76 1261.593 1350.62 1584.826 1969.842 1470.603 1231.604 1359.699 1230.682 1230.682 1102.587 1464.658 1073.535 1185.544 1426.639 1231.604 1102.587 1426.639 1969.842 1470.603 1231.604 1359.699 1102.587 1464.658 1073.535 1751.88 1185.544 1805.8 1426.639 1527.745 Theoretical mass (Da) 0.017 0.338 0.216 )0.016 )0.065 0.057 0.074 0.059 0.134 )0.023 0.485 )0.032 )0.037 )0.014 )0.072 0.024 0.91 0.026 0.016 )0.029 0.072 0.071 0.082 )0.047 0.471 )0.008 )0.043 )0.546 0.003 0.001 )0.066 0.004 0.44 )0.038 0.03 Match delta m ⁄ z (Th)f 10.3 8.26 6.71 8.85 9.57 12.8 10.9 8.03 11.9 9.81 9.25 11.3 6.36 8.68 7.14 11.7 8.96 8.04 6.31 7.59 9.69 8.01 10.7 12.8 9.42 8.08 6.85 13.4 6.5 9.28 7.09 7.62 10.1 8.97 7.62 Peptide z-scoreg Table LC-MS ⁄ MS analysis of candidate substrates in discovery of shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments 1.20 2.44 3.00 1.51 6.92 1.54 1.56 1.65 6.76 6.45 1.72 1.82 1.63 3.46 8.22 1.79 5.08 8.17 5.16 2.53 2.75 2.18 4.56 9.20 3.50 5.51 6.10 1.00 6.00 1.59 9.27 2.11 2.74 4.82 3.90 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 10)19 10)11 10)6 10)13 10)17 10)32 10)22 10)10 10)28 10)18 10)15 10)24 10)5 10)13 10)8 10)26 10)14 10)11 10)5 10)9 10)17 10)10 10)22 10)33 10)16 10)11 10)7 10)35 10)6 10)15 10)8 10)9 10)19 10)14 10)9 Peptide P-valueh D Ambort et al Meprin protease proteomics 4495 4496 CO5A2_HUMAN CO5A2_HUMAN 4202 1104 2104 CO5A2_HUMAN CLUS_CANFA SSPa EF2_RAT FLNA_MOUSE LMAN2_CANFA 5106 Protein identificationb Table Continued 7006 1802 602 ⁄ 9602 P49256 Q8BTM8 P05197 P05997 P05997 P05997 P25473 SwissProt accession number 12 3 2 Number of unique peptidesc Experimental m ⁄ z (Th) 985.781 616.785 680.757 552.235 537.738 903.272 714.215 1007.856 584.217 862.413 584.201 862.836 584.212 862.33 622.16 797.785 714.301 689.733 750.424 818.007 713.941 752.41 692.894 578.845 528.275 758.428 500.22 1235.101 713.858 448.599 537.316 869.483 916.184 516.781 Sequenced,e (22)G ⁄ DQAVSDTELQEM*STEGSK(40j) (57)K ⁄ TLIEQTNEER(67) (57)K ⁄ TLIEQTNEERK(68) (68)K ⁄ SLLSNLEEAK(78) (158)R ⁄ IDSLLENDR(167) (167)R ⁄ QQTHALDVM*Q#DSFNR(182) (182)R ⁄ ASSIM*DELFQDR(194) (198)R ⁄ EPQDTYHYSPFSLFQR(214) (1273)K ⁄ SLSSQIETM*R(1283) (1368)R ⁄ GSQFAYGDHQSPNTAITQM*TFLR(1391) (1273)K ⁄ SLSSQIETM*R(1283) (1368)R ⁄ GSQFAYGDHQSPNTAITQM*TFLR(1391) (1273)K ⁄ SLSSQIETM*R(1283) (1368)R ⁄ GSQFAYGDHQSPNTAITQM*TFLR(1391) (1406)K ⁄ NSVGYM*DDQAK(1417) (580)R ⁄ ETVSEESNVLC^LSK(594) (605)K ⁄ ARPFPDGLAEDIDKGEVSAR(625) (727)R ⁄ C^LYASVLTAQPR(739) (1891)K ⁄ DAGEGGLSLAIEGPSK(1907) (2089)K ⁄ VDINTEDLEDGTC^R(2103) (2264)R ⁄ EAGAGGLAIAVEGPSK(2280) (2346)K ⁄ VNQPASFAVSLNGAK(2361) (44)A ⁄ DITDGNSEHLKR(56j) (46)I ⁄ TDGNSEHLKR(56m) (47)T ⁄ DGNSEHLKR(56m) (126)K ⁄ NLHGDGIALWYTR(139) (141)R ⁄ LVPGPVFGSK(151) (151)K ⁄ DNFHGLAIFLDTYPNDETTER(172) (195)R ⁄ WTELAGC^TADFR(207) (207)R ⁄ NRDHDTFLAVR(218) (209)R ⁄ DHDTFLAVR(218) (223)R ⁄ LTVM*TDLEDKNEWK(237) (246)R ⁄ LPTGYYFGASAGTGDLSDNHDIISM*K(272) (272)K ⁄ LFQLM*VEH(280) ⁄ Tk 1969.842 1231.604 1359.699 1102.587 1073.535 1805.8 1426.639 2013.922 1166.56 2585.197 1166.56 2585.197 1166.56 2585.197 1242.518 1593.755 2142.07 1377.707 1499.746 1635.704 1425.746 1501.788 1383.674 1155.563 1054.515 1514.763 999.575 2467.129 1425.634 1342.674 1072.53 1736.829 2745.259 1031.511 Theoretical mass (Da) 0.148 0.025 0.1 0.066 0.037 0.636 0.112 0.113 0.071 0.327 0.087 )0.096 0.076 0.41 0.107 0.1 0.73 0.128 0.457 0.853 )0.06 )0.508 )0.049 )0.056 )0.01 )0.039 0.575 )0.529 )0.033 )0.034 )0.043 )0.061 )0.09 )0.018 Match delta m ⁄ z (Th)f 7.09 10.8 8.63 8.81 8.74 7.75 10.4 6.16 10.7 10.9 7.27 10.4 6.84 10 16.1 13.6 11.9 9.27 8.84 7.27 10.9 7.81 7.5 7.39 12 6.61 10.3 10.6 7.24 11.8 6.41 8.27 7.11 Peptide z-scoreg 8.33 5.30 4.85 1.19 2.17 6.29 1.42 8.72 1.43 3.60 1.26 4.86 2.84 1.10 2.68 1.90 5.50 2.03 1.43 1.46 2.85 5.55 4.37 1.23 2.74 2.25 7.88 5.12 3.30 5.41 5.80 9.90 2.00 1.13 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 10)8 10)22 10)13 10)13 10)13 10)10 10)20 10)5 10)21 10)7 10)22 10)8 10)20 10)6 10)18 10)53 10)37 10)27 10)15 10)13 10)8 10)23 10)10 10)8 10)8 10)28 10)6 10)20 10)21 10)8 10)27 10)6 10)11 10)7 Peptide P-valueh Meprin protease proteomics D Ambort et al FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS LMNA_RAT LOXL1_HUMAN 1602 5101 3502 NPM_RAT SERC_HUMAN STC1_HUMAN 2102 8110 4104 LYOX_RAT LMAN2_CANFA SSPa 403 ⁄ 9302 Protein identificationb Table Continued P52823 Q9Y617 P13084 P16636 Q08397 P48679 P49256 SwissProt accession number 9 Number of unique peptidesc Experimental m ⁄ z (Th) 692.816 578.799 757.873 394.734 1234.648 713.829 537.151 524.223 516.778 815.299 545.274 574.713 582.603 622.29 638.275 560.2 591.74 497.874 951.853 834.825 801.35 661.002 1119.092 892.899 784.81 735.34 1121.629 557.281 580.824 821.989 757.019 509.705 562.753 573.944 775.374 751.869 741.405 818.872 Sequenced,e (44)A ⁄ DITDGNSEHLKR(56j) (46)I ⁄ TDGNSEHLKR(56m) (126)K ⁄ NLHGDGIALWYTR(139) (143)V ⁄ PGPVFGSK(151m) (151)K ⁄ DNFHGLAIFLDTYPNDETTER(172) (195)R ⁄ WTELAGC^TADFR(207) (209)R ⁄ DHDTFLAVR(218) (237)K ⁄ NC^IDITGVR(246) (272)K ⁄ LFQLM*VEH(280) ⁄ Tk (28)R ⁄ LQEKEDLQELNDR(41) (50)R ⁄ SLETENAGLR(60) (62)R ⁄ ITESEEVVSR(72) (78)K ⁄ AAYEAELGDAR(89) (133)R ⁄ LKDLEALLNSK(144) (144)K ⁄ EAALSTALSEKR(156) (144)K ⁄ EAALSTALSEK(155) (156)R ⁄ TLEGELHDLR(166) (196)R ⁄ LQ#TLKEELDFQK(208) (400)K ⁄ C^LASTAYAPEATDYDVR(417) (540)K ⁄ YIVLESDFTNNVVR(554) (231)R ⁄ C^AAEENC^LASSAYR(245) (314)K ⁄ ASFC^LEDTSC^DYGYHR(330) (371)K ⁄ VSVNPSYLVPESDYSNNVVR(391) (395)R ⁄ YTGHHAYASGC^TISPY(411j) (32)K ⁄ VDNDENEHQLSLR(45) (33)V ⁄ DNDENEHQLSLR(45m) (80)K ⁄ M*SVQPTVSLGGFEITPPVVLR(101) (5)R ⁄ QVVNFGPGPAK(16) (61)R ⁄ ELLAVPDNYK(71) (169)K ⁄ GAVLVC^DM*SSNFLSK(184) (169)K ⁄ GAVLVC^DM*SSNFLSKPVDVSK(190) (213)R ⁄ DDLLGFALR(222) (222)R ⁄ EC^PSVLEYK(231) (323)K ⁄ ALELNM*LSLK(333) (342)R ⁄ ASLYNAVTIEDVQK(356) (119)R ⁄ M*IAEVQEEC^YSK(131) (139)K ⁄ RNPEAITEVVQ#LPNHFSNR(158) (165)R ⁄ SLLEC^DEDTVSTIR(179) 1383.674 1155.563 1514.763 787.422 2467.129 1425.634 1072.53 1046.517 1031.511 1628.8 1088.546 1147.572 1164.541 1242.718 1274.683 1118.581 1181.604 1491.782 1901.846 1667.851 1600.661 1979.778 2237.096 1783.762 1567.722 1468.654 2242.203 1112.597 1160.607 1642.769 2268.113 1018.544 1123.522 1146.631 1549.798 1501.642 2221.124 1636.761 Theoretical mass (Da) 0.029 )0.01 0.516 )0.015 )0.076 )0.004 0.122 