these 
 peptides appear to be part of the plant defense system , based on observations 
 that certain cyclotides exhibit insecticidal or antimicrobial activities .
their characteristic 
 structure comprises a head - to - tail cyclized cystine - knot motif , which 
 confers them great stability and resistance to enzymatic , thermal , 
 and chemical degradation .
hence , cyclotides 
 offer an interesting scaffold for peptide drug development , and they have been utilized as templates for 
 grafting of bioactive epitopes onto the stabilized framework .
for example , they are ligands of the human oxytocin 
 and vasopressin v1a receptors , both members of the g protein - coupled 
 receptor family , and they inhibit the 
 activity of human prolyl oligopeptidase , a serine - protease involved 
 in the processing of neuropeptides .
these 
 properties were discovered in bioassay - guided fractionation approaches 
 using plant - derived peptide extracts as starting materials ; needless 
 to say , a robust and efficient analytical workflow is crucial to determine 
 the molecular structure of each bioactive cyclotide .
therefore , advances 
 in the discovery and identification of cyclotides are of great interest 
 to provide novel peptide candidates for drug discovery and pharmacological applications .
cyclotide distribution within flowering plants has been explored 
 extensively , and today members of the angiosperm families cucurbitaceae , 
 fabaceae , solanaceae , poaceae , rubiaceae , and violaceae are known 
 to express these circular peptides .
all species 
 of violaceae examined hitherto are well - known as a rich source of 
 cyclotides .
the violets are the only plant family that ubiquitously 
 contain cyclotides in every species sampled to date . within the violaceae mnage the species viola tricolor
l. is well - known as a traditional medicinal 
 plant . in particular , herbal extracts 
 of v. tricolor
have been described to treat inflammatory 
 diseases such as inflammatory atopic dermatitis or psoriasis .
although plants contain a number of different bioactive substances , a recent study highlighted that a cyclotide - enriched v. tricolor extract inhibited the proliferation of activated 
 peripheral blood mononuclear cells .
earlier 
 it was demonstrated that cyclotides isolated from v. tricolor are antiproliferative agents against cancer cell lines .
cyclotides have also been discovered in other 
 members of violaceae , including v. odorata l. , v. yedoensis mak .
in addition , cyclotides isolated from species of violaceae have 
 been intensively investigated by mass spectrometry ( ms ) and nuclear 
 magnetic resonance ( nmr ) spectroscopy to provide structural data and 
 insight into their stability and chemical folding . as a consequence of the cyclic nature of cyclotides and therefore , 
 the lack of c- and n - termini , enzymatic processing is required to 
 open the backbone ring .
given that an enzymatic digest of a peptide / protein - enriched 
 plant extract yields thousands of peptides , the analysis of cyclotides 
 in nonpurified preparations is extremely challenging , in particular 
 due to the lack of appropriate nucleic acid / protein data sets available 
 for proteomics analysis .
to date , only laborious peptide purification 
 combined with manual de novo sequencing appears to 
 be suitable for precise sequence elucidation .
more recently , next generation sequencing and bioinformatics have 
 emerged as potential approaches for cyclotide discovery . in this study we combined transcriptome mining and ms to identify 
 and characterize novel vitri cyclotides from v. tricolor .
transcriptome analysis provided new insights about the structure 
 of cyclotide precursors , processing sites , and mature peptide sequences . 

furthermore , we characterized the cyclotide peptidome by malditof / tof 
 and lc  ms / ms .
systematic analysis and comparison of cyclotide 
 identification at the nucleic acid level with application of different 
 proteomics techniques provided the first comprehensive picture about 
 cyclotide diversity and their post - translational modifications ( ptm ) 
 in a single plant species .
the outcome of this work may serve as a 
 starting point for a routine high - throughput proteomics identification 
 of cyclotides and other ribosomally synthesized and post - translationally 
 modified natural peptide products .
acetonitrile ( acn ) , methanol ( meoh ) , dichlormethane , 
 trifluoroacetic acid ( tfa ) , and h2o were purchased as hplc 
 or lc - ms grade from carl roth ( karlsruhe , germany ) .
dithiothreitol 
 ( dtt ) , -cyano - hydroxy cinnamic acid , trypsin ( sequencing grade ) , 
 and iodoacetamide ( iaa ) were purchased as bioultra grade from sigma - aldrich 
 ( sigma - aldrich , vienna , austria ) .
endoproteinase glu c ( sequencing 
 grade ) was purchased from new england biolabs ( ipswich , ma ) .
v. tricolor was cultivated in a planting area near the entrance 
 of brywood , 16th avenue and nw 27th terrace , gainesville , florida 
 32605 ( coordinates 294000.6n 822148.2w ) . 

