The search for and identification of peptides from the moss Physcomitrella patens

ISSN 1068�1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 1, pp. 95–104. © Pleiades Publishing, Ltd., 2011.Original Russian Text © A.Yu. Skripnikov, N.A. Anikanov, V.S. Kazakov, S.V. Dolgov, R.Kh. Ziganshin, V.M. Govorun, V.T. Ivanov, 2011, published in BioorganicheskayaKhimiya, 2011, Vol. 37, No. 1, pp. 108–118.

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1 INTRODUCTION

Peptides are the most important class of low�molecular�weight bioregulators. Their role in the reg�ulation of various processes of cellular activity in ani�mals is well known. However, data on the physiologi�cal functions of peptides in plants are rather scarce.The diversity of regulatory, signaling, and protectiveplant peptides is restricted to two to three dozen com�pounds [1, 2]. In the past few years, families compris�ing numerous regulatory peptides have been identifiedin different taxonomic groups of angiosperms by usingbioinformatics approaches. These peptides are formedduring the proteolysis of proteins whose specializedfunction is to form a particular regulatory peptide afterthe termination of the fragmentation of the originalprotein molecule [2]. Structurally and functionally

Abbreviations: BSA, bovine serum albumin; HPLC–MS/MS,tandem mass spectrometry coupled with liquid chromatogra�phy; MALDI mass spectrometry, matrix�assisted laser desorp�tion/ionization mass spectrometry; NCBI, The National Centerfor Biotechnology Information; Rubisco, ribulose biphosphatecarboxylase/oxygenase.

1 Corresponding author: phone: +7 (495) 939�12�68; e�mail:[email protected].

similar peptides capable of interacting with receptor�like kinases are involved in important processes ofplant development, such as the control of the size ofthe apical meristem (CLE peptides from (CLV)3�endosperm surrounding region (ESR)�related pro�teins) and inflorescence drop�off (IDA peptides, frominflorescence deficient in abscission�like proteins) [3].

An active search for novel regulatory peptides hasbeen conducted in a number of laboratories. This newline of research, which has become a logical and meth�odological sequel of proteomics, received the namepeptidomics [4, 5]. Among the problems of peptidom�ics are the isolation, identification, and catalogizationof peptides present in cells, tissues, and organs, for thepurpose of their qualitative, quantitative, and systemsanalysis. Compounds containing no more than50 amino acid residues (М < 5–6 kDa) are tradition�ally designated as peptides. In some recent studies,amino acid polymers with molecular masses up to10 kDa, which appeared beyond the resolution of pro�teomic two�dimensional gel electrophoresis, wereclassified as peptides [2]. Some authors shift the pep�tide/protein boundary to 20 kDa [6].

The Search for and Identification of Peptides from the Moss Physcomitrella patens

A. Yu. Skripnikova, b, 1, N. A. Anikanova, b, V. S. Kazakova, S. V. Dolgovc, R. Kh. Ziganshina, V. M. Govoruna, d, and V. T. Ivanova, b

aShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho�Maklaya 16/10, Moscow, 117997 Russia

bBiological Faculty, Moscow State University, Moscow, 119899 RussiacShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Russian Academy of Sciences,

Pushchino, Moscow oblast, 142290 RussiadResearch Institute of Physicochemical Medicine, Russian Ministry of Health, Moscow

Received June 8, 2010; in final form, August 17, 2010

Abstract—The isolation and identification of peptides from the moss Physcomitrella patens (Hedw.) B.S.G.,which has been widely used in recent years as a model for studying plant biology, has been described. It wasshown for the first time that protoplasts, the protonemata, and gametophores of Ph. patens contain a variety ofpeptides. From gametophores, 58 peptides, which are the fragments of 14 proteins, and from the protonemata,49 peptides, the fragments of 15 proteins, were isolated and identified. It was found that the protonemata andgametophores of Ph. patens, which are the successive stages of the development of this plant, significantly differfrom each other in both the peptide composition and the spectrum of precursor proteins of the identified pep�tides. The isolation of protoplasts during the enzymatic destruction of the protonema cell wall is accompaniedby massive degradation of intracellular proteins, many of which are the proteins of the protosynthetic system,which is a characteristic response of higher plants to environmental stress factors. In all, 323 peptides, which arethe fragments of 79 proteins, were isolated and identified from moss protoplasts.

Keywords: peptidomics, plant peptides, Physcomitrella patens, protoplasts, Q�TOF

DOI: 10.1134/S1068162011010158

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A knowledge of plant peptides is not only impor�tant for systems biology, but can also be useful in vari�ous applied fields, agriculture, and the manufacture ofdietetic products and pharmaceutical preparations.For example, the plant peptide lunasin isolated fromsoybean and barley has an antioncogenic action [7]. Ithas been shown that short plant peptides of the groupof cyclotides having insecticide action also produceother biological effects: they release mental stress [8],suppress the replication of the immunodeficiencyvirus [9], and are capable of hemolytic [10] and cyto�toxic activities [11], indicating a potential use for plantpeptides in the development of novel drugs.