0.043 )0.015 0.109 0.007 0.081 0.675 0.077 0.074 0.098 0.07 0.394 0.078 0.108 )0.012 )0.069 0.464 )0.01 0.059 )0.005 0.48 0.025 0.487 0.403 0.026 0.575 0.016 0.379 0.533 )0.04 )0.023 0.516 Match delta m ⁄ z (Th)f 8.59 6.43 13.4 9.47 9.1 7.65 8.88 9.45 7.94 9.73 9.19 12.2 9.6 9.89 6.15 9.7 9.18 7.24 9.85 14.1 12.9 7.82 8.29 6.27 8.1 8.31 6.52 8.24 7.51 7.3 8.83 7.01 7.92 9.29 9.82 8.45 10.1 10.1 Peptide z-scoreg 6.59 1.04 2.85 3.28 4.37 3.25 1.30 3.29 4.22 1.57 8.26 2.80 1.52 3.58 6.67 2.86 3.50 5.12 8.28 2.75 3.00 5.07 6.36 2.29 3.99 7.53 7.52 3.43 1.14 2.52 1.93 4.97 2.03 2.90 6.97 2.22 1.04 2.41 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 10)13 10)5 10)36 10)16 10)15 10)9 10)13 10)16 10)10 10)17 10)15 10)29 10)16 10)18 10)5 10)17 10)15 10)8 10)18 10)40 10)33 10)10 10)12 10)5 10)11 10)12 10)6 10)11 10)8 10)7 10)13 10)7 10)10 10)15 10)18 10)12 10)18 10)19 Peptide P-valueh D Ambort et al Meprin protease proteomics 4497 SSP assigned by image analysis software PDQUESt, version 7.3.1 b CID spectra interpretation with public search engine PHENYX (version 2.1) on vital-it.ch against uniprot-SwissProt protein database (release 48.8) Taxonomy search space restricted to Mammalia (40 084 sequence entries) CANFA, Canis familiaris, dog; RAT, Rattus norvegicus, rat; HUMAN, Homo sapiens, human; RABIT, Oryctolagus cuniculus, rabbit; MOUSE, Mus musculus, mouse c For multiple peptide matches to same primary sequence, the top scoring peptide was listed d Modifications: C^, carbamidomethylation of cysteine; M*, oxidation of methionine; Q#, deamidation of glutamine e Numbers in parentheses indicate the P1 positions of cleavages [Correction added August 2008, after first online publication: in the preceding sentence ‘superscript numbers’ was corrected to ‘numbers in parentheses’.] f Match delta is the difference between theoretical m ⁄ z of matched peptide and observed m ⁄ z of parent ion g Peptide search criteria were set to a minimum peptide z-score of ‡ h Only protein identifications consisting of at least two unique peptides reaching a P-value of £ 0.00000001 were accepted i Normal tryptic peptide (dog protein ortholog with arginine in P1 position instead of glutamine) j N- and C-terminal half-cleaved peptides k Shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments l Half-cleaved peptides generated during in-gel digestion m In-source fragmentation products 7510 P18206 VINC_HUMAN 4302 D Ambort et al a 10)18 10)11 10)13 10)12 10)12 10)11 10)21 10)14 · · · · · · · · 3.01 1.43 1.02 1.08 2.86 1.93 8.42 1.02 9.93 8.3 8.82 8.53 8.52 8.2 10.5 9.06 0.095 )0.403 0.069 0.107 0.917 0.587 0.081 0.097 1029.488 2578.328 1061.55 1615.806 1228.71 1314.714 1172.64 1268.672 515.657 860.853 531.714 808.804 614.446 657.778 587.247 635.247 (50)K ⁄ GPDPSSPAFR(60) (60)R ⁄ IEDANLIPPVPDDKFQDLVDAVR(83) (74)K ⁄ FQDLVDAVR(83) (201)K ⁄ GGVNDNFQGVLQNVR(216) (199)K ⁄ ELLPVLISAM*K(210) (464)K ⁄ Q#VATALQNLQTK(476) (570)R ⁄ ALASQLQDSLK(581) (802)K ⁄ AVAGNISDPGLQK(815) TSP1_HUMAN SSPa P07996 Sequenced,e Match delta m ⁄ z (Th)f 4498 Protein identificationb Table Continued SwissProt accession number Number of unique peptidesc Experimental m ⁄ z (Th) Theoretical mass (Da) Peptide z-scoreg Peptide P-valueh Meprin protease proteomics or arginine residue) Additionally, this truncated peptide was not generated by in-source fragmentation because there was no co-eluting ion trace of the corresponding whole tryptic peptide LFQLMVEHTPDEENIDWTK VIP36 was described as a single-pass type I membrane protein with an extracellular carbohydrate recognition domain exactly terminating at those amino acids (residues 52–280) [23] Moreover, the amino acid sequence following the putative cleavage site corresponded to cleavage preference for meprina with the amino acids threonine and proline in the P1¢ and P2¢ positions [10] The targeted cleavage by hmeprin after this specific domain may indicate protein ectodomain shedding Nevertheless, the biological consequence of this remains to be elucidated Functional clustering into biological process and molecular function Next, the proteins identified by LC-MS ⁄ MS as putative meprin substrates were classified into functional groups according to the Human Protein Reference Database (Table 3) [24] Ten proteins could be assigned to the biological process of ‘cell growth and ⁄ or maintenance’ and four to ‘immune response’ (Fig 4) The remaining proteins were equally distributed into functional classes such as ‘transport’, ‘cell communication; signal transduction’, ‘metabolism; energy pathways’ and ‘protein metabolism’ In conclusion, these findings suggest possible functions for meprin in the regulation of cell homeostasis and the extracellular environment, and in the immune response Effect of in situ trypsin treatment Zymogen activation by limited trypsin treatment may lead to changes elicited by the trypsin and not by meprin To exclude such unspecific side effects caused by the trypsin treatment rather than by the effector (membrane-bound hmeprina ⁄ b), wild-type (WT) and meprina ⁄ b MDCK cells were treated in the same way Media of trypsin-treated and nontreated cells were prepared and then subjected to 1D SDS ⁄ PAGE and subsequent densitometric image analysis with aida software (Fig 5) We decided to perform a comparison between conditioned media of WT and meprina ⁄ b MDCK cells on 1D gels Quantitative assessment of protein bands revealed no significant differences between trypsin-treated and nontreated WT samples, whereas meprina ⁄ b samples showed substantial differences upon trypsin activation Moreover, the protein patterns of WT versus FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al Meprin protease proteomics Table BLASTP-based protein database searching and functional classification All peptide sequence tags (Table 2) were searched against the dog genome database using BLASTP, version 2.2.16 Database size was 33 527 dog RefSeq protein sequences The database is hosted at NCBI Functional classification according to Human Protein Reference Database Protein description Biological process Molecular function NCBI accession number SSPa Scoreb PREDICTED: similar to annexin A1 Clusterin Cell communication; signal transduction Immune response Calcium ion binding XP_533524 