BACKGROUND
antimicrobial peptides  are a diverse group of small, cationic peptides that can inhibit the growth of a broad range of microbes.
they can be found in plants as well as in animals and have been shown to play an important role in defense and innate immunity  <cit> .
the stable ectopic expression of amps in plants allows for the use of plants as biofactories or in the protection of crops against a wide range of pathogens  <cit> .
a universal method that could verify in planta amp stability and accumulation would allow for the rapid screening of different candidates to find novel amps for plant protection.
one of the first animal-peptides heterologously expressed in plants was cecropin b, a small amp from the giant silk moth hyalophora cecropia.
attempts to detect the peptide in transgenic tobacco and potato plants failed, indicating in planta instability  <cit> .
cecropin b has been shown to be extremely susceptible to endogenous plant peptidases and even modified versions of the peptide had half-lives of only few minutes when exposed to various plant extracts  <cit> .
finally, peptidases identified within the intercellular fluid of nicotiana tabacum plants  <cit> , were found to be responsible for peptide degradation, and remain a festering problem for the heterologous protein production in plants  <cit> .
recent studies repeatedly report peptide instabilities  <cit> , which has become the main focus for the de-novo design of amps for plant protection  <cit> .
most amps share a number of features: they are very small , highly cationic charged and have an even number of conserved cysteine residues , which are connected by intra-molecular disulfide bridges  <cit> .
cysteine-free amps are rarely described in plants, and among these, mainly glycine-rich peptides showed a similar antimicrobial activity  <cit> .
amps are typically produced as pre-proteins containing n-terminal signal peptides, essential for successful heterologous expression, as they avoid an undesired intracellular accumulation and allow the formation of disulfide bridges when passing through the endoplasmatic reticulum.
the secretion and extracellular accumulation of amps is also a natural prerequisite for a plant to “poison the apoplast” and protect the intercellular space against the invasion by microbial pathogens  <cit> .
the plant cell wall proteome  is insufficiently studied, as the extraction of cell wall proteins can be challenging  <cit> .
secreted proteins can bind the polysaccharide matrix or other cell wall components, and require specific methods for their release and simultaneously minimizing contaminations with intracellular proteins  <cit> .
destructive procedures are commonly performed for the extraction of amps from ground kernels  <cit> , whereas from leaf tissue proteins can also be released using a non-destructive vacuum infiltrations, in which amps are washed out of the apoplast with low intracellular contamination  <cit> .
due to their small size, amps are commonly overlooked and underrepresented in genome annotations of plants .
similarly, amps are also underrepresented in conventional, gel-based proteome studies, due to difficulties in detecting basic peptides with high pi level and small molecular sizes   <cit> .
small cysteine-rich peptides are not amenable for most methods routinely used for large proteins and even amps that accumulate to high levels in transgenic plants have been shown to be barely detectable on immunoblots  <cit> .
in the past, the production of efficient antibodies with affinity to the mature peptide has been shown to be problematic  <cit>  and their small size does usually not allow for tagging without negatively influencing their in vivo activity and likely artificially enhancing their stability.
recent progress and developments in mass spectrometry have expanded the field of proteomics from merely protein profiling to the accurate quantification of proteins.
the shift from gel-based to gel-free shotgun proteomics allows for high throughput and label-free quantitative comparison of biological samples, opening new research possibilities in plant sciences .
particular small, cysteine-rich peptides could benefit from this development, as these peculiar molecular features make them ineligible for most classical gel-based procedures.
however, such high throughput methods for the analysis of multiple amp families from plant tissue are lacking.
the wild tobacco  has been widely used as an ecological model plant and for field studies of gene function.
the development of a stable transformation procedure for this species  <cit>  allowed for the manipulation of different layers of plant defenses and revealed genes important for defense against herbivores under natural field conditions  <cit> .
we transformed wild tobacco plants with constructs for the ectopic expression of various amps to increase the plant’s resistance against microbes due to peptide accumulation in the apoplast.
as in planta stability cannot be predicted, we chose  <dig> different amps for ectopic expression, including peptides from avian and amphibian origin .table  <dig>
acronyms of the transgenic
nicotiana attenuata
lines and molecular properties of the ectopically expressed antimicrobial peptides
plant line
peptide name
peptide family
organism of origin
monoisotopic mass
p
i
genbank
def1
nicotiana attenuata
def2
nicotiana attenuata
vrd
vigna radiata
fab
vicia faba
ice
mesembryanthemum crystallinum
pna
ipomoea nil
esc
rana plancyi fukienensis
ssp
aptenodytes patagonicus
lea
leonurus japonicus
cap
capsella bursa-pastoris
here we describe the development of a peptide extraction method, capable of supporting high throughput plant screenings to confirm stable expression of a variety of different amps .
our goal was to develop a method that allows for the rapid processing of many samples with relatively small volumes without requiring complex purification or chromatographic steps.
the direct analysis of the intercellular fluid by nanouplc-mse allows for the  detection of extracellular amp deposition and even the  comparison of peptide amounts among the different transgenic plant lines.
furthermore, this method does not rely on the availability of antibodies and can be easily adapted to other plant species or could be used to analyze endogenous amp levels.
RESULTS
ectopic expression of amps in transgenic n. attenuata plants
for the ectopic expression of amps in the wild tobacco , ten different transformation constructs harboring ten different antimicrobial peptides  were constructed.
two of the peptides  were endogenous amps from n. attenuata and were ectopically expressed in all plant tissues.
most of the other peptides were derived from plants  and selected to span the range of diversity found in the various amp families .
additionally, two animal peptides  were tested for their suitability to be expressed in n. attenuata.
the stable transformation of n. attenuata was performed by agrobacterium mediated gene transfer  <cit>  and all peptides were expressed under the control of a constitutive 35s promoter.
to direct their channeling into the protein secretion pathway, all peptides contained their native n-terminal signal peptide .
only the animal derived esc and ssp constructs were fused to a plant signal peptide of the polygalacturonase-inhibiting protein  leader sequence from phaseolus vulgaris, which has been shown to target peptides for secretion in n. tabacum  <cit> .
the complete sequences of the pre-peptides and the composition of the disulfide bridges from all amps are illustrated in figure  <dig>  due to inconsistent naming of the peptides in the literature we use the acronyms of the plant lines from table  <dig> also as a synonym for the peptides or the peptide genes.
all transformed plants were thoroughly screened following the optimized protocol described in gase et al.