fresh leaves of v. tricolor were identified by d.s . 
 and colleagues and collected directly into liquid nitrogen .
a voucher 
 specimen ( soltis and miles 2930 ) was deposited at the university of 
 florida herbarium ( flas ) .
rna was isolated using a trizol / rnaqueous - midi 
 kit ( ambion , paisley , uk ) .
samples were sequenced as an indexed rnaseq 
 library on an illumina ga ii machine ( illumina , san diego , ca ) .
sequencing was paired - end ( 73 bp + 75 bp ) and 
 produced a total of over 2.3 gbp .
the sequences were assembled into 
 scaffolds using soapdenovo - trans ( beijing genomics institute , china ) .
transcriptome data of v. tricolor were accessed 
 via the 1kp - project ( www.onekp.com ) .
the v. tricolor data were accessed via tblastn using a set of published cyclotide 
 precursor sequences as queries ( www.cybase.org.au ) .
resulting 
 hits and contig sequences were translated and annotated based on homology 
 to known cyclotide precursors according to a similar methodology as 
 described recently .
briefly , hits were 
 collected and translated into their respective amino acid sequence 
 ( six - frame translation ) .
all transcriptome contig sequence ids , nucleotide 
 sequence data of precursors vitri 1 to vitri 99 , as well as protein 
 translations are listed in supplementary data s-1 .
novel full - length precursor 
 sequences are available in the genbank database under the accession 
 numbers kt203800kt203810 .
any ambiguous or erroneous assembly was analyzed by transrate to 
 identify mis - assembled contigs or assembly artifacts ( http://www.stevekellylab.com/software/transrate ) . dried v. tricolor total herbaceous plant material ( herba violae tricoloris plv
briefly , plant material was extracted in a dichloromethane / meoh 
 1:1 ( v / v ) solution for 24 h. afterward the addition of one - half volume 
 of ddh2o allowed liquid  liquid phase extraction . 

the meoh / water phase encompasses all moderately hydrophobic plant 
 constituents such as peptides , whereas lipophilic plant pigments are 
 enriched in the dichloromethane phase .
the meoh / water phase was further 
 treated in batch by solid phase extraction .
zeoprep 60  , c18 irregular 4063 m material ( zeochem , uetikon , 
 switzerland ) was used according to the manufacturer s recommendation 
 with surface activation by meoh , equilibration with 0.1% tfa and peptide 
 elution with acn / ddh2o / tfa 80/20/0.1% ( v / v / v ) .
fractionation of vitri extract was 
 performed using a dionex ultimate 3000 hplc system ( thermo fisher 
 scientific , waltham , ma ) equipped with a binary pump , autosampler , 
 multiwavelength detector , and a fraction collector .
chromatography was 
 performed using a preparative dichrom kromasil column ( dichrom gmbh , 
 marl , germany ; 250  20 mm , spherical c18 modified 
 particles , 10 m , 110  ) .
mobile phase eluent a consisted 
 of 0.1% tfa ( aqueous ) and mobile phase eluent b consisted of acn / ddh2o / tfa 90/10/0.08% ( v / v / v ) .
peptide separation on column was achieved with linear stepwise gradients 
 ( 0-5% b , 5-5% b , 12.5-20% b , 62.5-70% 
 b , 63.5-99% b , 65-99% b , 66-5% b and 72-5% 
 b ) .
fractions of 40 ml ( 5 min ) each , starting at 20% eluent b , were 
 collected and lyophilized .
the dried samples were kept at 20 
 c until further analysis , e.g. , mass deconvolution of cyclotide 
 mass signals or automated ms / ms sequencing and database search of 
 cyclotides for peptide identification in the proteomics workflow . for ms - based 
 analysis via malditof or lcms
vitri extract was dissolved 
 in 0.1% tfa ( aqueous ) at a concentration of 3 mg / ml .
for proteolytic 
 digest the extract was dissolved in 0.1 m nh4hco3 ( ph 7.8 ) at a concentration of 3 mg / ml .
any insoluble material was 
 removed by centrifugation at 12000 g for 10 min , and 
 the supernatant was used for further analysis . for proteomics analysis 
 by ms / ms
an aliquot of peptide solution ( 2040 l ) was 
 used for reduction , alkylation , and proteolytic digest .
briefly , disulfides 
 were reduced with 20 mm dithiothreitol ( dtt ) for 1 h at 37 c . 

free thiols were carbamidomethylated using 100 mm iodoacetamide for 
 20 min at 23 c in the darkness .
this reaction was quenched with 
 1 l of 100 mm dtt for 10 min at 37 c .
the reduced and 
 alkylated samples were digested with trypsin ( 0.3 g ) or endoproteinase 
 gluc ( 0.6 g ) for 1216 h at 37 c .
double digests 
 were performed accordingly , starting with trypsin digestion overnight , 
 followed by transient trypsin deactivation with dtt and addition of 
 endoproteinase gluc .
for all samples , proteolytic digests were stopped 
 by adding tfa to a final concentration of 0.5% ( v / v ) .
vitri extracts 
 and fractions were analyzed via maldi - tof to evaluate the samples 
 for peaks in the cyclotide specific mass range of 25004000 
 da .
maldi - tof analysis was performed on a 4800 reflector tof / tof ( time - of - flight ) 
 analyzer from absciex ( framingham , ma ) .