1703 57.1 Complement activity NP_001003370 PREDICTED: similar to collagen alpha 2(V) chain precursor PREDICTED: similar to elongation factor PREDICTED: similar to filamin A isoform PREDICTED: similar to fructose-bisphosphate aldolase A isoform PREDICTED: similar to lamin A ⁄ C isoform Lectin, mannose-binding Cell growth and ⁄ or maintenance Extracellular matrix, structural constituent XP_535998 Protein metabolism Translation regulator activity Cytoskeletal anchoring activity Lyase activity XP_533949 502 1405 1502 4202 1104 2104 5106 7006 99 61.3 125 103 50.4 50.4 62.8 62 XP_867537 1802 60.8 XP_849434 8111 117 3.00 · 10)27 Cell growth and ⁄ or maintenance Transport Structural molecule activity Transporter activity XP_864487 5101 104 2.00 · 10)23 NP_001003258 PREDICTED: similar to macrophage capping protein PREDICTED: similar to nucleophosmin isoform 12 PREDICTED: similar to phosphoserine aminotransferase isoform PREDICTED: similar to protein-lysine 6-oxidase precursor isoform PREDICTED: similar to stanniocalcin-1 precursor PREDICTED: similar to thrombospondin precursor PREDICTED: similar to vinculin Cell growth and ⁄ or maintenance Protein metabolism Cytoskeletal protein binding Chaperone activity XP_540197 602 ⁄ 9602 1602 2208 219 129 48.6 8.00 · 10)58 7.00 · 10)31 4.00 · 10)7d XP_866781 2102 57.8 2.00 · 10)9 Metabolism; energy pathways Transaminase activity XP_533520 8110 72.4 7.00 · 10)14 Cell growth and ⁄ or maintenance Catalytic activity XP_859412 403 ⁄ 9302 3502 90.5 33.3 3.00 · 10)19 0.017d Cell communication; signal transduction Cell growth and ⁄ or maintenance Cell growth and ⁄ or maintenance Calcium ion binding XP_543238 4104 79.7 5.00 · 10)16 Extracellular matrix, structural constituent Cytoskeletal protein binding XP_544610 4302 72 1.00 · 10)13 XP_536395 7510 42.6 7.00 · 10)5d Cell growth and ⁄ or maintenance Metabolism; energy pathways Expected valuec 1.00 · 10)9d 9.00 7.00 1.00 2.00 3.00 3.00 6.00 1.00 · · · · · · · · 10)22 10)11d 10)29 10)23 10)7 10)7 10)11 10)10 2.00 · 10)10 SSP assigned by image analysis software PDQUEST, version 7.3.1 b Only the top scoring significant hit was accepted c Search parameters: word size 3, filter low complexity, expect value 0.01, score matrix BLOSUM62 d Failed searches were repeated with settings for ‘short and nearly exact matches’: word size 2, filter off, expect value 20 000, score matrix PAM30 a meprina ⁄ b differed as well (Fig 5A) and indicated that overexpression of hmeprina ⁄ b per se causes differences that are independent from zymogen activation Finally, triplicate image analysis of gel lanes confirmed these findings (Fig 5B) but, more importantly, revealed a trend towards the appearance of low molecular weight proteins in media of meprina ⁄ b MDCK cells Hence, the triplicate assessment of data generated unambiguously pointed to reproducible differences triggered by the activation and not by the overexpression of meprina ⁄ b (Fig 5C) Obviously, activation of meprina ⁄ b results in the release of proteins into the culture medium Validation of direct or indirect effects by immunoblotting follow-up experiments Proteomics is a very powerful tool for proteasesubstrate identification, but the data obtained need to be verified by means of alternative techniques Western FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4499 Meprin protease proteomics D Ambort et al Fig Functional classification of identified proteins Pie chart showing the distribution of 22 identified proteins into their functional classes Functional classification was performed according to the Human Protein Reference Database For details, see Table blotting experiments revealed not only direct effects exhibited by the activity status of meprin (activated meprin versus non-activated meprin), but also indirect effects mediated by overexpression of meprin (WT versus meprina ⁄ b) Direct effects were observed for vinculin, lysyl oxidase and collagen type V (Fig 6A–C) Indirect effects were noted for annexin A1 (Fig 6D) The cytosolic actin-binding protein vinculin was found in the culture medium of MDCK cells (Fig 6A) The 116 kDa full-length form was detected in all samples, whereas putative cleavage products with molecular weights of 75 and 85 kDa [25], respectively, were visualized exclusively in media of MDCK cells expressing activated meprina ⁄ b The ECM stabilizing protein lysyl oxidase was reported to be synthesized as a 46 kDa precursor that is processed in the extracellular environment to the catalytically functional 32 kDa form by bone morphogenetic protein (BMP-1) ⁄ tolloid (TLD)-like metalloendopeptidases [26] We observed the presence of a 25 kDa protein species in media of MDCK cells expressing activated meprina ⁄ b (Fig 6B) Type V collagen is a quantitatively minor component of predominantly type I collagen fibrils in most noncartilage tissues and is required for collagen fibril nucleation [27] The monoclonal antibody 1E2E4 ⁄ Col5 did not recognize collagen type V on blots of denaturing, reducing SDS gels (Fig 6C) [28] Repetition of the experiment under nondenaturing, nonreducing conditions on a dot blot confirmed the absence of native collagen type V in media of WT and meprina ⁄ b MDCK cells (data not shown) We then systematically 4500 mapped all peptide sequences of collagen type V identified by LC-MS ⁄ MS to the full-length sequence as deposited in uniprot-SwissProt protein database Interestingly, all tryptic peptides from the three independent protein spots, SSP 1104, SSP 2104 and SSP 5106, matched the C-terminal propeptide region (residues 1227–1496) of collagen type V (Table 2) These findings suggest a putative role for hmeprin in the regulation of collagen assembly Annexin A1 is a calcium ⁄ phospholipid-binding protein that provides a link between calcium signalling and membrane functions [29] Two bands of 32 kDa and 35 kDa in size were found in conditioned media of WT MDCK cells (Fig 6D) In media of MDCKa ⁄ b cells, the 32 kDa form was not detectable Obviously, overexpression of hmeprina ⁄ b in MDCK cells abolished the 32 kDa band There was no marked difference between the trypsin-treated and nontreated cells This finding could indicate an indirect effect exerted by overexpression of meprin per se and not by the activity