<cit>  to find homozygous, single copy lines with stable transgene expression confirmed by qrt-pcr and excluding epigenetically silenced plant lines  <cit> .
although gene expression analysis confirms the functional expression of a transgene, it provides no information about actual protein levels or stability of the ectopically expressed peptide within a plant.
therefore we extend the screening procedure with a method that allows for the comparison of peptide abundances.figure  <dig>
acronyms of the transgenic
n. attenuata
lines and the amino acid sequences of the ectopically expressed antimicrobial peptides .
the n-terminal signal peptides are indicated in red, the mature peptide sequences are shown in blue and c-terminal or other domains in black.
cysteine residues which are connected by disulfide bridges are indicated.
the simulated 3d structures of the def <dig>  lea and ice peptides were retrieved from swiss-model  and drawn with pymol softwarepackage  <dig> rc <dig> .
selective peptide isolation by intercellular fluid extraction
the subcellular localization of the amps requires specific methods for a selective extraction.
we modified a vacuum infiltration/centrifugation protocol  <cit>  for the extraction of the apoplastic or intercellular fluid  from n. attenuata leaves .
icf samples should theoretically contain only proteins and peptides from the apoplast and loosely bound cell wall proteins, as the cytoplasmic membrane remains undamaged during processing.
to specifically enhance the solubility of basic peptides we used two different infiltration buffers, both containing high concentrations of salt and both with acidic ph .
the infiltration of about 5– <dig> leaves per plant allowed the recovery of  <dig> – <dig> ml yellowish icf.
the overall yield among all plants was relatively homogenous with a mean value of  <dig> μl icf per g fresh mass  .
by using a gentle centrifugation force  tissue damage and intracellular protein contamination could be avoided, which would be indicated by a greenish color of the icf.
for all downstream ms based applications a rigorous desalting of the icf samples was necessary.
we initially used small volume  ultrafiltration devices with a  <dig> kda cut-off and analyzed samples by maldi-tof mass spectrometry .
to also target extremely small < <dig> kda peptides and simultaneously exclude > <dig> kda proteins, we switched to reversed phase spe cartridges for desalting and used a three-step elution to sequentially elute peptides by their charge for a higher purification and enrichment of basic peptides .
with this procedure small volume samples could be rapidly desalted, reduced in sample complexity and enriched for amps and allowed the processing of multiple samples in parallel for nanouplc-mse analysis.figure  <dig>
schematic representation of the workflow used for sample preparation of antimicrobial peptides .
intercellular fluid  was extracted by vacuum infiltration and desalted using reversed phase solid phase extraction cartridges .
the samples were spiked with bovine serum albumin  which served as internal standard, tryptically digested and analyzed by nanouplc-mse.
final peptide quantity was calculated and expressed as pmol per g fresh mass .
amp mass mapping by maldi-tof mass spectrometry
for an initial comparison of the peptide mass pattern of transgenic with those of wt plants, the desalted crude icf extracts were subjected to analysis by matrix-assisted laser desorption/ionization – time-of-flight mass spectrometry .
this approach was chosen as it is well suited for the rapid screening of peptide samples of low complexity due to its simplicity.
samples were analyzed in linear ion mode in the m/z range of  <dig> – <dig>  to cover the expected masses of all peptides .
only in two of the transgenic lines, we found a peak within the expected mass range of the expressed peptides for ice –  <dig> .
<dig> da  and lea –  <dig> .
<dig> da  .
this was a strong indication for amp accumulation and successful localization within the apoplast.
the peak masses indicated full mature peptide length without truncations or proteolytic loss.
however, with this method we found no evidence of peptide accumulation for most of the other transgenic lines, regardless of type of ultrafiltration device used .
to test for an eventual leakage of the peptides during icf processing, we also concentrated and analyzed the used infiltration buffer  which remains after leaf removal following the vacuum infiltration .
even the analysis of the supernatant revealed a peak for the lea line, indicating the partial release of this peptide into the supernatant during the vacuum infiltration process .figure  <dig>
comparison of the maldi-tof mass spectra acquired from the intercellular fluid of wt and transgenic ice and lea lines.
icf was extracted with citrate buffer , desalted by ultrafiltration  and analyzed in linear ion mode in the mass range 1– <dig> kda.
peaks within the mass ranges of the expressed peptides are highlighted.
the inset shows the maldi-tof ms analysis of the supernatant from wt and lea lines .
amp identification by nanouplc–mse
to confirm amp accumulation on the sequence level, icf samples were tryptically digested and the obtained peptides were separated by nanoflow ultra-performance chromatography  for the detection by tandem mass spectrometry using mse analysis known as data-independent acquisition   <cit> .
the chosen mass spectrometric approach relies on the acquisition of alternating low/high collision energy data.
the high sampling rate in mse data acquisition enables collection of sufficient data points to quantify peak ion intensities and was implemented in the label-free quantification of proteins based on observation that the intensity of three most intense  tryptic peptides  of a protein can be used as a measure of its abundance  <cit> .
for nanouplc-mse analysis, icf samples were desalted by reversed phase spe according to our flowchart  and  <dig> μl of the final eluted fraction was spiked with  <dig> pmol bovine serum albumin , followed by digestion with trypsin.
since bsa does not occur in plants, it could function as an internal standard for quantification.
to assess the applied quantification method, linear response and analytical reproducibility were considered.
to this end serial dilutions were injected, corresponding to  <dig> - <dig> μl icf sample containing bsa amounts ranging from 50- <dig> fmol.
among all identified tryptic peptides several could be reliably matched to the sequences of the overexpressed amps .
as most of the expressed amps do not naturally occur in n. attenuata, the appearance within the transgenic plants could confirm amp expression, not only for the ice and lea lines, but also for the def <dig>  def <dig>  vrd, fab and pna genotypes.
with this method overall  <dig> of  <dig> n. attenuata genotypes could be tested positive regarding peptide expression and showed peptide secretion into the apoplast.
from the lipid-transfer protein of the lea line up to  <dig> tryptic peptides could be identified, resembling 88% of the mature peptide sequence.
although most amps result only in a small number of tryptic peptides , due to their small sizes, the sum of all detectable peptides resulted in more than 50% sequence coverage  .
in comparison, from the internal standard  up to  <dig> tryptic peptides could be recovered resembling  <dig> % sequence coverage.
all tryptic peptides were unique and could unmistakably be matched to the respective amps.
the defined amount of bsa spiked into the samples, allowed for the calculation of the molar concentration of each amp per ml icf or per g fresh mass , based on the comparison of the internal standard to the peptides of interest  <cit> .
in this way the absolute abundance of a peptide could be calculated for each sample.table  <dig>
tryptic peptides of overexpressed amps detected by nanouplc-ms
e
in the intercellular fluid of
n. attenuata
plants
line
pep score
calc.
exp.
rt
Δ ppm
sequence
sequence coverage
+
+
def1
def2
vrd
lea
pna
fab
ice
carbamidomethylated cysteine indicated as c*; Δ ppm = 106mtn
− <dig>
amp quantification by nanouplc–mse
although peptide abundance could be confirmed for the pna, fab, def <dig> and vrd lines, the quantitative comparison indicated relatively low peptide amounts within these lines with  <dig> – <dig> pmol g− <dig> fm .
in particular the pna peptide was very low abundant and on the limit of detection since it could only be detected in  <dig> out of  <dig> biological replicates.