samples were mixed with a 
 saturated matrix solution of -cyano - hydroxy cinnamic acid dissolved 
 in acn / ddh2o / tfa 50/50/0.1% ( v / v / v ) at a ratio of 1:6 , 
 and 0.5 l of this mixture was transferred onto the target plate 
 to allow air - drying in the darkness .
mass accuracy was ensured by 
 daily calibration using peptide mix 1 [ ( bradykinin fragment 839 
 ( 572.7 da ) , angiotensin ii ( 1046.2 da ) , neurotensin ( 1672.9 da ) , adrenocorticotropin 
 fragment 1839 ( 2465.7 da ) and bovine insulin chain b ( 3495.9 
 da ) ] ( laserbiolabs , sophia - antipolis , france ) .
ms spectra were recorded 
 using the reflector in positive ionization mode in a spectral range 
 between 2500 and 4500 da .
vitri extracts were evaluated via lc  ms 
 scan and mass signal deconvolution of ms scans . ms scans 
 were processed with deconvolution to evaluate mass signals in the 
 cyclotide typical mass range of 25004000 da .
sample desalting and preconcentration were performed 
 with a trap column , and peptides were separated with a pepmap acclaim 
 capillary column ( thermo fisher scientific ; 150  0.3 mm , 2 m 
 100  ) using a dionex ultimate 3000 uplc system . the mobile phase 
 for the loading pump consisted of 0.1% tfa ( aqueous ) ; 0.1% formic 
 acid ( aqueous ) and acn / ddh2o / formic acid 80/20/0.08% ( v / v / v ) 
 were used as mobile phases for chromatographic separation .
the flow 
 rates were set to 25 l / min for the loading pump and 6 l / min 
 for the separation pump .
the gradient for peptide separation was as 
 follows : 0-4% b , 10-4% b , 120-45% b , 125-99% 
 b , 132-99% b , 135-4% b and 150-4% b. the lc 
 qqtof compact system was equipped with a captive nanoflow ionization 
 source , and sample ionization was achieved in the positive electron 
 ionization mode .
ion transfer parameters were kept constant for the 
 duration of one run : prepulse storage 5 s , collision energy 
 transfer 9 ev , quadrupole energy 4 ev , funnel 1 400 vpp , funnel 2 
 600 vpp transfer time , and hexapole rf 400 vpp . for collision energy 
 rf and for ion transfer time stepping from 200 to 1500 vpp and 30150 
 s
external mass calibration was 
 achieved via direct infusion of the low concentration tuning mix from 
 agilent technologies ( santa clara , ca ) .
furthermore , internal calibration 
 via lock mass was applied for each analytical run using calibrant 
 hexakis ( 1h , 1h , 4h - hexafluorobutyloxy)phosphazine ( agilent 
 technologies ) .
base peak chromatograms in a signal window of 8002200 
 da were processed with the  compound find  tool of the 
 data analysis software 4.21 ( build 393 ) ( bruker daltonics ) .
tool was applied to each identified compound using the following settings : 
 deconvolution of masses between 25004000 da , abundance cutoff 
 5% , maximum charge 4 , only consider [ m + h ] ions of proteins or peptides .
evidence by charge and/or 
 isotope pattern was recorded for each signal . for ms / ms experiments 
 of vitri extracts or vitri fractions
f1f9 the chromatographic 
 conditions were identical as described for the mass signal deconvolution . 

peptide sequencing and automated identification by subsequent database 
 search were performed by ms / ms experiments on the qqtof mass spectrometer 
 using a bottom - up shotgun proteomics approach .
the mass spectrometer 
 was tuned and preoptimized to achieve best sequence coverage for precursors 
 up to 2200 da , as commonly observed from proteolysis of cyclotides . 

ms / ms sequencing was performed with a cycle time of 3 s and exclusion 
 criteria to reduce triggering of background signals .
scan range for 
 precursor recording was set to 502200 da with a minimal precursor 
 mass of 200 m / z .
peptides carrying 
 charges from 2 to 5 were set to trigger ms / ms . 

high energy collision induced dissociation was performed under the 
 regime of a set energy set .
this set applies optimized collision energy 
 for precursor signals based on the parameters : isolation m / z , isolation mass range width , and charge state 
 of the ion .
the set followed the manufacturer s recommendations , 
 and it was not further modified by the experimenter .
the  proteinscape  
 software package v. 3.1.5 474 ( build 20140711 - 1459 ) from bruker daltonics 
 together with the mascot algorithm ( matrixscience , ma ) was used for 
 data analysis and database search .
the available cyclotide sequence 
 database era was updated and customized 
 with the most recent data set of circular peptide sequences ( www.cybase.org.au ) and from recent cyclotide discovery studies to yield in total 376 individual peptide sequences , including 55 
 novel precursors from the vitri transcriptome analysis of this study . 

additionally , contaminants were implemented into the custom database , 
 e.g. , proteolytic fragments of keratins , bovine serum albumins and 
 proteases .
the publicly available contaminant database ( http://maxquant.org/contaminants.zip ) was provided by the max planck institute of biochemistry ( martinsried , 
 germany ) .
peptide search parameters were set to peptide mass tolerance 
 10 ppm , ms / ms tolerance 0.05 da , number of possible c atoms one , charge states 2  5 and considered up to one missed cleavage in the peptide search . 