status of meprin Discussion Establishment of a simple 2D IEF/SDS/ PAGE-based protease proteomic approach To date, some MMPs and ADAMs have been characterized on a system-wide level by means of protease proteomics [14–17] Two protease proteomic approaches defined the substrate repertoire of membrane-type 1-MMP (MT1-MMP ⁄ MMP-14) in a cell culture system-based environment or using human plasma as a polysubstrate [15,17] The other two studies described the substrate protease proteome of tumor necrosis factor-a converting enzyme ⁄ ADAM-17 in a cell culture system [14,16] These protease proteomes were defined using multi-dimensional LC-MS ⁄ MS with ICAT labelling or 2D IEF ⁄ SDS ⁄ PAGE with (or without) lectin-affinity pre-fractionation and cyanine dye labelling None of these studies systematically grouped the putative substrates into specific, functional categories In addition, no systematic display of data on biological replicates was presented Moreover, applying pre-fractionation techniques (i.e selecting for cysteine-containing peptides or glycoproteins) may allow for higher resolution capacity but at the cost of information loss For example, using ICAT labelling, only pairs of intact peptides are compared between two different conditions and therefore it is not possible to find shortened (non-trypsin-generated) N- or C-terminally truncated peptide fragments Furthermore, it is not possible to capture nonglycosylated FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al A Meprin protease proteomics B C Fig Effect of in situ trypsin treatment (A) Representative 1D SDS ⁄ PAGE separation of conditioned medium protein (20 lg per lane) from trypsin-treated (+) and nontreated ()) WT and meprina ⁄ b MDCK cells in a 12.5% SDS gel under reducing conditions Optimized Ruthenium staining: migration positions of molecular mass standards are shown on the gel In total, three independent technical gel replicates were produced (B) Densitometric analysis of profile scans from a representative 1D gel For each lane, a rectangular densitometric window was used to graphically display pixel intensity (quantum levels, QL) versus migration position (pixel) Peaks were subdivided into integrable areas and numbered WT (upper graph) and meprina ⁄ b profiles (lower graph) were then superimposed (C) Averaged quantitative comparison of WT (left hand side) and meprina ⁄ b peaks (right hand side) from three independent analyses Intensity of peak areas (QL) was backgroundcorrected (Bkg) proteins and peptide fragments with lectin affinity pre-fractionation In the present study, we demonstrate the applicability of a simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure to analyse candidate substrates for meprin in a cell culture system-based approach (Figs and 2; Table 1; see Fig S1) Despite previous reports on the limited resolution capacity of 2D gels to find putative cleavage products, we identified novel meprin substrates with cleaved (non-trypsin-generated) N- and C-termini in peptide fragments upon LC-MS ⁄ MS analysis [14,16] In a previously described 2D FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4501 Meprin protease proteomics D Ambort et al A B C D Fig Validation experiments by western blotting Conditioned medium protein of trypsin-treated (+) and nontreated ()) WT and meprina ⁄ b MDCK cells was separated according to mass as described in Fig Immunoblotting with antibodies against (A) vinculin, (B) lysyl oxidase, (C) collagen type V and (D) annexin A1 The migration positions of molecular mass standards and protein loading amounts are indicated IEF ⁄ SDS ⁄ PAGE-based investigation, a commercially available colloidal Coomassie stain was used that lacked the sensitivity of our house-made fluorescent dye [14] The improved detection sensitivity of Ruthenium staining helped to identify candidate substrates Further progress was achieved by the systematic use of 2D gel replicates (three pooled biological gel replicates and two more technical gel replicates), which enabled the design of a simple image analysis procedure that unmasked step-by-step qualitative differences among gel replicates The modular character of this type of analysis allowed the integration of all unique protein spots into one big 2D reference map Due to the poor representation of dog proteins (664 sequence entries) in the uniprot-SwissProt protein database (release 51.3), unambiguous protein and species identifications were inferred from peptide sequence tags searched with blastp (version 2.2.16) against the NCBI dog genome database (33 527 dog RefSeq protein sequence entries) (Table 3) [22,30] This valuable strategy was used in similar cases where the species of interest was underrepresented in protein databases [31] Moreover, visual inspection of the peptide sequence list revealed a hith4502 erto unkown shortened (non-trypsin-generated) peptide, LFQLMVEH (residues 273–280) of VIP36 (Table 2), which matched the reported cleavage preference for meprina [10] This finding points to a shortened C-terminus (not bearing a lysine or arginine residue) and overcomes the technical limitations of ICAT-based approaches [15] Although the possibility cannot be excluded that trypsin treatment of MDCK cells may activate other proteases in the system, we have reduced this to an absolute minimum Two potential N-terminally shortened half-cleaved peptides, TDGNSEHLKR (residues 47–56) and DGNSEHLKR (residues 48–56), which were truncated by only one or two amino acids, were found in two different protein spots (SSP 1602 and SSP 602 ⁄ 9602, respectively) of VIP36 (Table 2) However, these two N-terminally shortened peptides were generated from the corresponding tryptic peptide DTGNSEHLKR (residues 45–56) of the mature N-terminus during the ionization process and were therefore ‘in-source’ fragmentation products Hence, proteolytic processing of these two cleavage fragments by aminopeptidases or possibly dipeptidyl-peptidases may be excluded The classifica- FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al tion of protein identifications into functional groups with the Human Protein Reference Database facilitated the interpretation of the data generated (Fig and Table 3) [24] Hence, the metalloendopeptidase meprin may be