in contrast, the def <dig>  ice and lea lines indicated very high peptide amounts with 92– <dig> pmol g− <dig> fm .
this confirmed the desired high extracellular peptide accumulation within the apoplast, as it would be required for these transgenic plants.
to estimate the accuracy of the quantification method, the linear response of amps to the internal standard bsa  was determined by analyzing serially diluted samples.
for the high abundant peptides  as well as the low abundant peptides  the mse based quantification revealed a wide linear dynamic range among the injected concentrations, which reached for the lea peptide up to  <dig> fmol.
since we worked with native concentrations from biological samples we could not further exceed these values to reach possible saturation limits.
to confirm repeatability of the quantitative results we analyzed  <dig> additional replicates from the plant lines with high peptide abundance .
for all  <dig> biological replicates a high amp accumulation could be confirmed and showed among all individual quantifications a small technical error .
the averaged relative standard deviation  was  <dig> % for all the measured peptides and best for the lea peptide with only  <dig> %.figure  <dig>
comparison of peptide abundance calculated from lc-ms
e
data of different transgenic
n. attenuata
lines.
intercellular fluid  was extracted with mes buffer  and desalted using reversed phase cartridges.
the samples were analyzed by nanouplc-mse and the peptide abundance calculated based on the relation between the averages of the intensity of the three most intense peptides of the internal standard  to the peptides of interest  <cit> .
peptide abundances are shown as pmol per g fresh mass  ± sem from  <dig> biological replicates per genotype ; n.d. = not detected.
linear dynamic range of nanouplc − ms
e
measurements of amps.
to determine the linear dynamic range of quantification, the calculated peptide amounts  from 3– <dig> technical replicates were plotted against the corresponding amount of bsa in the sample ; bsa was linear in the full range tested.
linear regression  shown for the high abundant amps .
linear regression  shown for the low abundant amps .
as the def <dig> and def <dig> peptides were endogenous defensins of n. attenuata, peptide levels can be directly compared to native levels within untransformed wt plants.
the def <dig> peptide could indeed be detected in the icf of wt, as well as most other transgenic plants .
the def <dig> over-expression line showed the highest peptide amounts, which was about 16-fold higher than the average found in all other lines.
this correlated with the expectations from gene expression data, where these lines showed on average a 16-fold increase in transcript level compared to wt.
the def <dig> plants showed much higher transcript levels, which were on average 450-fold higher compared to wt .
this was as well consistent with the observed peptide amounts, which were 350-fold elevated compared to the basal amount found in some transgenic lines.figure  <dig>
comparison of endogenous def <dig> and def <dig> peptide abundance with strength of gene expression.
the def <dig> overexpressing lines showed about 16-fold higher peptide amounts compared to the average found in all other lines.
the def <dig> overexpressing lines showed about 350-fold higher amounts compared to the average found in all other lines.
calculation of fold differences in gene expression compared to wt using the comparative ct method  with actin as reference gene .
icf sample composition and protein localization
to illustrate general differences in protein composition of icf extracts to total leaf extracts, we compared raw icf samples  with total soluble leaf proteins by sds-page .
both extraction methods showed distinct protein profiles.
very large proteins  seem to be absent in the icf samples whereas total soluble protein extracts were dominated by protein bands at around ~ <dig> kda and ~ <dig> kda which belong to the large  and small subunit  of ribulose- <dig> -bisphosphate carboxylase .
the lack of these bands within the concentrated icf samples indicates that these samples did not contain major intracellular contaminations and that cell lysis played only a minor role during the vacuum infiltration process.
furthermore we evaluated if the icf samples were enriched in endogenous apoplastic peptides and performed database searches with the mse datasets.
since the abundance of non-target proteins was relatively low we used a  <dig> times higher concentration, than usually used for amp quantification.
since the sample preparation method was specific for small cationic peptides , we commonly found endogenous amps within the icf samples, belonging to the non-specific lipid-transfer protein , snakin or the plant defensin family .
this shows that this method is suitable for the analysis of endogenous amps which are expected to be present in apoplastic fractions.
but we also observed peptides belonging to the rubisco ssu and plastocyanin within most samples, which are both chloroplast proteins and indicate contamination from intracellular pools.
still, in a quantitative comparison intracellular proteins showed only 10–20% the abundance levels of the low abundant amps , whereas compared to the high abundant amps  they were only  <dig> – <dig> % as abundant .
thus it is unlikely that the expressed amps merely leaked from intracellular pools.
as we had evidence of peptide release into the infiltration buffer during icf processing we also analyzed the remaining supernatants after the extractions .
we concentrated  <dig> ml supernatant using spe cartridges and analyzed 5% of the eluted fraction .
most amps could be detected in the supernantant as well and the quantitative comparison revealed a similar pattern as observed from the icf samples.
the highest peptide amounts were found in the def <dig>  ice and lea lines  and smaller amounts found for the def <dig>  fab and vrd lines, indicating that peptides are released into the buffer nearly proportional to the overall peptide amount found in the apoplast.
discussion
the facile absolute quantification of plant proteins has the potential to substantially advance many research areas, however sample complexity still thwarts robust quantifications, particularly for cationic amps.
in this study, we developed a high throughput method for extracting and processing intercellular fluid from leaf tissue, generating samples suitable for mass spectrometric analysis and allowing the detection and quantification of different ectopically expressed amps in transgenic n. attenuata plants.
we adapted a vacuum infiltration method for n. attenuata and tested different desalting procedures to analyze peptide abundances with nanouplc-mse in a high throughput fashion .
as a result we could confirm the accumulation of heterologously expressed peptides within the apoplast and could quantify their abundance in comparison to endogenous amps.
amps require specific extraction methods
many purification methods make use of the unique biochemical properties of amps, such as their small size, their positive charge, their tolerance to acids and heat or even the presence of disulfide bridges, as done recently by hussain et al.
<cit> .
we took advantage of the subcellular localization within the apoplast and the selectivity of extraction during vacuum infiltration.
the obtained intercellular fluid , also commonly called apoplastic wash fluid  or intercellular washing fluid , shows a tremendously reduced complexity compared to crude, whole cell fractions, containing cytoplasmic and chloroplast proteins.
particular dominant proteins of the photosystem  were strongly reduced in the icf extracts  similar as shown in delannoy et al.