fixed modification was carbamidomethylation of cysteine side chains , 
 and variable modifications included methionine oxidation , tryptophan 
 modification to kynurenine and 3-hydroxykurenine , deamidation of asparagine 
 and glutamine and ethylation as well as methylation of glutamate or 
 aspartate residues , respectively .
all peptides with more than one 
 possible precursor were assigned to the rank one candidate . identified 
 proteins with a probability score > 50 of two independent analyses 
 of at least one digest ( as described in the section sample preparation for mass spectrometry ) were considered 
 to be valid ; this threshold score has been found by manual ms / ms validation 
 to yield acceptable peptide assignment . nevertheless , ion score cutoff 
 was set to > 15 to consider and exclude low quality peptides from multiple 
 ms / ms triggering events
. cyclotides with a partial sequence and those 
 giving rise to more than one candidate peptide were evaluated manually 
 for assignment of one hit , and they were cross validated with the 
 vitri extract for native peptide mass signals of the reported ms / ms 
 precursor peak .
the ms proteomics data have been deposited at the 
 proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository with the data set identifier pxd002867 
 ( project doi : 10.6019/pxd002867 ) .
since the discovery of  kalata  plant 
 peptides in oldenlandia affinis ( r&s ) dc . by 
 lorentz gran in the 1970s and the appreciation 
 of cyclotides as active substances in medical preparations for uterotonic 
 purpose ,
these ultrastable 
 peptides have been the focus of numerous investigations to delineate 
 their biological diversity , structural arrangement , and phylogenetic 
 distribution within the plant kingdom . with an estimated 50  000 
 cyclotides present in rubiaceae alone , the current rate and performance 
 of standard techniques of cyclotide discovery are clearly behind expectations , 
 and it needs further optimization .
so 
 far laborious purification of peptides has been necessary to obtain 
 accurate sequence information .
hence , not surprisingly , the number of novel cyclotides reported 
 by peptide sequencing in single studies to date is 18 and 24 , respectively , and 
 the overall number of cyclotides deposited in cybase from > 20 years 
 of research is currently at 922 . in the 
 era of high - throughput nucleic acid sequencing ,
new tools have become 
 available to examine cyclotide diversity on a genome / transcriptome 
 level .
therefore , we aimed to discover as many cyclotides 
 as possible expressed in a single plant species and to examine their 
 molecular diversity by a combination of sequence analysis at the nucleic 
 acid level and ms studies at the peptide level .
in addition , we performed 
 a state - of - the - art bottom - up proteomics approach using plant extracts 
 as well as hplc fractionated samples to identify the sequences and 
 ptms of many novel cyclotides present in v. tricolor and to establish a robust workflow for cyclotide analysis . to date , only two precursor sequences have been reported for v. tricolor , and these were identified by 
 pcr - based cdna sequencing .
little is known about 
 the variety of precursor sequences and how many cyclotides one plant 
 is capable of expressing .
therefore , it is of interest to gain insight 
 into the diversity of cyclotide precursors .
v. tricolor transcriptome data were available via the 1kp - project ( www.onekp.com ) and identified by tblastn searches using known cyclotide precursor 
 sequences as queries ( www.cybase.org.au ) .
contigs from 
 resulting homologous sequences have been translated and annotated 
 based on published cyclotide precursors .
this approach led to the 
 identification of 98 unique precursor proteins encoding 108 peptide 
 sequences ( supplementary table s-1 and supplementary data s-1 ) .
these findings nearly double the number of precursors 
 compared to a very recent report suggesting that a single viola species contains 53 cyclotide precursor sequences .
most cyclotide precursors that have been discovered 
 only contain one cyclotide domain , but it is well - known that precursor 
 molecules may contain multiple cyclotide domains .
in fact we identified 
 eight precursor proteins with two cyclotide domains ( vitri precursor 
 18 , 22 , 27 , 42 , 72 , 85 , 91 , and 94 ) and one precursor , vitri precursor 
 9 , encoding for three cyclotide domains ( figure 1a ) .
four cyclotide sequences ( vitri 24/28 , 
 36/37 , 43/44 and 9a/53 ) have been found to be encoded 
 by two different precursor proteins .
analysis of precursor architecture 
 as well as putative processing sites provides further insight into 
 cyclotide biosynthesis in v. tricolor ( figure 1b , supplementary data s-2 ) and may also help to distinguish cyclotides from 
 other cysteine - rich plant peptides .
the 
 majority of identified precursor proteins , namely , 54 precursors contain 
 an asparagine ( or aspartic acid ) at the c - terminus of the cyclotide 
 domain followed by a highly conserved sl , or rarely si , gl or ai in 
 the positions p1/p2 . surprisingly , 10 precursors differ 
 significantly in these common processing sites ; vitri precursors 15 , 
 16 , 17 , 19 , 27a , 35 , 40 , 66 , 76 , and 79 lack the common asparagine 
 ( aspartic acid ) in their cyclotide domain vital for cyclization ( supplementary data s-1 ) , and another seven peptides 
 completely lack a c - terminal tail sequence altogether ( vitri precursors 
 8 , 25 , 32 , 33 , 46 , 51 and 52 ) . hence this raises the question of whether 
 these peptides are being cyclized via different mechanisms or whether 
 they represent acyclic homologues ( supplementary table s-1 , supplementary data s-1 ) .