involved in processes of ‘cell growth and ⁄ or maintenance’ and ‘immune response’ Taken together, the novel strategies and applications presented herein may help to understand more precisely the function of a protease in a complex environment Novel roles for hmeprin in homeostasis of cell, cellular environment and in immune response BMP-1, mammalian TLD and hmeprin belong to the same metzincin subfamily of metalloendopeptidases, the astacin family [32] The main functions of BMP1 ⁄ TLD-like metalloendopeptidases are ascribed to the proteolytic removal of C-propeptides from fibrillar procollagens and to activation of lysyl oxidase [26,27,33] Upon activation, lysyl oxidase mediates the oxidative deamination of lysine residues to highly reactive aldehydes that spontaneously cross-link processed collagen monomers [26] Cross-linkage in self-assembling fibrous collagen is essential for its structural integrity Quantitatively minor type V collagen mainly exists as a1(V)2a2(V) heterotrimers that are incorporated into type I collagen fibrils and initiate collagen fibril assembly in regions of new tissue formation [27,28,33] Interestingly, we identified collagen type V in three individual protein spots and all peptides identified matched the C-terminal propeptide region (residues 1227–1496) (Table 2) The three protein isoforms found may represent differently glycosylated variants because collagen type V (SwissProt entry P05997) exhibits two N-glycosylation sites at amino acid residues 1262 and 1400, respectively In addition, the monoclonal antibody 1E2-E4 ⁄ Col5 did not detect native collagen type V in cell culture supernatants of WT and meprina ⁄ b MDCK cells under nondenaturing, nonreducing conditions Moreover, a single 25 kDa protein form of lysyl oxidase was solely found in media of trypsin activated MDCKa ⁄ b cells (Fig 6B) As previously described, lysyl oxidase acts only on processed collagens and not on its precursors; thus, the 25 kDa form potentially exhibits amine oxidase activity [26] Hence, we may speculate that hmeprin has activity similar to BMP-1 ⁄ TLD-like metalloendopeptidases in that it acts as a procollagen C protease as well as an activator of lysyl oxidase Therefore, an important role for hmeprina ⁄ b may be ascribed to tissue remodelling processes through the targeted regulation of ECM assembly Vinculin is an actin-binding protein localized on the cytoplasmic face of integrin-mediated cell-ECM junc- Meprin protease proteomics tions designated as focal adhesions [25] Vinculin stabilizes focal adhesions and thereby suppresses cell migration This effect is relieved by transient changes in local concentrations of inositol phospholipids It thus serves a regulatory, dynamic linkage between the ECM and intracellular actin cytoskeleton It was demonstrated that acidic phospholipids inhibit intramolecular association between the N- and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites on serines 1033 and 1045 [34,35] Upon activation of hmeprina ⁄ b in stably transfected MDCK cells, the 116 kDa full-length form of vinculin and truncated forms (75 and 85 kDa) were detected in cell culture supernatants (Fig 6A) These findings raise the question how meprin elicits such effects because the catalytic protease domain is localized extracellularly [32] One possible explanation is that meprin exerts its functions by intracellular signalling Indeed, hmeprinb possesses a C-terminal cytoplasmic domain with a protein kinase C phosphorylation site on serine 687 [36] Hence, meprinb, a single-pass type I membrane protein, may function as a signalling receptor [32] This feature provides a link to another metzincin subfamily, the ADAMs, because ADAM-15 was reported to interact specifically with Src family protein-tyrosine kinases upon phosphorylation on tyrosines 715 and 735 [37] Although the exact mechanisms underlying the secondary or downstream intracellular proteolytic events are not yet clear, hmeprin might mediate important intracellular signalling via its C-terminal domain, leading to regulation of cytoskeletal rearrangement during tissue remodelling processes The leguminous lectin-like vesicular integral-membrane protein VIP36 was originally identified as a component of glycolipid rafts and exocytic carrier vesicles in epithelial cells [38] Due to its homology to the mannose-selective lectin ERGIC-53, it was suggested that VIP36 operates in quality control of glycosylation in the Golgi [39] However, in MDCK cells, VIP36 is also localized to the apical plasma membrane and appears to be involved in intracellular transport and secretion of glycoproteins containing N-linked glycans [40] The meprins are extensively glycosylated, comprising approximately 25% carbohydrates, which are N-linked in meprina and both N- and O-linked in meprinb [41,42] Hence, VIP36 potentially interacts with meprina and ⁄ or meprinb via N-linked glycans Because there is no specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines the apical sorting of mouse meprina [41], VIP36 may direct the apical targeting Upon detailed analysis of peptides from two separate protein spots corre- FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4503 Meprin protease proteomics D Ambort et al sponding to VIP36, all the peptide sequences that were found consistently matched the extracellular carbohydrate recognition domain (Table 2) Because VIP36 is a single-pass type I membrane protein, and halfcleaved peptides terminating exactly at the end of the carbohydrate recognition domain were found, hmeprina appears to shed VIP36 from the plasma membrane [23] Analogous protein ectodomain shedding processes were described for MT-MMPs and ADAMs [14–16] The biological consequence of this event remains elusive In conclusion, subsequent to the introduction of the term protease proteome, various novel technological approaches have emerged and been successfully applied to decipher the substrate repertoire of a given protease on a system-wide level [13–17] The present study comprises the first protease proteomic approach implemented on an astacin family member of metalloendopeptidases On the basis of our findings, hmeprin may be considered as a signalling protease mediating direct and indirect cleavage and signal