Báo cáo khoa học: Identification of the N-termini of NADPH : protochlorophyllide oxidoreductase A and B from barley etioplasts (Hordeum vulgare L.) ppt

A pentapeptide motif that has been characterized as responsible for binding of protochlorophyllide to the transit peptide of PORA [Reinbothe C, Pollmann S, Phetsarath-Faure P, Quigley F,

Identification of the N-termini of

NADPH : protochlorophyllide oxidoreductase A and B

from barley etioplasts (Hordeum vulgare L.)

Matthias Plo¨scher1, Bernhard Granvogl1, Veronika Reisinger1and Lutz A Eichacker2

1 Department of Biology I, Ludwig-Maximilians-University Munich, Germany

2 Center for Organelle Research (CORE), Universitetet i Stavanger, Norway

The first step of plant greening is catalysed by

NADPH : protochlorophyllide oxidoreductase (POR),

which is one of the most abundant enzymes found in

etioplasts The enzyme catalyses the light-activated

reduction of protochlorophyllide (Pchlide) to

chloro-phyllide [1] In Arabidopsis thaliana, three isoforms of

the protein, PORA, PORB and PORC, are known

[2,3] In barley (Hordeum vulgare L.), only PORA and

PORB are found, and only one isoform of POR is

present in pea (Pisum sativum L.) [4,5] The isoforms

accumulate as membrane-associated extrinsic proteins

in the prolamellar body and to a lesser extent in

prothylakoids [6] The photoactive POR comprises a

stable ternary NADPH–Pchlide–POR complex that

may assemble into higher-molecular-weight oligomers

in vivo[7]

Although the various POR proteins appear to be structurally very similar [3], their expression has been shown to be differentially regulated by light Expres-sion of PORA mRNA is dependent on darkness, whereas PORB mRNA is continuously expressed after illumination [2,8] Upon illumination, the prolamellar body and prothylakoids are transformed to thylakoids, the concentration of PORA decreases, and only PORB remains in the chloroplasts [4]

This differential accumulation of PORA and PORB

in the inner etioplast membrane system has attracted considerable scientific attention The nucleus-encoded precursor proteins of POR (pPOR) are expressed in the cytosol Experiments have centred on study of the regulation of protein transport into the plastid Two hypotheses have been published [9] The first

hypothe-Keywords

etioplast; N-terminus; PORA;

protochlorophyllide oxidoreductase; transit

peptide

Correspondence

L A Eichacker, Center for Organelle

Research (CORE), Universitetet I Stavanger,

Kristine Bonnevis vei 22, N-4036 Stavanger,

Norway

Fax: +47 518 31860

Tel: +47 518 31896

E-mail: lutz.eichacker@uis.no

(Received 11 October 2008, revised 7

December 2008, accepted 10 December

2008)

doi:10.1111/j.1742-4658.2008.06850.x

The N-termini of the NADPH : protochlorophyllide oxidoreductase (POR) proteins A and B from barley and POR from pea were determined by acet-ylation of the proteins and selective isolation of the N-terminal peptides for mass spectrometry de novo sequence analysis We show that the cleav-age sites between the transit peptides and the three mature POR proteins are homologous The N-terminus in PORA is V48, that in PORB is A61, and that in POR from pea is E64 For the PORB protein, two additional N-termini were identified as A62 and A63, with decreased signal intensity

of the corresponding N-terminal peptides The results show that the transit peptide of PORA is considerably shorter than previously reported and predicted by ChloroP A pentapeptide motif that has been characterized as responsible for binding of protochlorophyllide to the transit peptide of PORA [Reinbothe C, Pollmann S, Phetsarath-Faure P, Quigley F, Weis-beek P & Reinbothe S (2008) Plant Physiol 148, 694–703] is shown here to

be part of the mature PORA protein

Abbreviations

Pchlide, protochlorophyllide; POR, NADPH : protochlorophyllide oxidoreductase; pPOR, precursor of NADPH : protochlorophyllide

oxidoreductase; SPP, stromal processing peptidase; TNBS, 2,4,6-trinitrobenzoesulfonic acid; UPLC, ultra performance liquid chromatography.