It was shown for animal systems that peptides thatare the derivatives of proteins (as opposed to peptidesobtained by direct synthesis on the template of theenzyme) can be separated into two main groups: bio�active peptides formed as a result of the selectiveaction of peptidases on specialized precursor proteins,and proteins resulting from the proteolytic degrada�tion of other proteins possessing their own, often wellstudied function.

It can be assumed that there are two analogousgroups of peptides in plant systems. Peptides that playa key role in growth processes and are involved inintercellular signaling, the resistance of plants topathogens, and detoxication of heavy metals should beassigned to the first group [1, 2]. These peptides oftencontain a characteristic functional motif in theirsequence. For example, some peptides that take partin plant protection have a motif of eight cysteines intheir structure [2, 12].

For the second as yet poorly studied group of plantpeptides, no pronounced cellular effects have beenestablished. Thus, it was shown that fragments ofRubisco (EC 4.1.1.39) can serve as a nitrogen sourcein protein synthesis [13]. It was found that the degreeof Rubisco proteolysis increases under stressful condi�tions of light, water, and nitrogen deficiency, at hightemperature, and upon damage by phytopathogens[13]. In this case, Rubisco fragments can be trans�ported along the phloem to actively growing organs ofthe plant [14].

At present, a number of publications on plant pep�tidomics describe studies aimed at a search for and theidentification of peptides with a particular biologicalactivity, e. g., antimicrobial action. These studies arebased as a rule on the use of gel chromatography,HPLC, and the determination of peptide structure byautomatic sequencing using the method of Edman andtandem mass spectrometry [15, 16]. For assigningnovel peptides with biological activity to the first orsecond group, additional studies may be needed, sincethe peptides of the first group are, as a rule, involved inthe intricate regulatory chain, and it will be necessaryto demonstrate its functioning. The attempts to detectnovel endogenous plant peptides, moreover, to analyzethe entire peptide pools in the plant material, often failbecause of difficult methodological problems. These

are associated with the fact that peptides are present inplant cells and tissues in minute amounts against thebackground of great quantities of ballast compounds,e. g., phenols, from which peptides are difficult to sep�arate by physicochemical methods [17].

Mass spectrometry of peptides is currently rapidlydeveloping as the methodological basis of proteomics,in terms of both qualitative and quantitative analysis.As distinct from proteomics, which is based on thecontrolled cleavage of proteins into peptides with“predictable” terminal amino acids, the goal of massspectrometry in peptidomics is the analysis of nativepeptides [2]. For their identification, special algo�rithms should be developed. The available method�ological arsenal of a search for novel regulatory pep�tides seems to be insufficient for the effective solutionof the problems of plant peptidomics.

The present study is devoted to the development ofmethodological approaches to the extraction, separa�tion, and identification of endogenous peptides of thegreen moss Physcomitrella patens. Mosses are of con�siderable basic interest for biology, owing to the phylo�genetic position they occupy as representatives of thefirst and the oldest (450 million�year�old) branches ofterrestrial higher plants. The molecular biology studiesof mosses significantly contribute to the reconstruc�tion of evolutionary changes accompanying such alarge event as the conquest of dry land by plants [18].Mosses are characterized by a great variety of species;after angiosperms, they have the second largest num�ber of species. Mosses possess a wide range of bio�chemical and physiological plasticity, which allowsthem to adapt to extreme temperature, humidity, andlight regimes in the terrestrial environment and form�ing symbiotic associations with various organisms,first of all nitrogen�fixing cyanobacteria. The resis�tance of mosses to drought and cold attract consider�able interest in connection with the urgency of studieson the molecular mechanisms of the resistance ofhigher plants to extreme environmental factors.Mosses have unique physiological mechanisms ofgravireception and phototropism, which have beenelaborated during the adaptation to the land habitatand are of interest from the viewpoint of evolutionary,gravitational, and space biology [19].

After the publication of nucleotide sequences ofthe genomes of seed plants of arabidopsis (Arabidopsisthaliana L.), rice, and poplar, the nuclear genome ofthe moss Ph. patens (Hedw.) B.S.G. was sequencedowing to the efforts of an international consortium in2008 [18]. Knowledge of the genetic basis of the mossopened up new possibilities for the peptidomics ofplants. In the present study, a combination of high�performance mass spectrometry with the bioinformat�ics analysis of peptides made it possible to revealendogenous peptides of predominantly plastid originin the moss Ph. patens.