the occurrence of linear 
 cyclotides has infrequently been reported for species of the rubiaceae , 
 violaceae , and poaceae plant families .
in fact , it was possible to confirm occurrence of 
 acyclic cyclotide homologues in v. tricolor ( see 
 section ms / ms peptide sequencing of cyclotides ) .
these acyclic peptides not only lack the n - terminal glycine and/or 
 the c - terminal asparagine , but the precursor proteins may also be 
 significantly shortened .
besides containing 
 unusual processing motifs , vitri precursors 24 and 28 exhibit a long 
 c - tail region , which has not been observed previously in a cyclotide 
 precursor .
the biosynthesis of cyclotides is still not fully understood ; 
 however research has addressed the role of processing enzymes and 
 the involvement of single residues at the processing sites within 
 the peptide precursors ( supplementary text s-1 ) .
( a ) 
 a generic cyclotide precursor is ribosomally synthesized and contains 
 an er signal , followed by a n - pro - region , n - terminal repeats ( ntr ) , 
 the cyclotide domain , and a c - terminal tail region .
cyclotide precursors 
 may encode up to three peptide domains as it has been demonstrated 
 for the v. tricolor tricyclon a precursor .
novel 
 vitri precursors discovered by transcriptome mining contain one , two , 
 or three cyclotide domains .
as indicated each precursor domain may vary in length . for 
 instance , vitri precursor 24 comprises an extended c - tail , whereas 
 vitri precursor 52 completely lacks this domain .
( b ) sequence comparison 
 of vitri precursor processing site has been provided by a sequence 
 logo .
three adjacent residues downstream of the c - terminus of the 
 mature cyclotide domain in positions p1 ,
p2 , and p3 , 
 and upstream of the n - terminus of the mature cyclotide domain in positions 
 p1 , p2 , and p3 have been considered for being essential for precursor 
 processing and cyclization in planta .
loop six comprises 
 the highly conserved ligation sites , namely , glycine and asparagine 
 or aspartic acid as highlighted in green .
these novel vitri precursor sequences encode up to 108 putative 
 cyclotides of v. tricolor , and many of them exhibit 
 novel or very rare features ( supplementary table s-1 ) .
they have been discussed in the supporting information as extended discussion of the sequence information 
 provided by the presented transcriptome analysis ( supplementary figure s-1 , supplementary text s-1 ) .
these new 
 and rare sequence properties underline the structural plasticity of 
 cyclotides , and they may be of future interest for studies utilizing 
 cyclotides in peptide engineering and drug development .
although transcriptome data analysis revealed 108 peptide sequences , 
 only a single known v. tricolor cyclotide ( vitri 
 d ) has been identified at the transcript level . moreover ,
the reported 
 vitri precursor tricyclon precursor a could not be verified by transcriptome 
 mining .
it is known that cyclotide expression levels 
 appear to be strongly determined by environmental conditions or seasonal 
 changes .
in fact , certain cyclotide precursors 
 are exclusively expressed in certain plant tissues , and biotic and abiotic factors may influence cyclotide 
 transcription .
most importantly , the major limitation 
 of in silico sequence mining is the erroneous prediction 
 of processing sites by homology , and hence certain cyclotides may 
 be assigned alternatively ( see footnotes supplementary table s-1 ) . only peptide analysis at the proteome level using 
 ms will be able to verify and confirm the data obtained from transcriptome 
 analysis .
having established a comprehensive view of the expression of cyclotide 
 peptides at the nucleic acid level , we were interested in cyclotides 
 present at the protein level . to elucidate the cyclotide proteome 
 of v. tricolor ms was used to collect evidence of 
 the expression of cyclotides in v. tricolor .
starting 
 with powdered herb material , hydrophobic compounds were extracted 
 and purified using well established protocols .
capillary liquid chromatography coupled to a qqtof mass spectrometer 
 was used to record base peak chromatograms of all specimens in a signal 
 range between 8002200 da ( figure 2a ) .
lc  ms survey scans were evaluated 
 for putative cyclotide masses using deconvolution tools ( supplementary table s-2 ) .
lc  ms resolved 
 126 peptide signals by the detection of the isotopic pattern , charge 
 state deconvolution , or both .
most notably , the lc  ms system 
 takes advantage of its high resolution power by chromatographic separation 
 and hence in total higher capacity in the scan mode .
in contrast malditof 
 ms analysis of vitri plant extract using the 4800 ms analyzer suffers 
 resolution problems due to multiple overlapping signals .
this is why 
 maldims only yielded 36 peptide signals ( figure 2b ) . on the other hand , co28 
 was only detected by maldims , but not by lc 
lc  ms revealed mass signals of vitri peptides kalata s , varv 
 peptide d , e , f , h , he , hm , vitri a , d , e , f , tricyclon a and cycloviolacin 
 o2 .
mass signals for vitri b , c and tricyclon b were not found with 
 either technique . obviously the lc  ms scan provided better 
 resolution , which resulted in a higher number of identified cyclotide 
 masses .
nevertheless , the combination of qqtof and malditof 
 ms appeared to be more powerful for increasing the number of identified 
 cyclotides . to gain deeper insight into cyclotide diversity of v. tricolor , in particular with regard to molecular structure 
 and ptms , we utilized ms - based peptide fragmentation and database 
 searching to identify cyclotides by an automated proteomics workflow .