<cit> .
to achieve an optimized infiltration process, the icf extraction protocol needs to be adapted to each plant species  <cit> .
the salt concentrations and the ph of the infiltration buffer also have a large influence on the protein extraction efficiency  <cit> .
in general, mild acids are commonly used for the extraction of amps as shown for the isolation of floral defensins from the ornamental tobacco, n. alata  <cit> .
in addition, has the use of acidic buffers the advantage of reducing phenolic browning of the extracts, which is a common problem for other protein extraction buffers used for n. attenuata and other tobacco species, e.g.
for trypsin protease inhibitor extraction  <cit> .
for the selective enrichment of amps we tested the pre-cleaning of large proteins with a 30k cut-off ultrafiltration step or heat clearance prior to desalting  and could confirm the heat stability of the ice and lea peptides.
but we generally omitted these steps as they did not improve the overall sample quality, in fact the manufacturer and type of the ultrafiltration device had rather a strong influence on icf sample composition .
ultrafiltration can separate proteins only by size, but allows no further purification.
desalting with reversed phase spe cartridges allowed not only size exclusion, but also separation by charge, which could remove contaminants .
as the sequentially elution steps during spe processing resulted in a further reduction of the icf sample complexity and could enrich basic peptides in the final fraction, it was the preferred method for all nanouplc-mse measurements.
the whole method was developed as a universal extraction and purification of cationic peptides, and has been also proven to be useful for the extraction of endogenous amps.
since the method was stringent for cationic peptides, not many other proteins could be found within these samples and the degree of intracellular contamination was overall very low.
only intracellular proteins < <dig> kda  could co-elute and were commonly observed in most spe desalted samples, whereas parts of the rubisco large subunit could only be detected in about half of the samples .
considering that proteins from the photosystem are the most abundant proteins in plants, the up to  <dig> orders of magnitude higher concentrations of the overexpressed amps show that intracellular contamination was basically negligible.
since there is no all-round method which could cover conditions of all amps, it was not surprising that the method was not optimal for the cap peptides.
these glycine-rich peptides were not cleavable by trypsin and likely need specific modifications regarding the desalting process or the use of different digestion enzyme to increase the chances of later detection.
nanouplc-mse based amp quantification
although amps have been expressed in various plant species there have rarely been attempts to quantify amp accumulation in transgenic plants.
in vitro test have shown potential for the use of rp-hplc and nmr based methods, but only for the quantification of pure fractions of cyclotides, and showed limitations for spectrophotometric methods for these peptides  <cit> .
for the direct analysis of cyclotides from plant extracts even maldi-tof ms based quantitative methods have been developed  <cit> .
we used maldi-tof analysis for peptide mapping and could only detect two very abundant peptides, probably due to the limited resolution and sensitivity of this method for peptides at molecular masses above  <dig> kda.
furthermore one of the biggest disadvantages is the lack of sequence information.
through technical advances in high-performance lc separation of peptides and development of modern mass spectrometer with high resolution and scanning rates, label-free quantification of proteins has been implemented in proteomic routine  <cit> .
this simple and cost-efficient method enables simultaneous protein quantification across many samples without tedious protein or peptide derivatization.
hi <dig> nanouplc-mse based quantification of proteins, used in this study, combined advantages of ultra-performance liquid chromatography that provides high reproducibility in nanouplc runs with high sampling rate of mse data acquisition required for accurate quantitative analysis  <cit> .
instead of analyzing secreted proteins from cell culture media  <cit> , we injected desalted and tryptically digested icf samples derived from plant tissue for a direct quantification.
despite the achieved in vitro precisions, variability among samples prepared from complex tissues is the major limitation in the application of quantitative proteomics  <cit> , which is particularly true for cell wall bound peptides.
despite the variability among biological replicates resulting from separate infiltration procedures , we found consistent patterns of peptide abundance and, among the highly abundant peptides, a remarkable large linear dynamic range .
it should be noted that the small size of most amps strongly limits the options in selecting best ionizable tryptic peptides for quantification measures  <cit> , in contrast to very large and abundant plant proteins, which yield a much broader variety of tryptic peptides and allow more precision in quantification  <cit> .
when necessary, we also included miss-cleaved tryptic peptides to be able to perform the hi <dig> peptide quantification for all amps.
this was the most appropriate method as it resulted in good linear ranges for most amps compared to bsa.
but the defensins  would show a higher linearity if the sum of intensity of all matched peptides would be used for quantification.
however, as this procedure decreased accuracy for the lea and ice peptides, we used the hi <dig> method for quantification of all peptides to maintain comparability among all the different amps.
another possible way improving further accuracy could be achieved by using a peptide standard of a similar size as the amps.
amp localization and expression in plants
in the ornamental tobacco  two floral defensins had been previously reported to be localized only in the vacuole, suggesting that their carboxyl-terminal pro-domains have a protein trafficking function  <cit> .
the orthologous def <dig> peptide of n. attenuata has 100% amino acid similarity to n. alata nad <dig> and we expected an accumulation within the vacuole.
however, in transgenic n. attenuata plants ectopically expressing this peptide large amount was detectable within the icf samples, consistent with their secretion into the apoplast .
although the def <dig> peptide shared 86% protein sequence similarity with def <dig>  their expression strength and the amount of accumulated peptide differed dramatically between these lines.
def <dig> was much more over-expressed than def <dig>  an observation that strongly calls into question the ability to predict suitable candidates for over-expression studies based merely on sequence data.
the overall tremendous differences in amp accumulation amongst all plant lines emphasize the value of a direct assessment of peptide amounts.
in fact, the pna and esc lines were initially among our most promising candidates, as for these peptides a successful expression has been reported in n. tabacum  <cit> .
but the extreme low detectability and the c-terminal pro-domain of the pna peptide are indicators that this peptide might be intracellular localized, whereas the amphibian esculentin- <dig> peptide was undetectable in the esc line and has been reported to show signs of degradation by exopeptidases in n. tabacum  <cit> .
however, the lack of amp detectability could either indicate instability or amounts below the detection limit, both valuable reasons to exclude the plant lines from further studies.
amps usually need to accumulate to large amounts, as was found in the def <dig>  ice and lea lines, to exert a biological function.
interestingly, most of the peptides could also be found within the supernatant, which remained after vacuum infiltration .
more strikingly, the overall pattern of peptide abundance was very similar among icf and supernatant samples.
this suggests that either the peptides readily diffuse out of the apoplast during the infiltration process, or were washed from the leaf surface.
the analysis of a pure leaf surface wash would be a promising future experiment, which could further clarify this hypothesis.
a leaf surface deposition by glandular trichomes is in particularly likely for the def <dig> and def <dig> peptides as the concentrations  were only 10– <dig> times lower in the supernatant than the concentrations  from the icf samples.
in contrast, the concentrations of the other peptides were 44– <dig> times lower in the supernatant.
however, the active secretion of these peptides from the roots could not be confirmed.
we harvested hydroponic solutions of the transgenic plants and concentrated it using spe cartridges.
from the eluted fractions 10% were analyzed , showing no match for any of the expressed amps.
CONCLUSIONS
bio-analytical technology has recently made tremendous progress in the development of peptide quantification techniques and opens many opportunities for applications  <cit> .
the analyses of peptide fluctuations within the plant cell wall, after wounding or infection, are possible examples.
the most limiting factor for peptide quantification is perhaps the bias resulting from sampling and sample preparation.
accurate quantifications of absolute in vivo concentrations are challenging due to different chemical properties of different peptides which result in diverging affinities for extraction and/or purification.
further improvement is expected if digestion methods other than trypsin-assisted proteolysis will be tested for small polypeptides with a limited number of lys and arg in the chain.
here we show that a relatively simple extraction procedure can efficiently release a diverse set of antimicrobial peptides from leaf tissues to provide the basis for a universal method that achieves reliable peptide quantification results by nanouplc-mse that applies label-free quantification.