THE SEARCH FOR AND IDENTIFICATION OF PEPTIDES FROM THE MOSS 97

RESULTS AND DISCUSSION

Essential in the peptidomic analysis is the elimina�tion of nonspecific proteolysis during the isolation ofpeptide samples. With this in mind, we performed allextraction procedures at 4°С (Fig. 1). In addition, inorder to prevent possible proteolytic artifacts, a mix�ture of inhibitors of plant proteases (4�(2�amino�ethyl)benzylsulfonylfluoride, bestatin, pepstatin A,inhibitor E�64, leupeptin, and 1,10�phenanthroline)was added to a strongly acidic extraction solution. Apeptide–protein extract, after removal of the cell walldebris, was separated by chromatography on a gel fil�tration column. The chromatography conditions werechosen so that large protein molecules (with massesover 6 kDa) were eluted in the free volume of the col�umn, whereas low�molecular fractions containingpeptides were subjected to chromatographic separa�tion. Three chromatographic fractions were taken foranalysis (Fig. 2). For further separation, each of thethree peptide fractions was subjected to anion�exchange fractionation on microcolumns into sevensubfractions. The quality of fractionation on microcol�umns was controlled by MALDI mass spectrometry.

The protonemata and gematophores of the mossand protoplasts isolated from the protonemata weresubjected to the peptidomic analysis. In the extractfrom gametophores, 58 peptides were identified,which consist of 6–24 amino acid residues and belongto the sequences of 14 different proteins, which weredetermined, similar to peptides, from the genomedatabase using the program Spectrum Mill (Table 1).

Of these, 15 peptides belong to five proteins that areinvolved in photosynthesis processes (components ofphotosystems I and II and cytochrome b559), 13 pep�tides are the Rubisco fragments (the large subunit andthree isoforms of the small subunit), and 30 peptides

are the fragments of five predicted proteins2 with

unknown functions.

In the protonemata, 49 peptides were identified,which consist of 7–24 amino acid residues and belongto the sequences of 15 proteins (Table 2). Of these,23 peptides belong to the sequences of six proteins thatare involved in the photosynthesis processes (compo�nents of photosystems I and II, cytochrome b559, andchlorophyll�binding proteins), 15 peptides are theRubisco fragments (the large subunit and three iso�forms of the small subunit), eight peptides belong tofour predicted proteins, and three peptides are thefragments of peptidyl�propyl cis–trans�isomerase.

From protoplasts, 323 peptides were isolated,which consist of 7–27 amino acid residues and appearin the sequences of 79 proteins (the data are not shownbecause of the large size of the table but are availablefrom the authors). Of these, 24 peptides are the frag�ments of eight proteins of the photosynthetic appara�tus (components of photosystems I and II, cyto�chrome b559, and plastocyanine), 62 peptides belongto the sequences of seven Rubisco subunits (the largesubunit and six isoforms of the small subunit),

2 A predicted protein is the protein whose sequence was proposedbased on the analysis of the sequence of the genome.

Isolation ofprotoplasts fromthe protonemata

Freezing inin liquid nitrogen

Solid�phaseextraction(concentrating andsalting out)

Harvesting

protonemata andgametophores

Anion�exchangechromatographyon microcolumns

Concentrating andand salting outon reverse�phasemicrocolumns

Homogenization

Gel filtration,sampling of fractions

Preparation ofsamples and recordingof mass spectra

Fig. 1. General scheme of the experiment.

of biomass:

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280220120 17070

III III

Time, min

A280

Fig. 2. Gel chromatography of a peptide preparation. Time intervals of sampling the fractions for further fractionation are shownby Roman numerals.

154 peptides belong to the sequences of 45 predictedproteins, and 83 peptides are the fragments of 19 pro�teins responsible for the execution of various func�tions. Among these are the protein S12 of the large sub�unit of the ribosome, the elongation factor 1 alpha, andthe following enzymes: carboanhydrase, lipoxygenase,peptidyl�propyl cis–trans�isomerase, isoforms ofmalate dehydrogenase, nonsymbiotic hemoglobin,and others.

Comparison of the peptides isolated from the threeforms of the moss (Tables 1 and 2; the data for proto�plasts are not given) shows a significant contribution ofthe large and small Rubisco subunits and cytochromeb559 to the generation of peptides and the formation ofidentical peptides in all three forms. Interestingly, cor�mophyte gametophores (Table 1) and the filamentousprotonemata (Table 2) differ markedly by the numberof precursor proteins of peptides, which suggests thatthe proteolysis of proteins and the generation of pep�tides are regulated by different mechanisms, which areprobably associated with morphogenetic processes ofthe two different life forms of the moss.

It is interesting to note that the amount of peptidesidentified in protoplasts is almost six times greaterthan in the protonemata from which they are isolatedand five times greater than in gametophores. Proto�plasts are isolated from the protonemata using theenzymic preparation Driselase produced from basidi�omycetes. For controlling the potential proteolyticactivity of Driselase, BSA was incubated in the solu�tion for isolation of protoplasts. Electrophoresis andMALDI mass spectrometry of the components of thesolution after incubation showed the absence of BSA

proteolysis products (data not shown). The isolation ofprotoplasts was also carried out in the presence of theinhibitors of proteases and BSA. The results showedthat the addition of the inhibitors and BSA does notlead to any decrease in the number of identified pep�tides (data not shown). Thus, the enzymatic activity ofDriselase during the isolation of protoplasts does notdirectly affect the intracellular protein degradation.