sis states that translocation across the envelope

mem-brane is mediated by the general import pathway,

utilizing the translocons of the outer and inner

chloro-plast envelope membrane, TOC and TIC [10–13] The

second hypothesis proposes that only pPORB is

imported by the general import pathway, whereas

pPORA requires an additional mechanism, as import

was described as being dependent on Pchlide binding

to the precursor peptide [14–17] Various

protochloro-phyllide-dependent translocon proteins have been

described [18–21] Recently, a pentapeptide motif was

described for binding of Pchlide to the transit peptide

of pPORA [22]

The N-terminus of mature PORA from Hordeum

vulgareL was first determined by Edman degradation

[23], and resulted in identification of G75 as the first

amino acid of the mature protein Shortly thereafter, a

tryptic peptide, with G68 as the N-terminus was

identi-fied [24] The N-terminus of PORB is described in the

SwissProt database, and is classified as ‘potential’

(Q42850) The database entry refers to an older

charac-terization of the N-terminus of POR from pea [5], but

this N-terminus shows no homology to the N-terminus

of PORA described by Schulz et al [23] Despite

dis-agreement between the results of the scientific studies,

the highly tentative determinations of these N-termini,

especially of PORA, have not been challenged

experi-mentally using modern mass spectrometric techniques

We therefore used modern methods for precise

determination of the N-terminal cleavage sites of

PORA and PORB from barley and POR from pea

We modified a published method to enable selective

LC-MSMS based sequencing of N-terminal peptides at

low concentration or if blocked at the N-terminus [25]

We show that the N-termini of PORA and PORB of

barley and POR from pea are homologous, and that

Pchlide cannot bind to the PORA transit peptide of

pPORA as recently proposed [22]

Results

Acetylation of the POR protein and selective

isolation of the N-terminal peptide

Proteins extracted from plant or animal tissue are well

separated by polyacrylamide gel electrophoresis to

decrease the complexity of the sample Proteins of

equal molecular weight are concentrated in a gel band

or spot where they are accessible for identification and

further investigations Here, we used mass

spectrome-try-based protein identification of the N-terminal

peptides of POR separated by SDS–PAGE to compare

the precursor cleavage site of various POR proteins

For experimental determination of the mature N-ter-minus of the gel-separated POR proteins, we modified

an experimental procedure for proteome-wide analysis

of N-terminal peptides to be used after gel separation

of proteins [25]

We used acetic anhydride for in-gel acetylation of primary amino groups of the proteins and OMX-S reaction tubes for efficient in-gel digestion The a-amino group at the N-termini and the e-amino group of lysines were found to be completely acety-lated, whereas serines and threonines were only partially acetylated Partial acetylation of the hydroxyl groups was avoided by incubation of gel-trapped pro-teins in hydroxylamine Acetylated propro-teins were then in-gel-digested by a rapid protocol as described in the OMX-S instruction manual Instead of Tris buffer, a disodium tetraborate buffer was used to avoid side reactions during the following reaction steps After in-gel digestion, exclusively peptide sequences with arginine at the C-terminus were identified throughout After extraction of the peptide solution from the poly-acrylamide gel, the sample volume was split into two equal parts One part was directly separated by liquid chromatography (UPLC), and the second part was modified using trinitrobenzoesulfonic acid (TNBS) before UPLC separation As TNBS selectively modifies the N-terminal amino groups of internal peptides, the hydrophobicity of the corresponding peptides is increased, leading to a delay in the retention time during chromatographic separation In contrast, the N-terminal peptides were not modified at the peptide level and hence could be easily identified as no reten-tion shift was observed for these peptides (Fig 1) Finally, the exact amino acid sequence of the N-termi-nal peptides was determined from the MS⁄ MS spectra recorded from peptides with unchanged chromato-graphic separation