 antimicrobial peptides are a diverse group of small, cationic peptides that can inhibit the growth of a broad range of microbes. they can be found in plants as well as in animals and have been shown to play an important role in defense and innate immunity. the stable ectopic expression of amps in plants allows for the use of plants as biofactories or in the protection of crops against a wide range of pathogens. a universal method that could verify in planta amp stability and accumulation would allow for the rapid screening of different candidates to find novel amps for plant protection. one of the first animal-peptides heterologously expressed in plants was cecropin b, a small amp from the giant silk moth hyalophora cecropia. attempts to detect the peptide in transgenic tobacco and potato plants failed, indicating in planta instability. cecropin b has been shown to be extremely susceptible to endogenous plant peptidases and even modified versions of the peptide had half-lives of only few minutes when exposed to various plant extracts. finally, peptidases identified within the intercellular fluid of nicotiana tabacum plants, were found to be responsible for peptide degradation, and remain a festering problem for the heterologous protein production in plants. recent studies repeatedly report peptide instabilities, which has become the main focus for the de-novo design of amps for plant protection. most amps share a number of features: they are very small, highly cationic charged and have an even number of conserved cysteine residues, which are connected by intra-molecular disulfide bridges. cysteine-free amps are rarely described in plants, and among these, mainly glycine-rich peptides showed a similar antimicrobial activity. amps are typically produced as pre-proteins containing n-terminal signal peptides, essential for successful heterologous expression, as they avoid an undesired intracellular accumulation and allow the formation of disulfide bridges when passing through the endoplasmatic reticulum. the secretion and extracellular accumulation of amps is also a natural prerequisite for a plant to “poison the apoplast” and protect the intercellular space against the invasion by microbial pathogens. the plant cell wall proteome is insufficiently studied, as the extraction of cell wall proteins can be challenging. secreted proteins can bind the polysaccharide matrix or other cell wall components, and require specific methods for their release and simultaneously minimizing contaminations with intracellular proteins. destructive procedures are commonly performed for the extraction of amps from ground kernels, whereas from leaf tissue proteins can also be released using a non-destructive vacuum infiltrations, in which amps are washed out of the apoplast with low intracellular contamination. due to their small size, amps are commonly overlooked and underrepresented in genome annotations of plants. similarly, amps are also underrepresented in conventional, gel-based proteome studies, due to difficulties in detecting basic peptides with high pi level and small molecular sizes. small cysteine-rich peptides are not amenable for most methods routinely used for large proteins and even amps that accumulate to high levels in transgenic plants have been shown to be barely detectable on immunoblots. in the past, the production of efficient antibodies with affinity to the mature peptide has been shown to be problematic and their small size does usually not allow for tagging without negatively influencing their in vivo activity and likely artificially enhancing their stability. recent progress and developments in mass spectrometry have expanded the field of proteomics from merely protein profiling to the accurate quantification of proteins. the shift from gel-based to gel-free shotgun proteomics allows for high throughput and label-free quantitative comparison of biological samples, opening new research possibilities in plant sciences. particular small, cysteine-rich peptides could benefit from this development, as these peculiar molecular features make them ineligible for most classical gel-based procedures. however, such high throughput methods for the analysis of multiple amp families from plant tissue are lacking. the wild tobacco has been widely used as an ecological model plant and for field studies of gene function. the development of a stable transformation procedure for this species allowed for the manipulation of different layers of plant defenses and revealed genes important for defense against herbivores under natural field conditions. we transformed wild tobacco plants with constructs for the ectopic expression of various amps to increase the plant’s resistance against microbes due to peptide accumulation in the apoplast. as in planta stability cannot be predicted, we chose different amps for ectopic expression, including peptides from avian and amphibian origin.table acronyms of the transgenic nicotiana attenuata lines and molecular properties of the ectopically expressed antimicrobial peptides plant line peptide name peptide family organism of origin monoisotopic mass p i genbank def1 nicotiana attenuata def2 nicotiana attenuata vrd vigna radiata fab vicia faba ice mesembryanthemum crystallinum pna ipomoea nil esc rana plancyi fukienensis ssp aptenodytes patagonicus lea leonurus japonicus cap capsella bursa-pastoris here we describe the development of a peptide extraction method, capable of supporting high throughput plant screenings to confirm stable expression of a variety of different amps. our goal was to develop a method that allows for the rapid processing of many samples with relatively small volumes without requiring complex purification or chromatographic steps. the direct analysis of the intercellular fluid by nanouplc-mse allows for the detection of extracellular amp deposition and even the comparison of peptide amounts among the different transgenic plant lines. furthermore, this method does not rely on the availability of antibodies and can be easily adapted to other plant species or could be used to analyze endogenous amp levels.  ectopic expression of amps in transgenic n. attenuata plants for the ectopic expression of amps in the wild tobacco, ten different transformation constructs harboring ten different antimicrobial peptides were constructed. two of the peptides were endogenous amps from n. attenuata and were ectopically expressed in all plant tissues. most of the other peptides were derived from plants and selected to span the range of diversity found in the various amp families. additionally, two animal peptides were tested for their suitability to be expressed in n. attenuata. the stable transformation of n. attenuata was performed by agrobacterium mediated gene transfer and all peptides were expressed under the control of a constitutive 35s promoter. to direct their channeling into the protein secretion pathway, all peptides contained their native n-terminal signal peptide. only the animal derived esc and ssp constructs were fused to a plant signal peptide of the polygalacturonase-inhibiting protein leader sequence from phaseolus vulgaris, which has been shown to target peptides for secretion in n. tabacum. the complete sequences of the pre-peptides and the composition of the disulfide bridges from all amps are illustrated in figure  due to inconsistent naming of the peptides in the literature we use the acronyms of the plant lines from table  also as a synonym for the peptides or the peptide genes. all transformed plants were thoroughly screened following the optimized protocol described in gase et al. to find homozygous, single copy lines with stable transgene expression confirmed by qrt-pcr and excluding epigenetically silenced plant lines. although gene expression analysis confirms the functional expression of a transgene, it provides no information about actual protein levels or stability of the ectopically expressed peptide within a plant. therefore we extend the screening procedure with a method that allows for the comparison of peptide abundances.figure acronyms of the transgenic n. attenuata lines and the amino acid sequences of the ectopically expressed antimicrobial peptides. the n-terminal signal peptides are indicated in red, the mature peptide sequences are shown in blue and c-terminal or other domains in black. cysteine residues which are connected by disulfide bridges are indicated. the simulated 3d structures of the def lea and ice peptides were retrieved from swiss-model and drawn with pymol softwarepackage rc. selective peptide isolation by intercellular fluid extraction the subcellular localization of the amps requires specific methods for a selective extraction. we modified a vacuum infiltration/centrifugation protocol for the extraction of the apoplastic or intercellular fluid from n. attenuata leaves. icf samples should theoretically contain only proteins and peptides from the apoplast and loosely bound cell wall proteins, as the cytoplasmic membrane remains