The abrupt increase in the number of peptidesidentified in protoplasts may be related to the physiol�ogy and biomechanics of the protoplast isolation pro�cess, which rapidly results in the substantial reorgani�zation of cell architectonics. By the action of the cel�lulolytic preparation Driselase, the cell wall of theprotonema degrades locally, usually in the apical partof the tip cell of the main or the side axis of the pro�tonema. The protoplast is separated from the maininner side of the cell wall very quickly, within a few sec�onds and minutes (data not shown), enters the hyper�tonic solution through a small (5–10 μm) hole, andacquires a spherical shape.

Together with the great number of peptides identi�fied in protoplasts, our attention was attracted to the factthat Rubisco fragments amount to almost a fifth of allpeptides. The large subunit undergoes the strongest deg�radation; it gives about 70 peptide fragments. Figure 3shows identified fragments of the large and smallRubisco subunits, which are indicated in proteinsequences. It is important to note that peptide frag�ments of chloroplast proteins dominate in the pepti�dome of the moss. We assume that the degradation ofchloroplast proteins occurs owing to the activity ofintraorganelle proteases since plastid membranes may



Table 1. Peptides isolated from gametophores. Amino acids flanking the sequence of the identified peptide are given in paren�theses

Sequence Fragment Precursor protein

(L)FVSTGLAYDVFGSPRPNEY(F) 37–55 Cytochrome b559, alpha�subunit(L)AYDVFGSPRPNEY(F) 43–55(N)SLEQVDEFTRSF(–) 70–83(F)NSLEQVDEFTRSF(–) 71–83(N)SLEQVDEFTRSF(–) 72–83(F)LSVRVYL(G) 63–69 Photosystem I, iron�sulfur center(R)VYLGAETTRSMGLAY(–) 67–81(L)IEINRFFP(D) 27–34 Subunit IX of the reaction center of photosystem I(L)IEINRFFPDA(L) 27–36(L)IEINRFFPDAL(V) 27–37(L)IEINRFFPDALVLP(L) 27–40(Q)EIRAAEDPEFETF(Y) 303–315 Protein D2 of photosystem II(N)LVFPEEVLPRGNAL(–) 340–353(F)YVITWESPKEQIF(E) 56–68 Predicted protein 110797(F)YVITWESPKEQIFE(M) 56–69

(A)FFNFAGERPDYL(G) 20–31 Predicted protein 114376(A)FFNFAGERPDYLG(V) 20–32

(G)MSNVEKIQLA(F) 25–34 Predicted protein 161667(L)INIINAPLQG(S) 52–61(L)INIINAPLQGSKIAEG(F) 52–67(L)INIINAPLQGSKIAEGF(A) 52–68(L)INIINAPLQGSKIAEGFAKIISA(I) 52–74(N)IINAPLQGSKIAEGF(A) 54–68(K)IAEGFAKIISA(I) 64–74(K)IAEGFAKIISAIAEY(T) 64–78(G)FAKIISAIAEY(T) 68–78(A)IAEYTIKINE(G) 75–84(A)IAEYTIKINEGG(V) 75–96(E)YTIKINEGG(V) 78–96(L)LNVVIGKHGLL(T) 117–127(L)LNVVIGKHGLLTL(I) 117–129(L)NVVIGKHGLLTL(I) 118–129(N)VVIGKHGLLTL(I) 119–129(T)LIPFFEPIR(L) 129–137(T)LIPFFEPIRL(S) 129–138(T)LIPFFEPIRLS(L) 129–139(L)IPFFEPIR(L) 130–137(L)IPFFEPIRL(S) 130–138(L)IPFFEPIRLSL(V) 130–140(L)IAEIPTQKPAADVQFGSL(S) 155–172(L)LQIPGDDRPIEF(T) 23–34 Predicted protein 180791(L)LQIPGDDRPIEFT(N) 23–35(D)FVDILR(H) 154–159 Predicted protein 75080(F)IYIEQDEII(–) 16–23 Protein PSI I(G)LVFPAITM(A) 28–36(T)YYTPDYQTKDTDIL(A) 24–37 Large subunit of Rubisco(T)YYTPDYQTKDTDILA(A) 24–38(T)YYTPDYQTKDTD(I) 34–35(L)FTSIVGNVFGFKA(L) 117–129(L)FTSIVGNVFGFKAL(R) 117–130(N)VFGFKAL(R) 124–130(C)TIKPKLGLSAKNY(G) 173–185(D)YLTGGFTANTSLAHY(C) 269–283(Y)LTGGFTANTSLAHY(C) 270–283(F)SYLPPLSDDQIARQVD(Y) 81–96 Small subunit of Rubisco (fragment) 8310(F)SYLPPLSDDQIARQVDY(M) 81–97(C)GFLVARPN(–) 177–184 Small subunit of Rubisco 201973(F)DTVGAVSRTNFSGSGSSGYYDGRY(W) 13–36 Small subunit of Rubisco 226715

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Table 2. Peptides isolated from the protonemata. Amino acids flanking the sequence of the identified peptide are given in parentheses