Neutral solvents decrease the appearance

of alkali metal adducts Interestingly, acetylated peptides showed significantly increased mass signals of sodium and potassium alkali metal and various di- and tri-alkali metal adducts if standard solvents with 0.1% formic acid were used for the UPLC separation (Fig 2A) In addition, the alkali metal adducts showed very low quality MS⁄ MS spectra, leading to difficulties for de novo sequencing analysis In order to increase the signal intensity of the protonated signals, we exchanged the acidified solvent containing 0.1% formic acid for a neutral solvent con-taining 10 mm ammonium formate (Fig 2B) Ammo-nium adducts were eliminated completely compared to

the standard method by decreasing the cone voltage to

35 V and increasing the capillary voltage to 3500 V

(see Experimental procedures)

Identification of the N-terminal amino acids from

various POR proteins

First, the N-terminal peptide of PORA from barley was

determined A peak at 6.55 min appeared, with no

hydrophobic shift, in both chromatograms (Fig 1) MS

analysis revealed a peptide with m⁄ z 871.11 [M + 3H]3+, and fragmentation analysis resulted in a corre-sponding amino acid sequence of VATAPSPVTT SPGSTASSPSGKKTLR The N-terminal amino acid valine and both lysines in the sequence were acetylated Sequence comparison to the corresponding annotated barley sequence identified V48 as first amino acid of the mature PORA protein In contrast, determination of the N-terminal amino acid of PORB from barley resulted in identification of not one but three N-termi-nal peptides A61 was identified as the first amino acid

of a peptide with m⁄ z 693.31 [M + 3H]3+ This pep-tide showed the highest signal intensity and had the sequence AAAVSAPTATPASPAGKKTVR (Fig 3) Interestingly, we also found a second N-terminal pep-tide signal with m⁄ z 669.69 [M + 3H]3+ and amino acid sequence AAVSAPTATPASPAGKKTVR, and a third N-terminal peptide signal with m⁄ z 646.02 [M + 3H]3+ and a corresponding amino acid sequence AVSAPTATPASPAGKKTVR All three N-terminal peptides were acetylated at the N-terminal amino acid, and the signal intensity decreased with lower molecular masses We therefore concluded that all three proteins were acetylated exclusively at the level of the mature protein, indicating that the various proteins resulted from three different cleavages by the processing prote-ase All experimental repeats revealed an equal ratio among the three N-terminal PORB peptides

The N-termini of various POR proteins are homologous

During identification of the N-terminus from POR of pea, we also found only one N-terminal peptide, with

TOF MS ES + BPI 1.02e3

TOF MS ES + BPI

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

min

0

100

%

0

100

A

B

%

N-terminal peptide

Fig 1 Base peak-intensity chromatogram of N-terminal and

internal peptides of PORA Peptides of PORA were isolated from

barley etioplasts and separated by UPLC using neutral solvents

containing 10 m M ammonium formate One half of each sample

was separated by UPLC after in-gel acetylation and digestion

with-out further modification (A) The second part of the sample was

separated after further modification of internal peptides using

TNBS, resulting in increased hydrophobicity of internal peptides (B).

In (B), only the N-terminal peptide of PORA remains unaltered and

elutes at the same retention time of 6.55 min as in (A).

0

100

A

B

%

TOF MS ES + 1.11e3

TOF MS ES + 7.77e3

[M + 3H]3+

[M + 2H + Na]3+

[M + 2H + K]3+

[M + H + 2Na]3+

[M + 3H]3+

[M + 2H + Na]3+

[M + 3Na]3+

[M + H + K + Na]3+

870 872 874 876 878 880 882 884 886 888 890 892 894

m/z

0

100

%

878.40

883.71

878.44 871.44

871.41

891.06 885.73

Fig 2 Mass spectra of the N-terminal pep-tide from barley PORA Mass spectra of the N-terminal peptide (871.11 [M + 3H] 3+ ) were recorded after peptide ionization in standard solvents containing 0.1% formic acid (A) and neutral solvents containing