transduction functions as well as degrading of a number of ECM proteins Although the detailed mechanisms need to be determined, hmeprin appears to be involved in ‘cell growth and ⁄ or maintenance’ and in ‘immune response’ Fascinatingly, all protease proteomic approaches employed on metzincin metalloendopeptidases, namely MMP-14 and tumor necrosis factor-a converting enzyme ⁄ ADAM-17 [14–17], lead to the same conclusion: metalloendopeptidases are the most crucial entities in the regulation of cell homeostasis and its environment and in innate immunity Therefore, future protease proteomic studies will aim to expand our current knowledge on astacin family members and their relatives Experimental procedures Materials ImmobilineÔ DryStrips pH 3–10 NL and PharmalyteÔ 3–10 were purchased from Amersham Biosciences (Uppsala, Sweden); ammonium persulfate was obtained from Bio-Rad Laboratories (Richmond, CA, USA); CoomassieÒ Brilliant Blue G 250, iodoacetamide and thiourea were obtained from Fluka (Buchs, Switzerland); ZOOM urea was from Invitrogen (Carlsbad, CA, USA); dithioerythritol, EDTA, formic acid, glycerol (approximately 87%) and paraffin oil were purchased from Merck (Darmstadt, Germany); ProtoGel acrylamide stock solution was obtained from National Diagnostics (Atlanta, GA, USA); sequencing grade modified trypsin was obtained from Promega (Madison, WI, USA); acetonitrile was from Riedel-de-Haen Fluka; SDS ă 4504 was obtained from Serva (Heidelberg, Germany); Phenylmethanesulfonyl fluoride and Tween 20 were obtained from Sigma-Aldrich (St Louis, MO, USA); Chaps was obtained from USB corporation (Cleveland, OH, USA) Cell culture and meprin activation by in situ trypsin treatment Meprina ⁄ b MDCK cells were grown in minimum essential medium with Earle’s salts supplemented with 5% (v ⁄ v) fetal bovine serum, 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin [6,43] For serum-free conditions, the same medium composition was used without fetal bovine serum 1.15 · 106 cells were plated in 100 mm dishes and incubated for approximately days at 37 °C in an atmosphere of 5% CO2 until cells were confluent For limited trypsin treatment, cells were washed twice with mL of serum-free medium [5] Cells were then treated with 40 lL of trypsin solution (1 mgỈmL)1 in 50 mm Tris–HCl, pH 7.5) diluted in mL of serum-free medium for 30 at 37 °C For inactivation of trypsin, cells were washed twice with mL of serum-free medium Cells were then incubated with 40 lL of soja bean trypsin inhibitor solution (2 mgỈmL)1 in water) diluted in mL of serum-free medium for 30 at 37 °C After inactivation, cells were washed twice with mL of serum-free medium Negative controls were treated in the same way but without trypsin Upon activation, cells were conditioned in mL of serum-free medium for 22 h at 37 °C Sample preparation of culture medium Sample was prepared as previously described [44] The culture medium of 18 experimental replicates per condition (70–72 mL) was collected and defined as pooled biological replicate Protease inhibitors (1 mm EDTA, mm phenylmethanesulfonyl fluoride) were immediately added and the conditioned medium clarified by centrifugation for h at 100 000 g at °C in a fixed-angle rotor (TFT 70.38) on a Kontron Centrikon T-2060 ultracentrifuge (Kontron Instruments AG, Zurich, Switzerland) Supernatants were ă concentrated 300-fold by ultrafiltration in CentriconÒ Plus70 centrifugal filter devices (Millipore Corporation, Billerica, MA, USA) at °C Concentrates were washed three times in sample solubilization buffer (20 mm Tris, pH 9.0, mm EDTA, mm phenylmethanesulfonyl fluoride) Final protein concentration was determined with the BCAÔ Protein Assay Kit (Pierce, Rockford, IL, USA) 2D IEF/SDS/PAGE 2D IEF ⁄ SDS ⁄ PAGE was performed essentially as described [45] Three pooled biological replicates and two more technical replicates were run to have in total five 2D gels per condition (activated meprin versus non-activated FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al meprin) for subsequent analytical image analysis For analytical gels, 250 lg of concentrated medium protein from trypsin activated and non-activated MDCKa ⁄ b cells was solubilized in 450 lL of buffer containing m urea, m thiourea, 4% (w ⁄ v) Chaps, 1% (w ⁄ v) dithioerythritol and 2% (v ⁄ v) Pharmalyte 3–10 for h on a rotary shaker at room temperature Sample-containing buffer was then centrifuged for 30 at 16 100 g before application to IPG strips (pH 3–10 NL, 24 cm; Amersham Biosciences) Strips were rehydrated overnight in sample-containing buffer on the ImmobilineÔ DryStrip Reswelling Tray (Amersham Biosciences) under paraffin oil Focusing was always started at 300 V, and the voltage was slowly increased in a linear gradient to 3500 V until a final volthour product of 63 kVh was reached Focusing was performed on a MultiphorÔ II horizontal electrophoresis apparatus (Amersham Biosciences) under paraffin oil at 20 °C After focusing the strips were equilibrated in m urea, 30% (v ⁄ v) glycerol, 2% (w ⁄ v) SDS, 50 mm Tris–HCl, pH 8.8, with 1% (w ⁄ v) dithioerythritol and 4.8% (w ⁄ v) iodoacetamide, respectively, with each step being performed for 15 For the second dimension, strips were transferred to 12.5% acrylamide (% T), 2.6% crosslinker (% C) SDS ⁄ PAGE gels (255 · 205 · 1.5 mm) using the EttanÔ Daltsix multiple vertical electrophoresis apparatus (Amersham Biosciences) with running conditions: 15 mA per gel for h 30 and 50 mA per gel for 5–6 h at 15 °C For preparative gels, mg of protein was solubilized in mL of buffer for h at room temperature, centrifuged for 30 at 16 100 g, concentrated ten-fold in CentriconÒ YM-3 centrifugal filter devices (Millipore Corporation) and then diluted with buffer to a final volume of 450 lL The total volthour product was increased to approximately 80 kVh The cathodic paper wick was immersed in 1% (w ⁄ v) dithioerythritol before the run to counteract depletion effects in the basic part of the strip 1D SDS/PAGE Three technical replicates (for image analysis) of one pooled biological replicate per conditioned medium sample were run through a polyacrylamide gel according to previously documented methods [46] Two technical gel replicates were produced for immunoblotting A 12.5% T, 