undamaged during processing. to specifically enhance the solubility of basic peptides we used two different infiltration buffers, both containing high concentrations of salt and both with acidic ph. the infiltration of about 5– leaves per plant allowed the recovery of – ml yellowish icf. the overall yield among all plants was relatively homogenous with a mean value of μl icf per g fresh mass. by using a gentle centrifugation force tissue damage and intracellular protein contamination could be avoided, which would be indicated by a greenish color of the icf. for all downstream ms based applications a rigorous desalting of the icf samples was necessary. we initially used small volume ultrafiltration devices with a kda cut-off and analyzed samples by maldi-tof mass spectrometry. to also target extremely small < kda peptides and simultaneously exclude > kda proteins, we switched to reversed phase spe cartridges for desalting and used a three-step elution to sequentially elute peptides by their charge for a higher purification and enrichment of basic peptides. with this procedure small volume samples could be rapidly desalted, reduced in sample complexity and enriched for amps and allowed the processing of multiple samples in parallel for nanouplc-mse analysis.figure schematic representation of the workflow used for sample preparation of antimicrobial peptides. intercellular fluid was extracted by vacuum infiltration and desalted using reversed phase solid phase extraction cartridges. the samples were spiked with bovine serum albumin which served as internal standard, tryptically digested and analyzed by nanouplc-mse. final peptide quantity was calculated and expressed as pmol per g fresh mass. amp mass mapping by maldi-tof mass spectrometry for an initial comparison of the peptide mass pattern of transgenic with those of wt plants, the desalted crude icf extracts were subjected to analysis by matrix-assisted laser desorption/ionization – time-of-flight mass spectrometry. this approach was chosen as it is well suited for the rapid screening of peptide samples of low complexity due to its simplicity. samples were analyzed in linear ion mode in the m/z range of – to cover the expected masses of all peptides. only in two of the transgenic lines, we found a peak within the expected mass range of the expressed peptides for ice –. da and lea –. da. this was a strong indication for amp accumulation and successful localization within the apoplast. the peak masses indicated full mature peptide length without truncations or proteolytic loss. however, with this method we found no evidence of peptide accumulation for most of the other transgenic lines, regardless of type of ultrafiltration device used. to test for an eventual leakage of the peptides during icf processing, we also concentrated and analyzed the used infiltration buffer which remains after leaf removal following the vacuum infiltration. even the analysis of the supernatant revealed a peak for the lea line, indicating the partial release of this peptide into the supernatant during the vacuum infiltration process.figure comparison of the maldi-tof mass spectra acquired from the intercellular fluid of wt and transgenic ice and lea lines. icf was extracted with citrate buffer, desalted by ultrafiltration and analyzed in linear ion mode in the mass range 1– kda. peaks within the mass ranges of the expressed peptides are highlighted. the inset shows the maldi-tof ms analysis of the supernatant from wt and lea lines. amp identification by nanouplc–mse to confirm amp accumulation on the sequence level, icf samples were tryptically digested and the obtained peptides were separated by nanoflow ultra-performance chromatography for the detection by tandem mass spectrometry using mse analysis known as data-independent acquisition. the chosen mass spectrometric approach relies on the acquisition of alternating low/high collision energy data. the high sampling rate in mse data acquisition enables collection of sufficient data points to quantify peak ion intensities and was implemented in the label-free quantification of proteins based on observation that the intensity of three most intense tryptic peptides of a protein can be used as a measure of its abundance. for nanouplc-mse analysis, icf samples were desalted by reversed phase spe according to our flowchart and μl of the final eluted fraction was spiked with pmol bovine serum albumin, followed by digestion with trypsin. since bsa does not occur in plants, it could function as an internal standard for quantification. to assess the applied quantification method, linear response and analytical reproducibility were considered. to this end serial dilutions were injected, corresponding to - μl icf sample containing bsa amounts ranging from 50- fmol. among all identified tryptic peptides several could be reliably matched to the sequences of the overexpressed amps. as most of the expressed amps do not naturally occur in n. attenuata, the appearance within the transgenic plants could confirm amp expression, not only for the ice and lea lines, but also for the def def vrd, fab and pna genotypes. with this method overall of n. attenuata genotypes could be tested positive regarding peptide expression and showed peptide secretion into the apoplast. from the lipid-transfer protein of the lea line up to tryptic peptides could be identified, resembling 88% of the mature peptide sequence. although most amps result only in a small number of tryptic peptides, due to their small sizes, the sum of all detectable peptides resulted in more than 50% sequence coverage. in comparison, from the internal standard up to tryptic peptides could be recovered resembling % sequence coverage. all tryptic peptides were unique and could unmistakably be matched to the respective amps. the defined amount of bsa spiked into the samples, allowed for the calculation of the molar concentration of each amp per ml icf or per g fresh mass, based on the comparison of the internal standard to the peptides of interest. in this way the absolute abundance of a peptide could be calculated for each sample.table tryptic peptides of overexpressed amps detected by nanouplc-ms e in the intercellular fluid of n. attenuata plants line pep score calc. exp. rt Δ ppm sequence sequence coverage + + def1 def2 vrd lea pna fab ice carbamidomethylated cysteine indicated as c*; Δ ppm = 106mtn − amp quantification by nanouplc–mse although peptide abundance could be confirmed for the pna, fab, def and vrd lines, the quantitative comparison indicated relatively low peptide amounts within these lines with – pmol g− fm. in particular the pna peptide was very low abundant and on the limit of detection since it could only be detected in out of biological replicates. in contrast, the def ice and lea lines indicated very high peptide amounts with 92– pmol g− fm. this confirmed the desired high extracellular peptide accumulation within the apoplast, as it would be required for these transgenic plants. to estimate the accuracy of the quantification method, the linear response of amps to the internal standard bsa was determined by analyzing serially diluted samples. for the high abundant peptides as well as the low abundant peptides the mse based quantification revealed a wide linear dynamic range among the injected concentrations, which reached for the lea peptide up to fmol. since we worked with native concentrations from biological samples we could not further exceed these values to reach possible saturation limits. to confirm repeatability of the quantitative results we analyzed additional replicates from the plant lines with high peptide abundance. for all biological replicates a high amp accumulation could be confirmed and showed among all individual quantifications a small technical error. the averaged relative standard deviation was % for all the measured peptides and best for the lea peptide with only %.figure comparison of peptide abundance calculated from lc-ms e data of different transgenic n. attenuata lines. intercellular fluid was extracted with mes buffer and desalted using reversed phase cartridges. the samples were analyzed by nanouplc-mse and the peptide abundance calculated based on the relation between the averages of the intensity of the three most intense peptides of the internal standard to the peptides of interest. peptide abundances are shown as pmol per g fresh mass  ± sem from biological replicates per genotype; n.d. = not detected. linear dynamic range of nanouplc − ms e measurements of amps. to determine the linear dynamic range of quantification, the calculated peptide amounts from 3– technical replicates were plotted against the corresponding amount of bsa in the sample; bsa was linear in the full range tested. linear regression shown for the high abundant amps. linear regression shown for the low abundant amps. as the def and def peptides were endogenous defensins of n. attenuata, peptide levels can be directly compared to native levels within untransformed wt