10 m M ammonium formate (B) In standard solvents, distinct sodium ([M + 2H + Na] 3+ ), disodium ([M + H + 2Na]3+), trisodium (M + 3Na] 3+ ) and potassium adducts ([M + 2H + K] 3+ and [M + H + K + Na] 3+ ) appeared (A) In neutral solvents, the signal intensity of 871.11 [M + 3H] 3+

increased and only the sodium adduct [M + 2H + Na]3+was detectable, with a significantly lower signal intensity (B).

m⁄ z 804.39 [M + 3H]3+ and amino acid sequence

ETAAPATPAVNKSSSEGKKTLR (data not shown)

The first amino acid of the mature protein was

deter-mined to be E64 This finding appeared at first to be

in conflict with the SwissProt database entry, in which

T65 is denoted as the N-terminal amino acid of the

mature POR (SwissProt entry Q01289) However, in

the reference publication cited by SwissProt, E64 has

been determined by Edman degradation to be the first

amino acid of the mature protein, corroborating

our finding [5] The sequences of PORA and PORB

from barley and POR from pea were aligned using

clustal w (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_auto

mat.pl?page=npsa_clustalw.html) (Fig 4) Sequence

homology in the N-terminal region was found,

indicat-ing that the N-terminus of POR from pea is positioned one amino acid upstream in comparison to PORA and the main PORB protein identified in barley

When we performed a theoretical calculation of the N-terminus of PORA protein using the program chlorop (http://www.cbs.dtu.dk/services/ChloroP/), A91 was predicted to be the first amino acid of the mature protein (Fig 4) Hence, there is a difference of

43 amino acids from the N-terminus determined here Previous descriptions of the N-terminus of PORA also differ significantly from our results Schultz et al described G75 as the first amino acid of the mature pro-tein This processing site was determined by a method based on Edman degradation [23] (Fig 4) Later, the same group described a tryptic peptide with G68 as the first amino acid [24] (Fig 4) For PORB of barley, Chlo-roP predicts A59 as the first amino acid This prediction

is close to A61, which was experimentally determined to

be the first amino acid of the most intense signal of the three N-termini of PORB In the case of mature POR of pea, chlorop predicted A63 as the first amino acid This prediction differs by only one amino acid from E64, which is the first amino acid of the mature POR protein according to our experimental determination

Discussion

Pchlide binding motifs are only found in the mature PORA

In contrast to previous publications [26], we found that the N-termini of the various POR proteins show strong sequence homology (Fig 4), and our results also indicate a significantly shorter transit peptide for PORA This finding is of importance with respect to a hypothesis proposed regarding Pchlide-dependent import of pPORA [15,17] In favour of this hypothesis,