2.6% C resolving gel was first prepared and allowed to polymerize before overlaying with a 5% T, 2.6% C stacking gel; 10–30 lg protein was heated for in a reducing buffer [20 mm Tris–HCl, pH 6.8, 10% (w ⁄ v) glycerol, 2% (w ⁄ v) SDS, 100 mm dithiothreitol] and then centrifuged for at 16 100 g prior to loading The proteins were electrophoresed at mA per gel for h 30 and 50 mA per gel for 50 on a MiniPROTEAN Electrophoresis Cell (Bio-Rad Laboratories) Three microlitres (for fluorescence staining) or 10 lL (for immunoblotting) of Precision Plus ProteinÔ All Blue Standards (Bio-Rad Laboratories) were loaded per gel Meprin protease proteomics Staining and imaging Analytical 2D and 1D gels were visualized by post-electrophoretic fluorescence staining with ruthenium II tris (bathophenanthroline disulfonate) Ruthenium was synthesized exactly as described previously [47] Staining was performed according to the improved protocol [48] In addition to the standard procedure, gels were incubated in 20 mm Tris for 30 with slow agitation (80 r.p.m.), washed twice in deionized water for 10 and, finally, destained again in 40% (v ⁄ v) ethanol, 10% (v ⁄ v) acetic acid overnight with slow agitation (80 r.p.m.) The next day gels were rinsed twice in deionized water for 10 before scanning on a Fuji Film Fluorescent Image Analyzer FLA-3000R (Fuji Film, Tokyo, Japan) with control software basreader, version 3.01 (Raytest Isotopenmessgerate GmbH, Straubenă hardt, Germany) Images were digitized using the parameters: 473 nm excitation, orange filter O580, sensitivity 1000, 16 bits per pixel, 50 lm pixel size Images saved in Fuji BAS file format were converted to 16 bit per pixel Tagged Image File Format images with aida, version 3.11 (Raytest Isotopenmessgerate GmbH, Straubenhardt, ă Germany) Preparative 2D gels were stained by colloidal Coomassie brilliant blue G-250 and used for subsequent protein identification [49] Image analysis A simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure was designed to circumvent cumbersome quantitative comparison and statistical evaluation The procedure is based on qualitative differences among reference gels (level match-sets) of each group of five gel replicates In total, three pooled biological gel replicates (from 18 dishes per pooled sample) and two more technical gel replicates (of one representative pooled sample) were produced per condition (activated meprin versus non-activated meprin) 2D IEF ⁄ SDS ⁄ PAGE-based image analysis was performed using the program pdquest, version 7.3.1 (Bio-Rad Laboratories) Data were inverted and images displayed as black spots on white background Each gel replicate was cropped into four quadrants of same image size (2157 · 1682 pixels) Image cropping of same areas among gel replicates was realized by reference to highly conserved landmark spots present in all gels In total, four independent analyses were performed on each of the four quadrant sections Therefore, corresponding quadrant sections of gel replicates from the same condition were grouped into level matchsets Gel sections were filtered (median, · pixels) to remove image noise Spots were detected as follows: sensitivity 20.00, size scale 9, minimum peak 4230 Background was subtracted with floater method (radius size = 67 pixels) Spot editing was conducted only within same condition members Spot matching was conducted within same condition members of the same level match-set To determine FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4505 Meprin protease proteomics D Ambort et al qualitative differences between the two conditions, level match-sets were clustered into super level match-sets (higher-level match-sets) Matching was then performed on the reference gels of the level match-sets All higher-level match-sets were combined into one super higher-level match-set (combined higher-level match-set) Qualitative differences were then displayed as sets of unique spots One-dimensional SDS ⁄ PAGE-based image analysis was performed using the 1D Evaluation module of aida, version 3.11 In total, three technical gel replicates with pooled biological replicates (from 18 dishes) of each condition were produced The pooled biological replicates of each condition were loaded on the same gel Three independent quantitative analyses were performed Gel image attributes were defined in quantum levels and pixel (65 536 quantum levels per pixel) Rectangular densitometer windows (100 pixels in width over entire lane) were used to generate profile scans of each gel lane In each profile scan, vertical peak borders were defined to subdivide the whole gel lane into integrable major peaks After baseline correction, the averaged signal intensity integrated over each defined peak was plotted against its number Protein identification by LC-MS/MS and protein database searching Gel plugs containing protein spots of interest were excized and the proteins were subjected to in-gel tryptic digestion and peptide extraction as described [50] Twenty microlitres of protein digest was loaded onto a self-made microbore column (inner diameter 0.15 mm, length 80 mm) at a flow rate of approximately lLỈmin)1 of solvent A [0.1% (v ⁄ v) formic acid in water ⁄ acetonitrile (98 : 2)] Columns were packed with GROM-SIL 300 Octyl-6 MB, mm, reversed-phase material (Grom GmbH, Rottenburg-Haiflingen, Germany) Columns were developed by a bi-phasic acetonitrile gradient of 0–5% solvent B [0.1% (v ⁄ v) formic acid in water ⁄ acetonitrile (4.9 : 95)] for followed by 5–40% solvent B for 20 at a flow rate of approximately lLỈmin)1 The column effluent was directly coupled to an Esquire3000+ ion trap mass spectrometer from Bruker Daltonics (Bremen, Germany) via a capillary ESI source operated at 3700 V CID was triggered on the two most abundant not singly charged peptide ions in the m ⁄ z range of 360–1400 Precursors were set in an exclusion list for 0.5 Peak lists from the raw data were created by data analysis, version 3.1 (Bruker Daltonics, Bremen) MS ⁄ MS compounds exceeding a total ion chromatogram intensity of 4000 ion counts were exported and all spectra from the same precursor eluting within a retention time window of 0.5 were compiled to one MS ⁄ MS peak list MS ⁄ MS peak detection was made