plants. the def peptide could indeed be detected in the icf of wt, as well as most other transgenic plants. the def over-expression line showed the highest peptide amounts, which was about 16-fold higher than the average found in all other lines. this correlated with the expectations from gene expression data, where these lines showed on average a 16-fold increase in transcript level compared to wt. the def plants showed much higher transcript levels, which were on average 450-fold higher compared to wt. this was as well consistent with the observed peptide amounts, which were 350-fold elevated compared to the basal amount found in some transgenic lines.figure comparison of endogenous def and def peptide abundance with strength of gene expression. the def overexpressing lines showed about 16-fold higher peptide amounts compared to the average found in all other lines. the def overexpressing lines showed about 350-fold higher amounts compared to the average found in all other lines. calculation of fold differences in gene expression compared to wt using the comparative ct method with actin as reference gene. icf sample composition and protein localization to illustrate general differences in protein composition of icf extracts to total leaf extracts, we compared raw icf samples with total soluble leaf proteins by sds-page. both extraction methods showed distinct protein profiles. very large proteins seem to be absent in the icf samples whereas total soluble protein extracts were dominated by protein bands at around ~ kda and ~ kda which belong to the large and small subunit of ribulose- -bisphosphate carboxylase. the lack of these bands within the concentrated icf samples indicates that these samples did not contain major intracellular contaminations and that cell lysis played only a minor role during the vacuum infiltration process. furthermore we evaluated if the icf samples were enriched in endogenous apoplastic peptides and performed database searches with the mse datasets. since the abundance of non-target proteins was relatively low we used a times higher concentration, than usually used for amp quantification. since the sample preparation method was specific for small cationic peptides, we commonly found endogenous amps within the icf samples, belonging to the non-specific lipid-transfer protein, snakin or the plant defensin family. this shows that this method is suitable for the analysis of endogenous amps which are expected to be present in apoplastic fractions. but we also observed peptides belonging to the rubisco ssu and plastocyanin within most samples, which are both chloroplast proteins and indicate contamination from intracellular pools. still, in a quantitative comparison intracellular proteins showed only 10–20% the abundance levels of the low abundant amps, whereas compared to the high abundant amps they were only – % as abundant. thus it is unlikely that the expressed amps merely leaked from intracellular pools. as we had evidence of peptide release into the infiltration buffer during icf processing we also analyzed the remaining supernatants after the extractions. we concentrated ml supernatant using spe cartridges and analyzed 5% of the eluted fraction. most amps could be detected in the supernantant as well and the quantitative comparison revealed a similar pattern as observed from the icf samples. the highest peptide amounts were found in the def ice and lea lines and smaller amounts found for the def fab and vrd lines, indicating that peptides are released into the buffer nearly proportional to the overall peptide amount found in the apoplast. discussion the facile absolute quantification of plant proteins has the potential to substantially advance many research areas, however sample complexity still thwarts robust quantifications, particularly for cationic amps. in this study, we developed a high throughput method for extracting and processing intercellular fluid from leaf tissue, generating samples suitable for mass spectrometric analysis and allowing the detection and quantification of different ectopically expressed amps in transgenic n. attenuata plants. we adapted a vacuum infiltration method for n. attenuata and tested different desalting procedures to analyze peptide abundances with nanouplc-mse in a high throughput fashion. as a result we could confirm the accumulation of heterologously expressed peptides within the apoplast and could quantify their abundance in comparison to endogenous amps. amps require specific extraction methods many purification methods make use of the unique biochemical properties of amps, such as their small size, their positive charge, their tolerance to acids and heat or even the presence of disulfide bridges, as done recently by hussain et al.. we took advantage of the subcellular localization within the apoplast and the selectivity of extraction during vacuum infiltration. the obtained intercellular fluid, also commonly called apoplastic wash fluid or intercellular washing fluid, shows a tremendously reduced complexity compared to crude, whole cell fractions, containing cytoplasmic and chloroplast proteins. particular dominant proteins of the photosystem were strongly reduced in the icf extracts similar as shown in delannoy et al.. to achieve an optimized infiltration process, the icf extraction protocol needs to be adapted to each plant species. the salt concentrations and the ph of the infiltration buffer also have a large influence on the protein extraction efficiency. in general, mild acids are commonly used for the extraction of amps as shown for the isolation of floral defensins from the ornamental tobacco, n. alata. in addition, has the use of acidic buffers the advantage of reducing phenolic browning of the extracts, which is a common problem for other protein extraction buffers used for n. attenuata and other tobacco species, e.g. for trypsin protease inhibitor extraction. for the selective enrichment of amps we tested the pre-cleaning of large proteins with a 30k cut-off ultrafiltration step or heat clearance prior to desalting and could confirm the heat stability of the ice and lea peptides. but we generally omitted these steps as they did not improve the overall sample quality, in fact the manufacturer and type of the ultrafiltration device had rather a strong influence on icf sample composition. ultrafiltration can separate proteins only by size, but allows no further purification. desalting with reversed phase spe cartridges allowed not only size exclusion, but also separation by charge, which could remove contaminants. as the sequentially elution steps during spe processing resulted in a further reduction of the icf sample complexity and could enrich basic peptides in the final fraction, it was the preferred method for all nanouplc-mse measurements. the whole method was developed as a universal extraction and purification of cationic peptides, and has been also proven to be useful for the extraction of endogenous amps. since the method was stringent for cationic peptides, not many other proteins could be found within these samples and the degree of intracellular contamination was overall very low. only intracellular proteins < kda could co-elute and were commonly observed in most spe desalted samples, whereas parts of the rubisco large subunit could only be detected in about half of the samples. considering that proteins from the photosystem are the most abundant proteins in plants, the up to orders of magnitude higher concentrations of the overexpressed amps show that intracellular contamination was basically negligible. since there is no all-round method which could cover conditions of all amps, it was not surprising that the method was not optimal for the cap peptides. these glycine-rich peptides were not cleavable by trypsin and likely need specific modifications regarding the desalting process or the use of different digestion enzyme to increase the chances of later detection. nanouplc-mse based amp quantification although amps have been expressed in various plant species there have rarely been attempts to quantify amp accumulation in transgenic plants. in vitro test have shown potential for the use of rp-hplc and nmr based methods, but only for the quantification of pure fractions of cyclotides, and showed limitations for spectrophotometric methods for these peptides. for the direct analysis of cyclotides from plant extracts even maldi-tof ms based quantitative methods have been developed. we used maldi-tof analysis for peptide mapping and could only detect two very abundant peptides, probably due to the limited resolution and sensitivity of this method for peptides at molecular masses above kda. furthermore one of the biggest disadvantages is the lack of sequence information. through technical advances in high-performance lc separation of peptides and development of modern mass spectrometer with high resolution and scanning rates, label-free quantification of proteins has been implemented in proteomic routine. this simple