( a ) plant peptide extract from v. tricolor was analyzed via lc  ms .
cyclotide masses 
 were found to carry + 2 , + 3 , and rarely + 4 h - ions . signals > 5.0% intensity 
 threshold were assigned to mass and charge deconvolution tool from 
 data analysis software ( bruker daltonics ) .
( b ) accordingly a malditof spectrum is shown between 2750 
 and 3500 da .
for cyclotide names , retention times and molecular weight 
 ( monoisotopic masses ) refer to supplementary table s-2 .
ms / ms fragmentation 
 data of vitri extract were recorded with a capillary lc - qqtof instrument 
 and queried against the customized vitri database . for this purpose , 

the existing era database was updated 
 to contain all vitri precursors or mature peptide sequences that were 
 discovered by transcriptome mining ( supplementary table s-1 and supplementary data s-1 ) .
the lack of a c- or 
 n - terminus requires enzymatic processing for opening the cyclotide 
 backbone , and linearization facilitates sufficient fragmentation using 
 collision induced dissociation since intact cyclotides exhibit poor 
 fragmentation .
the most advanced strategy 
 is to utilize a combination of multiple endopeptidases , i.e. , trypsin 
 and endoproteinase glu c. this methodology resulted 
 in multiple precursor peptides for one cyclotide with significant 
 hits using the database analysis .
identification of a unique full or partial 
 sequence in at least one of the digested samples with a mascot score 
 > 50 was considered valid , and each cyclotide hit was further supported 
 by peptide identification using malditof or lc 
38 cyclotides were determined , 26 with full and 12 with 
 partial sequence coverage ( table 1 ) . using this proteomics workflow ,
it was possible 
 to confirm the expression of vitri cyclotides varv peptide a , d , e , 
 f , h , vitri a , d , e , cycloviolacin o2 and tricyclon a. five cyclotides ( vitri 18b , 22a , 39 , 42a , and 94b ) were detected 
 from the novel transcriptome data set , and tricyclon b as well as 
 oak6 cyclotide 1 were verified at protein level for the first time .
further , this methodology yielded identification of the cyclotides 
 co13 , co22 , and co28 , kalata b1 , varv peptide b , c and g , vigno 4 
 and 5 , viba 30 and 32 , vodo m , vodo n. identified in trypsin digest . identified in endoproteinase gluc 
 digest .
best score out of two independent 
 analysis of each of the three digest experiments .
modifications recorded in at least 
 one peptide of assigned peptides of in total six independent analysis .
one advantage of using an automated proteomics workflow is the 
 possibility to determine ptms .
it was possible to 
 confirm many of them occur in cyclotides from v. tricolor .
the most commonly observed modifications of vitri cyclotides have 
 been illustrated , such as aspartate methylation , glutamate ethylation , 
 tryptophan oxidation to kynurenine and 3-hydroxykynurenine , as well 
 as deamidation of asparagine and glutamine ( figure 3a  d ) .
it is noteworthy that some peptides 
 differ only by one pivotal residue , for instance , vitri e ( n29 ) and 
 cycloviolacin o28 ( d29 ) . in this case
deamidation of the n29 residue 
 of vitri e would yield cycloviolacin o28 with a d29 residue .
judging 
 from the ms / ms data , both cyclotides were found side - by - side in v. tricolor , but nevertheless it was necessary to manually 
 reanalyze the automated assignments .
besides identification of common 
 ptms of amino acid side chains , proteomics is a powerful tool to determine 
 cyclization of the n- and c - termini .
analysis of endoproteinase gluc 
 digested samples revealed evidence for the presence of acyclic peptides 
 in v. tricolor , namely , acyclo - kalata s , -tricyclon 
 a , -vigno 5 -viba 30 , -varv peptide c , d , and e and -viba 30 . here , 
 the peptides from the c - terminal end to the first cleavage site of 
 the protease served as indicative fragments ( table 1 and figure 3e ) .
the acyclic peptide species have been additionally 
 confirmed by ms / ms analysis of reduced and alkylated samples . since 

cyclized peptides emerge stabilized during collision - induced dissociation , 
 only linear peptide species yielded ms / ms spectra with sufficient 
 fragmentation for database searching .
evaluation of post - translational modifications in cyclotides identified 
 by ms / ms sequencing and database analysis .
database searching of ms / ms 
 spectra of cyclotides revealed a number of peptides with posttranslational 
 modifications .
the most abundant ones have been illustrated exemplarily : 
 ( a ) aspartate methylation in vitri peptide 94b ; ( b ) tryptophan degradation 
 product kynurenine in varv peptide e ; ( c ) tryptophan oxidation to 
 3-hydroxy - tryptophan in kalata b1 ; and ( d ) glutamate ethylation and 
 glutamine or asparagine deamidation in vitri peptide 42a .