TOF MS ES + 621

640 645 650 655 660 665 670 675 680 685 690 695 700 705 710

m/z

0

100

%

[M – A + 3H] 3+

[M – 2A + 3H] 3+

[M + 3H] 3+

[M + 2H + Na] 3+

693.36

700.70 669.69

646.02

Fig 3 Mass spectrum of N-terminal peptides from PORB

Pep-tides were ionized in neutral solvents with 10 m M ammonium

for-mate Three peptides were identified with m ⁄ z 693.36 [M + 3H] 3+ ,

669.69 [M + 3H] 3+ and 646.02 [M + 3H] 3+ The highest signal

intensity at 693.36 [M + 3H] 3+ was identified by de novo sequence

analysis as a peptide form containing three N-terminal alanines The

minor peptide signals were identified as two alternative N-terminal

PORB peptides containing two alanines with m ⁄ z 669.69

[M ) A + 3H] 3+ and one alanine with m ⁄ z 646.02 [M ) 2A + 3H] 3+

Fig 4 Sequence alignment of pPORA and pPORB from barley and pPOR from pea Arrowheads indicate the cleavage sites between the transit peptide and the mature PORA protein The transit peptide as described here is shown in bold type The bold arrow ( ) indicates the position of the experimentally verified N-terminus of PORA Previous descriptions of the position of the N-terminus according to Benli et al [23] are marked by a narrower arrow ( ), and that according to Schulz et al [24] by a short arrow ( ) The arrowhead ( ) indicates the pre-dicted cleavage site according to the program CHLOROP (http://www.cbs.dtu.dk/services/ChloroP/) The pentapeptide motif proposed to be responsible for binding of Pchlide according to Reinbothe et al [22] is shown in bold, italic letters and is only present in PORA Identical resi-dues are indicated by asterisks, strongly similar resiresi-dues are indicated by colons, and weaker similarity is indicated by dots to illustrate the homology of the cleavage sites between PORA and PORB from barley and POR from pea The complete protein sequence alignment was performed using CLUSTAL W [37].

it has been proposed that the transit peptide contains

a Pchlide binding site [22,26], and that binding of

Pch-lide to pPORA is essential for import into the etioplast

stroma The transit peptide of PORA is the only

exam-ple of a precursor protein for which a substrate

bind-ing site has been proposed to regulate import in

addition to the general import pathway [19–21] This

hypothesis was based on chimeric fusion proteins in

which the transit peptide of pPORA was functionally

exchanged with the transit peptide of pPORB and vice

versa, and the transit peptides were fused to a reporter

protein of mouse It was found that only the isolated

transit peptide of PORA bound Pchlide, with a

stoichi-ometry of 1 : 1 [26] Recently, amino acids T56–G60

have been defined as a pentapeptide motif that is

responsible for binding Pchlide and for the import of

PORA [22] (Fig 4) However, according to our

find-ings, amino acids T56–G60 of PORA are located

exclusively in the mature part of the PORA protein

and not in the transit peptide The position of the

motif in the N-terminal region of the mature protein is

in conflict with a function of the motif in a

Pchlide-responsive transit peptide as described previously [22]

Pchlide-dependent import would require a binding of

Pchlide to the mature part of PORA, which is

C-termi-nal of the processing site It remains open whether

Pchlide binding to the proposed motif in mature

PORA is of importance for regulation of PORA

import [22] In addition to a regulatory function in

protein import, binding of Pchlide at this binding site

could be important for transient stabilization of the

PORA protein in the plastid stroma after import and

before the protein is assembled into an enzymatically

active form As a number of groups have found that

accumulation of PORA in the plastid stroma is a

substrate-independent process, close inspection of

pub-lished data and development of new experimental

set-ups is essential to clarify this interesting topic

[6,10–13,27]

Alternative N-termini of PORB

In contrast to the one unique processing site that we

describe here for the PORA protein, we found three

possible N-termini for the PORB protein The

N-ter-minal amino acid of the corresponding peptide signal

with the most intense signal is homologous to the

N-terminal amino acid of PORA (Fig 4) The two

additional N-terminal peptides of PORB both start

with the amino acid alanine In parallel with the loss

of one and two amino groups, the signals of the

N-ter-minal peptides decrease in intensity This could be

indicative of a correspondingly lower concentration of

these two alternative PORB proteins The reason for differential processing of PORB could be error-prone positioning of the processing peptidase at the cleavage site in the presence of three consecutive alanines, or could indicate that a second processing peptidase scans the N-termini after the first cleavage

The cleavage site is characterized by a conserved arginine within the transit peptide, which is located two amino acids upstream of the N-terminal cleavage site Aliphatic and non-polar amino acids are found N-terminal to the arginine Alanine or threonine is found C-terminal to the processing site Similar amino acids are present around the processing site in POR of pea Glutamine has been found to be the last amino acid of the transit peptide in PORA and B of barley, whereas POR from pea contains a glutamic acid at this position, which is the first amino acid of the mature protein The sequence homology around the amino acids of the processing site therefore leads to the con-clusion that the processing peptidase or peptidases might be the same for all POR proteins [28,29] Although a general stromal processing peptidase (SPP) has been characterized, and a preferred consensus sequence for cleavage between a basic amino acid (arginine or lysine) and a C-terminal alanine was described [30,31], it is open where exactly SPP cleaves Two alternative N-termini have been described for the stromal cysteine synthase [32], similar to the three N-termini of PORB described here SPP may therefore cleave specifically after R-58, with a second less specific processing peptidase cleaving C-terminal of A59, Q60 and A61⁄ 62 Then, V48 of PORA at the homologous position to A61 of PORB would position the second processing protease to yield one exactly defined N-terminus Alternatively, SPP may be the only pro-cessing peptidase In this case, the cleavage site down-stream of the SPP consensus motif has a lower specificity for PORB