and cost-efficient method enables simultaneous protein quantification across many samples without tedious protein or peptide derivatization. hi nanouplc-mse based quantification of proteins, used in this study, combined advantages of ultra-performance liquid chromatography that provides high reproducibility in nanouplc runs with high sampling rate of mse data acquisition required for accurate quantitative analysis. instead of analyzing secreted proteins from cell culture media, we injected desalted and tryptically digested icf samples derived from plant tissue for a direct quantification. despite the achieved in vitro precisions, variability among samples prepared from complex tissues is the major limitation in the application of quantitative proteomics, which is particularly true for cell wall bound peptides. despite the variability among biological replicates resulting from separate infiltration procedures, we found consistent patterns of peptide abundance and, among the highly abundant peptides, a remarkable large linear dynamic range. it should be noted that the small size of most amps strongly limits the options in selecting best ionizable tryptic peptides for quantification measures, in contrast to very large and abundant plant proteins, which yield a much broader variety of tryptic peptides and allow more precision in quantification. when necessary, we also included miss-cleaved tryptic peptides to be able to perform the hi peptide quantification for all amps. this was the most appropriate method as it resulted in good linear ranges for most amps compared to bsa. but the defensins would show a higher linearity if the sum of intensity of all matched peptides would be used for quantification. however, as this procedure decreased accuracy for the lea and ice peptides, we used the hi method for quantification of all peptides to maintain comparability among all the different amps. another possible way improving further accuracy could be achieved by using a peptide standard of a similar size as the amps. amp localization and expression in plants in the ornamental tobacco two floral defensins had been previously reported to be localized only in the vacuole, suggesting that their carboxyl-terminal pro-domains have a protein trafficking function. the orthologous def peptide of n. attenuata has 100% amino acid similarity to n. alata nad and we expected an accumulation within the vacuole. however, in transgenic n. attenuata plants ectopically expressing this peptide large amount was detectable within the icf samples, consistent with their secretion into the apoplast. although the def peptide shared 86% protein sequence similarity with def their expression strength and the amount of accumulated peptide differed dramatically between these lines. def was much more over-expressed than def an observation that strongly calls into question the ability to predict suitable candidates for over-expression studies based merely on sequence data. the overall tremendous differences in amp accumulation amongst all plant lines emphasize the value of a direct assessment of peptide amounts. in fact, the pna and esc lines were initially among our most promising candidates, as for these peptides a successful expression has been reported in n. tabacum. but the extreme low detectability and the c-terminal pro-domain of the pna peptide are indicators that this peptide might be intracellular localized, whereas the amphibian esculentin- peptide was undetectable in the esc line and has been reported to show signs of degradation by exopeptidases in n. tabacum. however, the lack of amp detectability could either indicate instability or amounts below the detection limit, both valuable reasons to exclude the plant lines from further studies. amps usually need to accumulate to large amounts, as was found in the def ice and lea lines, to exert a biological function. interestingly, most of the peptides could also be found within the supernatant, which remained after vacuum infiltration. more strikingly, the overall pattern of peptide abundance was very similar among icf and supernatant samples. this suggests that either the peptides readily diffuse out of the apoplast during the infiltration process, or were washed from the leaf surface. the analysis of a pure leaf surface wash would be a promising future experiment, which could further clarify this hypothesis. a leaf surface deposition by glandular trichomes is in particularly likely for the def and def peptides as the concentrations were only 10– times lower in the supernatant than the concentrations from the icf samples. in contrast, the concentrations of the other peptides were 44– times lower in the supernatant. however, the active secretion of these peptides from the roots could not be confirmed. we harvested hydroponic solutions of the transgenic plants and concentrated it using spe cartridges. from the eluted fractions 10% were analyzed, showing no match for any of the expressed amps.  bio-analytical technology has recently made tremendous progress in the development of peptide quantification techniques and opens many opportunities for applications. the analyses of peptide fluctuations within the plant cell wall, after wounding or infection, are possible examples. the most limiting factor for peptide quantification is perhaps the bias resulting from sampling and sample preparation. accurate quantifications of absolute in vivo concentrations are challenging due to different chemical properties of different peptides which result in diverging affinities for extraction and/or purification. further improvement is expected if digestion methods other than trypsin-assisted proteolysis will be tested for small polypeptides with a limited number of lys and arg in the chain. here we show that a relatively simple extraction procedure can efficiently release a diverse set of antimicrobial peptides from leaf tissues to provide the basis for a universal method that achieves reliable peptide quantification results by nanouplc-mse that applies label-free quantification. 
we coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanouplc-mse analysis using hi method and identified/quantified of expressed amps in the transgenic plants ranging from to amino acids in length. intercellular fluidcysteine-rich peptidesheterologous expressiontransgenic plantsvacuum infiltrationdata-independent acquisitiondefensinlipid-transfer proteinknottinissue-copyright-statement© the author 2015 the chosen mass spectrometric approach proved to be highly sensitive in the detection of different amp types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation. however, the in planta stability and subcellular localization of each amp should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pi ranges of amps which limits the utility of standard proteomic gel-based methods. the quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of to pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in n. attenuata. the method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species. we created transgenic nicotiana attenuata plants that independently express different amps under a constitutive 35s promoter and compared the extracellular accumulation of each amp using a universal and versatile protein quantification method.
 overexpressing novel antimicrobial peptides in plants is a promising approach for crop disease resistance engineering. however, the in planta stability and subcellular localization of each amp should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pi ranges of amps which limits the utility of standard proteomic gel-based methods. despite recent advances in quantitative shotgun proteomics, its potential for amp analysis has not been utilized and high throughput methods are still lacking.  we created transgenic nicotiana attenuata plants that independently express different amps under a constitutive 35s promoter and compared the extracellular accumulation of each amp using a universal and versatile protein quantification method. we coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanouplc-mse analysis using hi method and identified/quantified of expressed amps in the transgenic plants ranging from to amino acids in length. the quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of to pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in n. attenuata. the chosen mass spectrometric approach proved to be highly sensitive in the detection of different amp types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation.  heterologous expression of amps in plants can result in highly variable and non-predictable peptide amounts and we present a universal quantitative method to confirm peptide stability and extracellular deposition. the method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords intercellular fluidcysteine-rich peptidesheterologous expressiontransgenic plantsvacuum infiltrationdata-independent acquisitiondefensinlipid-transfer proteinknottinissue-copyright-statement© the author 2015 