( e ) the 
 ms / ms fragment spectrum of a c - terminal fragment of endoproteinase 
 gluc processed vitri peptide 22a has been shown to confirm the presence 
 of acyclic cyclotides in v. tricolor .
the major b- 
 and y - ion series have been labeled in all spectra . furthermore , peptide 
 mass , cleavage protease , mass error , and mascot score of the illustrated 
 peptides have been provided .
although cyclotide sequencing in nonfractionated vitri plant extract 
 using the automated proteomics workflow yielded several known and 
 novel cyclotides , many peptides lacked identification by sequence , 
 as compared to analysis by molecular weight ( table 1 ) . thus , to maximize the number of identified 
 cyclotides by sequence we introduced an hplc - assisted fractionation 
 approach with the aim to enrich cyclotides in a particular fraction 
 and subsequently increase sensitivity of the proteomics workflow compared 
 to the extract starting material .
the vitri extract 
 was subjected to preparative hplc , and nine fractions were collected 
 ( supplementary figure s-2 ) .
. v. tricolor fraction f1 ( vitri - f1 ) contained only minor amounts of cyclotide 
 masses in the range of 25004500 da , and vitri - f2 did not show 
 any cyclotide - related signals .
instead , analysis of vitri - f3f9 
 yielded significantly more molecular weight signals as compared to 
 malditof ms of nonfractionated vitri extract . according to 
 previous studies ,
fractionation has been confirmed as an efficient 
 tool for enrichment and increased identification of cyclotides .
the results of the proteomics study of the vitri fractions 
 have been summarized in table 1 . as compared to analysis of the vitri
extract , overall 82 
 peptides have been validated by proteomics of the fractionated samples . 

most notable , 20 novel vitri peptides ( 1 , 2 , 3 , 4 , 8 , 9a/53 , 14 , 17 , 18a , 20 , 21 , 23 , 24/28 , 27a , 29 , 30 , 36/37 , 38 , 42a , 
 50 ) have been unambiguously characterized by ms / ms sequencing . in 
 addition , vitri f , cycloviolacin o4 and o20 , chacur 1 , vaby c , e , 
 viba 9 , 11 , 15 , 19 vibi c , g , and vigno 6 , 7 , 9 , and 10 have all been 
 validated and identified by the proteomics approach . moreover , an 
 additional 11 acyclic peptides have been found as compared to the 
 analysis of the vitri extract ; these were acyclo - kalata b1 , -varv 
 peptide b , -f , -g , -vitri e , -vodo n , -vitri peptide 8 , 9a/53 , 22a , 39 and -cycloviolacin o22 .
most acyclic peptides were found 
 alongside the cyclic species , except for vitri 8 of which only the 
 acyclic peptide has been identified .
in fact , vitri precursor 8 encoding 
 for vitri 8 is missing the essential c - tail for successful cyclization in planta . in summary ,
sample enrichment by hplc fractionation 
 is very effective to enhance the number of total cyclotides identified 
 by about 2-fold . using proteomics
cyclotide 
 analysis is associated with challenges regardless if they are studied 
 at the nucleic acid or proteome level . to highlight the benefits and 
 drawbacks of each method
, it may be useful to compare the results , 
 i.e. the number of identified cyclotides , from transcriptome mining , 
 mass deconvolution , and ms / ms sequencing ( figure 4a ) . analyzing cyclotides at the transcript 
 level , by mass signals deconvolution or by ms / ms sequencing 108 , 127 , 
 and 82
essentially , 54 cyclotides 
 were identified by ms / ms sequencing as well as mass deconvolution , 
 31 by ms / ms as well as transcriptome analysis , and 17 were found by 
 both mass deconvolution and transcriptome mining ; 11 peptides were 
 identified by all three methods .
after all , 28% of 108 vitri cyclotides 
 identified by transcriptome mining were confirmed by ms / ms or molecular 
 weight ; 50% of cyclotides were assigned from partial sequences , 2% 
 constitute cyclotide - like peptides , and only 20% of all full - length 
 cyclotides determined by transcriptome mining remained unverified 
 by ms ( figure 4b ) . 
 considering full - length precursor molecules ,
importantly , 12 out of 14 
 previously published vitri peptides have been identified by ms / ms 
 sequence , which confirms the effectiveness of our approach , considering 
 that we worked with plant extracts and fractions but avoided using 
 purified cyclotides for proteomics analysis .
comparative analysis of vitri cyclotides identified by transcriptome 
 mining , mass deconvolution , and ms / ms sequencing .
( a ) an overview 
 of vitri peptides discovered in this study is summarized by a venn 
 diagram . the number of identified cyclotides by each approach ( transcriptome 
 mining , mass deconvolution or ms / ms sequencing ) is indicated by the 
 numbers in each of the three interwheeling circles .
the overlapping 
 regions represent the numbers of identified cyclotides by combination 
 of two or all three approaches .