Sequence Fragment Precursor protein

(E)VTDPIYPGGSFDPLGL(A) 187–202 Chlorophyl A�B�binding chloroplast protein(D)PIYPGGSFDPLGL(A) 190–202

(L)FVSTGLAYDVFGSPRPNEY(F) 37–55 Cytochrome b559, alpha�subunit(L)AYDVFGSPRPNEY(F) 43–55(A)YDVFGSPRPNEY(F) 44–55(R)FNSLEQVDEFTRSF(–) 70–83(F)NSLEQVDEFTRSF(–) 71–83(N)SLEQVDEFTRSF(–) 72–83(L)EQVDEFTRSF(–) 74–83(E)QVDEFTRSF(–) 75–83(M)VNPRVFF(D) 2–8

(M)VNPRVFFD(V) 2–9 Peptidyl�propyl cis–trans�isomerase 170556(M)VNPRVFFDVT(I) 2–11(F)LSVRVYL(G) 63–69

(R)VYLGAETTRSMGLAY(–) 67–81 Photosystem I, iron�sulfur center

(L)IEINRFFPD(A) 27–35

(L)IEINRFFPDA(L) 27–36 Subunit IX of the reaction center of photosystem I(L)IEINRFFPDAL(V) 27–37(L)IEINRFFPDALVLP(L) 27–40(L)IEINRFFPDALVLPL(–) 27–41(A)KLPEAYAIFDPIVDV(M) 22–36

(A)KLPEAYAIFDPIVDVM(P) 22–37 Protein K of the reaction center of photosystem II(F)VWQASVSFR(–) 50–58(A)VDQAPGVRYPVVVR(F) 59–72

(F)DKVNYAGVSTNNYSPDEL(E) 74–91 Predicted protein 133111(A)VTYDVPRWTVPA(A) 26–37

(A)VTYDVPRWTVPAA(A) 26–38 Predicted protein 170004(L)YGGPTPNVIRSILPLFE(A) 6–22

(F)SLADAFHTPYMN(W) 167–178 Predicted protein 177778(L)ADLSHLPYTY(L) 166–175

(L)ADLSHLPYTYL(L) 166–176 Predicted protein 183533(A)KYGEKSVYFDL(G) 46–56 PsaH�subunit of the reaction center of photosystem I 165481(A)KYGEKSVYFDLG(E) 46–57(T)YYTPDYQTKDTDIL(A) 24–37

(T)YYTPDYQTKDTDIL(A) 24–37 Large subunit of Rubisco(T)YYTPDYQTKDTDILA(A) 24–38(T)YYTPDYQTKDTDILA(A) 24–38(Y)AIEPVAGEENQYIAY(V) 86–100(G)MPIVMHDYL(T) 262–270(G)MPIVMHDYLT(G) 262–271(G)MPIVMHDYLT(G) 262–271(Y)LTGGFTANTSLAHY(C) 270–283(E)GERQVTLGFVDL(L) 337–348(F)SYLPPLSDDQIARQVD(Y) 81–96 Small subunit of Rubisco (fragment) 8310(F)SYLPPLSDDQIARQVDY(M) 81–97(F)DTVGSVSRTNFSGAGSSGYYDGRY(W) 106–129 Small subunit of Rubisco 221004

(F)DTVGAVSRTNFSGSGSSGYYDGR(Y) 13–35 Small subunit of Rubisco 226715

(F)DTVGAVSRTNFSGSGSSGYYDGRY(W) 13–36



be a substantial barrier for cytoplasmic proteases.Thus, an important problem of plant proteomics is adetailed investigation of the generation of the peptidepool formed during the degradation of chloroplastproteins.

In the structures of the peptides isolated, we per�formed a search for amino acid sequences of twoknown biologically active plant peptides. The first isrubiscolin�6, a fragment of the large subunit of spinachRubisco, which has the sequence YPLDLF and whichwas assigned to the group of δ�opioids owing to theanalgesic and neuroleptic action it produces on intra�venous and peroral administration [20]. The secondpeptide is rubimetide (sequence MRW) isolated froma pepsin–pancreatin hydrolysate of the Rubisco largesubunit, which has analgesic and hypotensive effects[21]. These two sequences were identified only in thecomposition of the larger fragments of the Rubiscolarge subunit, isolated from protoplasts: rubiscolin�6in (Y)VAYPLDLFEEGSVTNL(F) and rubimetide in(D)DENVNSQPFMRW(R). It cannot be excludedthat further fragmentation of the detected peptides canlead to the formation of rubiscolin�6 and rubimetidein moss protoplasts.