with the Apex peak finder algorithm using a FWHM of 0.1 m ⁄ z, a S ⁄ N of one, a relative to base peak intensity threshold of 2%, and an absolute intensity threshold of 10 ion counts as para- 4506 meters A mixed list of deconvoluted and nondeconvoluted MS and MS ⁄ MS signals, with an allowance for only the 200 most abundant peaks from nondeconvoluted MS ⁄ MS signals of each spectrum, were exported into mascot generic file format text (mgf) files MS signal deconvolution was set to ‘Auto’ for resolved isotope, and a maximum charge of four with minimally three peaks in set and a molecular weight agreement of 0.05% for related ion deconvolution, respectively MS ⁄ MS peak deconvolutions were allowed for a maximum charge of one only S ⁄ N and FWHM values were also exported into the mgf files CID spectra interpretation was performed with the public search engine phenyx (version 2.1) on the vital-it.ch server operated by GeneBio (Geneva, Switzerland) against the uniprot-SwissProt protein database (release 48.8) with fixed carbamidomethyl modification of cysteine residues, variable oxidation of methionine and variable deamidation of asparagine and glutamine Parent and fragment mass tolerances were set to Da Up to two missed cleavages and half tryptic peptides were allowed The taxonomic search space was restricted to Mammalia (40 084 sequence entries) Peptide search criteria were set to a minimum peptide z-score of ‡ and a maximum peptide P-value of £ 0.0001 All protein identifications consisting of at least two unique peptides reaching a P-value of £ 0.00000001 were accepted To double-check significant hits, the same spectra were interpreted with the web-based search engine mascot (version 2.1) operated by Matrix Science Ltd (London, UK) against same database and parameter settings as above [21] To identify proteins not previously described for dog, all significant peptide matches were searched with program blastp (version 2.2.16) against the dog genome database [22,30] Database size was 33 527 dog RefSeq protein sequences This database is hosted at NCBI (Bethesda, MD, USA) Search parameters comprised: word size 3, filter low complexity, expect value 0.01, score matrix BLOSUM62 Failed searches were repeated with settings for ‘short and nearly exact matches’: word size 2, filter off, expect value 20 000, score matrix PAM30 Only the top scoring significant hit was accepted Functional classification Proteins were clustered into functional groups according to the Human Protein Reference Database (http://hprd.org/) [24] The corresponding human orthologs were grouped and subgrouped into biological process and molecular function Immunoblotting Western blotting was performed as described [51] After 1D SDS ⁄ PAGE as detailed above the protein was transferred to a HybondÔ-P polyvinylidene fluoride membrane (Amersham Biosciences) by application of a constant potential for FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al 15 at 30 V and 50 at 80 V The membrane was then incubated overnight at °C in Tween 20 NaCl ⁄ Tris [20 mm Tris–HCl, pH 7.5, 137 mm NaCl, 0.1% (w ⁄ v) Tween 20] containing 5% (w ⁄ v) milk powder The membrane was washed twice in Tween 20 NaCl ⁄ Tris for and 10 and incubated for h with primary antibody prepared in Tween 20 NaCl ⁄ Tris containing 2% (w ⁄ v) milk powder Thereafter, the membrane was washed four times in Tween 20 NaCl ⁄ Tris The secondary antibody was horseradish peroxidase-linked donkey anti-rabbit or sheep anti-mouse (Amersham Biosciences) diluted : 10 000 in antibody solution The membrane was incubated for h and washed four times in Tween 20 NaCl ⁄ Tris Immunoblots were analysed using the ECL plus Western Blotting Detection System (Amersham Biosciences) Monoclonal antibodies against vinculin (diluted : 2000) and annexin A1 (diluted : 2000) were a generous gift of E B Babiychuk (Department of Cell Biology, Institute of Anatomy, University of Berne, Switzerland) Polyclonal antibody against lysyl oxidase (diluted : 4000) was purchased from Imgenex Corporation (San Diego, CA, USA) Monoclonal antibody 1E2-E4 ⁄ Col5 against collagen type V (diluted : 1000) was from Chemicon Australia Pty Ltd (Victoria, Australia) [28] Acknowledgements The authors wish to acknowledge and thank Ursula Luginbuhl for excellent technical assistance, Dr Eduard ă B Babiychuk for the generous gift of vinculin and annexin A1 antibodies, Professor Bernhard Erni for free access to the Fuji Film Fluorescent Image Analyzer FLA-3000R and aida software and Professor Robert Beynon for teaching MS-based techniques This work was funded by the Swiss National Science Foundation (SNSF) (grant 3100A0-100772 to E.E.S.) and the European Science Foundation (ESF) Integrated Approaches for Functional Genomics (grant 0341 to D.A.) 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D, Schlappritzi E, Hayn G, Matter U & Haeberli A (2005) Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins Proteomics 5, 2619–2630 51 Towbin H, Staehelin T & Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc Natl Acad Sci USA 76, 4350–4354 Supporting information The following supplementary material is available: Fig S1 Application of a simple 2D IEF ⁄ SDS ⁄ PAGEbased protease proteomic approach in substrate finding 2D IEF ⁄ SDS ⁄ PAGE-based image analyses of the second quadrant Fig S2 2D IEF ⁄ SDS ⁄ PAGE-based image analyses of the third quadrant Fig S3 2D IEF ⁄ SDS ⁄ PAGE-based image analyses of the fourth quadrant This supplementary material can be found in the online version of this article Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary material supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4509 ... Non-activated meprin In total Activated meprin Non-activated meprin In total Activated meprin Non-activated meprin In total Activated meprin Non-activated meprin In total 1 2 3 4 All All All... that hmeprin has activity similar to BMP-1 ⁄ TLD-like metalloendopeptidases in that it acts as a procollagen C protease as well as an activator of lysyl oxidase Therefore, an important role for. .. methionine and variable deamidation of asparagine and glutamine Parent and fragment mass tolerances were set to Da Up to two missed cleavages and half tryptic peptides were allowed The taxonomic search