( b ) a pie chart illustrates coverage 
 for confirmation of predicted vitri cyclotides by ms .
all predicted 
 108 vitri cyclotides identified by transcriptome mining were grouped 
 in cohorts : ( i ) confirmed by sequence or molecular weight ( 28% ) , ( ii ) 
 partial sequences from transcriptome mining ( 50% ) , ( iii ) cyclotide - like 
 molecules ( 2% ) and unverified full - length cyclotides ( 20% ) .
clearly , analyzing cyclotides by molecular weight only is the most 
 error - prone method for unambiguous characterization of cyclotides . 
 nevertheless , combined with transcriptome analysis , a simple ms run 
 may provide useful information about the diversity of cyclotides in 
 a given plant extract .
similarly , transcriptome mining by itself resulted 
 in the identification of many novel cyclotides , but the sequences 
 of many vitri precursors contained uncommon c- or n - terminal processing 
 sites , such as the missing of the conserved asparagine or aspartic 
 acid residues or complete lack of c - tail motifs .
in combination with 
 ms however , these powerful tools were able to verify some of these 
 peptides as acyclic cyclotide homologues ( supplementary table s-1 ) .
obviously ms / ms sequencing using an automated proteomics 
 workflow appears to be the most effective approach for identifying 
 and characterizing novel cyclotides from v. tricolor , since it is capable of providing evidence not only for the presence 
 of a cyclotide and further yields detailed information sequence and 
 ptms .
thus , we are confident that our results may provide a proof - of - principle 
 and motivation for continued efforts in cyclotide discovery in many 
 plant families using a combined transcriptome and proteome analysis 
 workflow .
the presented study combined cyclotide peptide analysis at both 
 the nucleic acid and the proteome level to obtain insights into the 
 diversity of circular cysteine - rich peptides of v. tricolor .
it provides a comprehensive view of the transcription and on the 
 translation of plant cyclotides and to our knowledge the largest study 
 to date in the field of cyclotide discovery and characterization . 

our results may provide a guideline for future cyclotide discovery 
 projects combining transcriptome and proteome analysis .
here we were 
 able to shed light to the unexplored diversity of plant - derived cyclotides 
 within the violaceae family , which comprises up to 
 31 genera and approximately 1000 species .
previously 10 cyclotides have been estimated as a realistic number 
 of peptides to be expressed per viola species .
a more recent study determined that a single 
 species may express up to 53 peptides . to date
, 161 individual cyclotides are known in violaceae , and in 
 total 922 cyclotides are published online in cybase ( www.cybase.org.au ) .
our finding that v. tricolor contains at least 
 164 different cyclotides significantly augments the number of cyclotides 
 present in violaceae .
thus , we determined that violaceous plants may 
 indeed exhibit a much higher cyclotide diversity than earlier anticipated and contain up to > 150  000 individual 
 cyclotides .
similarly , other plant families such as rubiaceae may 
 contain 150  000 cyclotides or more , determined by the number 
 of cyclotides per species and multiplied by the estimated number of 
 species known to express cyclotides .
this 
 simple calculation does not account for any peptide that may be ubiquitously 
 expressed in many species .
in addition other plant families of cucurbitaceae , 
 fabaceae , poaceae and solanaceae also contain cyclotides .
encompassing 
 the diversity of v. tricolor as a natural library 
 of bioactive peptides , this commercially available medicinal herb 
 may be a suitable starting point for future bioactivity - guided screening 
 studies , and the presented methodology may provide a guideline for 
 cyclotide analytics combining transcriptome and proteome analysis .
 cyclotides are plant - derived mini proteins . 
 they are genetically 
 encoded as precursor proteins that become post - translationally modified 
 to yield circular cystine - knotted molecules . 
 because of this structural 
 topology cyclotides resist enzymatic degradation in biological fluids , 
 and hence they are considered as promising lead molecules for pharmaceutical 
 applications . despite ongoing efforts to discover novel cyclotides 
 and analyze their biodiversity , 
 it is not clear how many individual 
 peptides a single plant specimen can express . 
 therefore , we investigated 
 the transcriptome and cyclotide peptidome of viola tricolor . 
 transcriptome mining enabled the characterization of cyclotide 
 precursor architecture and processing sites important for biosynthesis 
 of mature peptides . 
 the cyclotide peptidome was explored by mass spectrometry 
 and bottom - up proteomics using the extracted peptide sequences as 
 queries for database searching . in total 164 cyclotides 
 were discovered 
 by nucleic acid and peptide analysis in v. tricolor . 
 therefore , violaceous plants at a global scale may be the source 
 to as many as 150  000 individual cyclotides . 
 encompassing the 
 diversity of v. tricolor as a combinatorial library 
 of bioactive peptides , this commercially available medicinal herb 
 may be a suitable starting point for future bioactivity - guided screening 
 studies . 
Introduction
Experimental Section
Results and Discussion
Conclusion