It is shown on the visualized protein sequence(Fig. 3) that identified peptides cover a great part of

the sequence of the Rubisco large subunit. Taking intoconsideration reports [12, 13], which demonstrate themassive proteolytic degradation of chloroplast pro�teins under stress (heat, cold, and salt shock, phyto�pathogen attack), we assume that the isolation of pro�toplasts presents to plant cells a stress of the sameorder as the stress induced by natural unfavorable fac�tors. We have advanced a similar hypothesis in our pre�vious study [22]. During the proteomic analysis ofPh. patens, we have characterized for the first time theprotein composition of freshly isolated protoplastsfrom the moss protonema. We have detected proteinspots on two�dimensional electrophoregrams that wereidentified by mass spectrometry as Rubisco fragmentswith a mass of 10–20 kDa [22]. It may be suggested thatthe appearance of Rubisco polypeptide fragmentsdetected on two�dimensional electrophoregrams is thefirst stage of the formation of smaller (up to 2.5 kDa)peptide fragments, which were revealed in cells usingtandem mass spectrometry.

Thus, we showed here for the first time that proto�plasts, the protonemata, and gametophores of themoss Ph. patens in the cells and tissues, contain vari�ous endogenous peptides. We determined theirsequences and precursor proteins they are formedfrom. We showed that the cormophytic and filamen�

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(b)

(a)

Fig. 3. Amino acid sequences of Rubisco from Ph. patens (E C 4.1.1.39) (sequences of peptides isolated from protoplasts are indi�cated by lines). (a) The large subunit; index P34915 in the genome database. (b) The small subunit A9TYN7.

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tous life forms of the moss are characterized by differ�ent peptide composition and precursor proteins ofpeptides. The moss peptides detected can be consid�ered as the physiological products of proteolytic deg�radation, which are formed during the natural func�tioning of cells and are not the instrumental artifacts ofprotein fragmentation during the homogenization oftissues and cells. The isolation of moss protoplasts isaccompanied by the degradation of proteins amongwhich the proteins of the photosynthesis systemaccount for the greatest portion; this leads to the gen�eration of endogenous peptides, which is peculiar tostress responses of higher plants.

The data obtained supplement the characteristicsof plant protoplasts, which are widely known unicellu�lar model systems that can be used in the solution ofproblems of experimental plant biology. We assumethat the peptide pool of moss protoplasts changes dur�ing their regeneration. An analysis of the dynamics ofthe peptidome during the regeneration of moss proto�plasts is of interest from the viewpoint of the biology ofplant development and is the subject of our furtherstudies. We do not rule out that the peptides detectedmay be physiologically active compounds. The testingand study of their biological activity is within the scopeof our studies concerned with the plant peptidomicswith the use of Ph. patens as the model.

EXPERIMENTAL

Growing of the protonema and gametophores of themoss and preparation of protoplasts. The protonema ofthe moss Ph. patens was grown on modified Knopmedium (800 mg/l of Ca(NO3)2 ⋅ 4H2O, 250 mg/l ofMgSO4 ⋅ 7H2O, 125 μg/l of FeSO4 ⋅ 7H2O, and 5 g/l ofglucose supplemented with the following microele�ments: CuSO4 ⋅ 5H2O, ZnSO4 ⋅ 7H2O, H3BO3, MnCl2 ⋅4H2O, CoCl2 ⋅ 6H2O, KI, (NH4)2MoO4 ⋅ 2H2O) and500 mg/l of ammonium tartrate (medium PPNH4)[23, 24] under illumination by white light fromTL�D/827 luminescent lamps (Philips, Holland) witha photon flux of 61 μmol/m2 s under a 16�h photope�riod regime at 26°C. The agarized medium was pouredin Petri dishes 9 cm in diameter. After solidification,an aqueous suspension of fragments of protonema fil�aments 10–50 cells long, which was prepared using anUltra�Turrax T10 basic homogenizer (IKA, Ger�many), was applied by a plastic pipette with a cut tip toPetri dishes onto the surface of agar (1 ml to each). Onday seven of culture growth, protonema filaments wereharvested by a spatula from the surface of agar andplaced in a 150�ml glass with sterile distilled water(100 ml). The sterile working nozzle of the homoge�nizer (S10N�10G) was immersed into the water, andprotonema filaments were fragmented for 1 min at anozzle rotation speed of 2000 rpm. Then the suspen�sion was taken by a sterile plastic pipette with a cut tipand seeded onto fresh agarized medium. Passage was

carried out every seven days. For the peptidomic anal�ysis, a five�day�old protonemata was used.

Moss gametophores were grown on modified agar�ized Knop medium (medium PPNO3) [24, 25] in9�cm Petri dishes under illumination by white lightfrom TL�D/827 luminescent lamps (Philips, Holland)with a photon flow of 61 μmol/m2 s under a 16�h pho�toperiod regime at 26°С. Curtains of gametophoreswere separated by forceps into fragments containingfour to five shoots and transferred to fresh nutrientmedium once a month. For the peptidomic analysis,three�month�old gametophores were used.

Protoplasts were isolated from the protonematausing a 1% solution of the enzyme Driselase (Fluka,Switzerland) in 0.48 M solution of mannitol (Fluka,Switzerland) [25]. A five�day�old protonema was har�vested by a spatula from the surface of agar and placedin Petri dishes 9 cm in diameter containing a Driselasesolution (15 ml). The incubation was carried out for30 min under continuous gentle stirring, manually bya circular motion (60 rpm), under weak illumination(5 μmol/m2 s photons) at 25°С. Then a suspension ofisolated protoplasts was filtered through a steel meshwith a cell size of 80 μm, and protoplasts were furtherincubated in the Driselase solution for an additional15 min. Protoplasts were sedimented by centrifugationusing a bucket rotor in 50�ml plastic test tubes at 100 gfor 5 min. Then protoplasts were washed twice fromDriselase with an aqueous 0.48 M mannitol solutionby centrifugation at the same speed and sedimentedagain. Then the supernatant was taken, and the sedi�ment of protoplasts was frozen in liquid nitrogen andplaced to a freezer (–70°С) for 24 h for the subsequentextraction of peptides.

Extraction of peptides from protoplasts and tissuesof the moss. Frozen protoplasts were transferred to a10�ml test tube, 2 ml of 1 M CH3COOH in 10%СH3CN containing a cocktail of inhibitors of plantproteases was added, and the protoplasts were treatedfor 1 min with an Ultra�Turrax T10 basic homogenizer(IKA, Germany) using a S10N�10G nozzle at a rota�tion speed of 30 000 rpm at 4°C. Peptide extracts froma five�day�old protonema were prepared by transfer�ring the moss filaments, harvested by a spatula, fromthe surface of agarized medium to a porcelain mortarpreliminarily cooled to –70°C, where they wereimmediately frozen by liquid nitrogen. In order to pre�pare the peptide extracts from gametophores, three�month�old moss sprouts were cut by manicure scissorsat a height of 1 mm from the surface of the agarizedmedium and transferred by forceps to a porcelainmortar preliminarily cooled to –70°C, where theywere immediately frozen by liquid nitrogen. Frozenprotonema filaments and moss shoots were ground toa state of fine powder by a pestle preliminarily cooledto –70°C. Then a solution of 1 M acetic acid in 10%acetonitrile containing a cocktail of inhibitors of plantproteases (4�(2�aminoethyl)benzylsulfonylfluoride,bestatin, pepstatin A, inhibitor E�64, leupeptin,



1,10�phenanthroline) (Sigma–Aldrich) was added,and the plant material was treated for 1 min with anUltra�Turrax T10 basic homogenizer (IKA, Ger�many) using a S10N�10G nozzle at a rotation speed of30000 rpm. The suspension containing cell wall frag�ments as a ballast material was centrifuged at11000 rpm for 10 min at 4°C. The supernatant wastransferred into a clean test tube and centrifuged againat 11000 rpm for 10 min at 4°C, and the resulting sed�iments were thrown away. Samples of peptide extractswere immediately applied to a gel filtration column forthe isolation and fractionation of plant peptides.

Gel filtration was carried out on a column 2.5 cm indiameter and 30 cm long filled with Sephadex G�25superfine in 0.1 M CH3COOH. The elution was with0.1 M CH3COOH at a flow rate of 1 ml/min. Proteinsand peptides were detected on an LKB Bromma 2518Uvicord SD device at a wavelength of 280 nm. Thefractions of the preparation collected by an automaticLKB Bromma 2211 SuperRAC collector were storedat +4°С for no more than 48 h. For further analysis,fractions with molecular masses less than 6 kDa wereused. They were subjected to solid�phase extraction.

Solid�phase extraction was carried out on C18Oasis HLB VAC RC reverse�phase cartridges (Waters,United States) using a Vac�Elut vacuum chamber(Varian, United States). Preconditioning of cartridgeswas carried out by successively passing through eachcartridge 1 ml of methanol, 1 ml of a 7 : 3 СH3CN–isopropanol mixture, and 1 ml of 0.1% TFA. All pro�cedures were performed so as to exclude the overdry�ing of sorbent in the cartridge. Then a sample wasintroduced into the cartridge at a rate of 600 μl/min,and water (1 ml) was passed at the same rate. Sorbedsubstances were eluted from cartridges with 1.5 ml of80% СH3CN into 2�ml Eppendorf�like test tubes,concentrated on a SpeedVac Concentrator vacuumcentrifugal evaporator (Savant, United States) to avolume of 100 μl, transferred to microtest tubes with avolume of 200 μl, and evaporated to dryness.

Each peptide sample was fractionated on twoanion�exchange microcolumns. A microcolumn rep�resents a tip for an automatic pipette 200 μl in volumein which two layers of the Emporetm Extraction diskanion�exchange membrane (ANO�3482 company3M) 1.6 mm in diameter are located [26].

The preconditioning of microcolumns was carriedout as follows. Through each column, 30 μl of metha�nol and the solutions of 0.1% NH4OH, 80% СH3CN in0.1% NH4OH, 0.5 М CH3COONH4, and 0.1%NH4OH (20 μl each) were passed with a rate of20 μl/min. A peptide preparation was dissolved in60 μl of 0.1% NH4OH, separated into two parts, 30 μleach, and each part was applied to a separate micro�column. For this purpose, the solution was passedthrough a microcolumn and collected into a microtesttube. The procedure was repeated twice, followingwhich the eluate was stored in a separate microtesttube for the control analysis. After applying the pep�

tide preparation, 20 μl of 20% acetonitrile in 0.1%ammonium hydroxide was passed through the micro�columns to remove the nonspecifically sorbed mate�rial, which was collected as part of the eluate in a sep�arate microtest tube for control.

Then the fractionation by stepwise gradient ofCH3COONH4 (50, 100, 200, 300, and 500 mM; thevolume of a step 20 μl) was carried out. In order toneutralize NH4OH, 20 μl of 0.5% TFA was added toeach microtest tube with the collected sample. Thensamples were evaporated to dryness on a SpeedVacConcentrator vacuum centrifugal evaporator. Beforerecording the mass spectra, samples were salted out onreverse�phase C18 microcolumns. For each sample,one microcolumn was used, which was made on thebasis of a 200 μl tip for an automatic pipette with twolayers of the Emporetm Extraction disk reverse�phaseC18 membrane (ANO�3482 company 3M) 1.6 mm indiameter [26]. For the preconditioning, 20 μl ofCH3OH and 20 μl of 1% TFA were successively passedthrough microcolumns. Peptide preparations weredissolved in 20 μl of 3% CH3CN in 1% TFA. Theresulting solution was applied to a microcolumn at arate of 20 μl/min. The preparation on the microcol�umn was washed with 20 μl of 3% СH3CN in 0.1%TFA. Peptides were eluted with 20 μl of 50% СH3CNin 0.1% TFA. The salted out peptide preparations wereconcentrated on a SpeedVac Concentrator vacuumcentrifugal evaporator (Savant, United States) to avolume of 5 μl, diluted with 3% СH3CN in 1% TFA to20 μl, and transferred to microtest tubes of a system forautomatic sampling (7 μl each) of an Agilent 1200chromatograph (United States).

Q�TOF mass spectrometry analysis was carried out onan Agilent 6520 Acurat�Mass Q�TOF mass spectrometer(United States). Peptides were separated on a G4240�62002 chromatographic chip (5 μm, ZORBAX300SB�C18 300 Å, 40�nl precolumn, analytical column,75 μm × 150 mm) using a linear gradient of СH3CN(5–100% in 0.1% solution of formic acid in water) for60 min with a flow rate of 3 μl/min on a precolumn(pump Agilent 1200 Series Capillary Pump G1382 A)and 300 nl/min on an analytical column (pump Agilent1200 Series nanoflow LC pump G2226A).

The spectra of positively charged ions wererecorded in a centroid regime at a voltage on the cap�illary of 1850 V. The data were recorded in an extendeddynamic region (3200 m/z) with a frequency of 2 GHz.In the MS regime, the range of measurements of m/zwas 300–3200, and 50–3000 in the MS/MS regime. IfMS/MS spectra were recorded, three most intensiveprecursor ions exceeding the threshold of 1000 units,with the precursor ion charge of 2, 3, and more wereautomatically selected during one MS scanning. Theinternal standard for calibration was uninterruptedlyintroduced during the LC/MS run.

Analysis of Q�TOF mass spectrometry data. Thelists of peaks were obtained by the program SpectrumMill Data Extractor. The following parameters were

104


SKRIPNIKOV et al.

used during the formation of mass lists: integrating thescans of one and the same precursor ion into one; m/z± 1.4 when they occurred in the time “window” of±15 s. The signal/noise ratio for the precursor ion was25, and the maximum precursor ion charge for an ionwas seven. The lists of masses determined were sent tothe retrieval system Spectrum Mill (version RevA.03.03.084 SR4; United States).

Identification of peptides was carried out using thedatabase Uniprot (http://uniprot.org) with access forthe moss Ph. patens (the number of records in thedatabase was 35415). The accuracy of determining themass of the precursor ion was ±20 ppm; the mass offragments was ±50 ppm. The list of identified proteinswas automatically validated using the Spectrum Millsystem.

The visualization and quantitative analysis of thedistribution of the sequences of identified peptideswithin the sequence of the precursor protein were per�formed using the program BioTools 3.1 (Bruker Dal�tonics, Germany).

MALDI mass spectrometry. The intermediate con�trol of peptide preparations at the stages of solid�phaseextraction and anion�exchange chromatography andthe analysis of BSA preparations treated with Drise�lase were performed using an Ultraflex�TOF�TOFtime�of�flight mass spectrometer (Bruker Daltonics,Germany) equipped with a UV laser (337 nm). Positiveions were detected in the reflectron regime at the fol�lowing voltages: 25 kV on an ion source IS1, 21.75 kVon IS2, 9.5 kV on lenses, 26.43 kV on reflectron Ref1,and 13.80 kV on reflectron Ref2. Ions were detected in700–4000 m/z range.

ACKNOWLEDGMENTS

This work was supported by the RAS Presidium BasicResearch Program “Molecular and Cell Biology”.

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The search for and identification of peptides from the moss Physcomitrella patens