Experimental procedures

Chemicals All organic solvents and water used in this work were of HPLC gradient quality and purchased from Fisher Scien-tific (Schwerte, Germany) Acetic anhydride, ammonium formate and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were obtained from Fluka (Buchs, Switzerland), Coomassie brilliant blue R250 was obtained from Serva (Heidelberg, Germany), and disodium tetraborate decahydrate was obtained from Merck (Darmstadt, Germany) Sequencing grade modified trypsin was purchased from Promega (Mannheim, Germany)

Protein isolation and gel electrophoresis

Etioplasts were isolated from 4.5-day-old dark-grown

bar-ley seedlings (Hordeum vulgare L var Steffi) as described

previously [33] Membrane proteins were solubilized in SDS

buffer (3% w⁄ v SDS, 15% w ⁄ v sucrose, 100 mm sodium

carbonate, 0.04% w⁄ v bromophenol blue, 0.3% v ⁄ v

b-mer-captoethanol) by heating for 2 min at 72C, and separated

on 12.5% SDS–polyacrylamide gels containing 4 m urea in

a Protean II electrophoresis system (Bio-Rad, Hercules,

CA, USA) [34] For each lane, proteins from 1· 108

plast-ids were loaded Gels were stained with Coomassie brilliant

blue

POR from etiolated pea leaves (Pisum sativum L var

Violetta) was isolated from 14-day-old seedlings grown in

the dark on vermiculite Due to the small size of the leaves

(approximately 1 mm2), membrane-associated proteins were

prepared from the whole leaf material In brief, the leaves

were ground in TMK buffer (10 mm Tris⁄ HCl pH 6.8,

10 mm magnesium chloride, 20 mm potassium chloride) at

4C and centrifuged at 16 000 g for 5 min The

superna-tant was discarded, and the pellet was resuspended in TMK

buffer and centrifuged twice at 16 000 g for 5 min to

remove soluble proteins Then the pellet was resuspended in

SDS buffer and proteins were solubilized by heating for

5 min at 72C before separation on a 12.5%

SDS–poly-acrylamide gel containing 4 m urea Proteins were stained

with Coomassie brilliant blue and fixed in the gel matrix by

incubation in acetic acid (10%) Detection of POR was

carried out by gel-blot analysis [35]

In-gel acetylation and in-gel digestion

For sample preparation, the OMX-S tool for in-gel

diges-tion was used (OMX, Wessling, Germany) [36] Briefly, the

protein spot of interest was excised from the SDS gel and

the gel was ruptured by centrifugation at 13 000 g for 2 min

Proteins were destained in 50 mm ammonium bicarbonate

and 50% acetonitrile at 37C for 5 min Then the

orienta-tion of the OMX-S tool in the centrifuge was inverted, and

the solution was removed from the reaction chamber by

cen-trifugation at 2500 g For in-gel acetylation of intact

pro-teins, 22.5 lL of 50 : 50 (v⁄ v) acetonitrile ⁄ water and 2.5 lL

of acetic anhydride were added to the reaction chamber, and

the mixture was incubated at 37C for 30 min Thereafter,

acetic anhydride was removed completely by washing three

times with 25 lL of 50 : 50 (v⁄ v) acetonitrile ⁄ water for 5

min each Partial acetylation of serines and threonines was

avoided by adding 12 lL of a solution containing 0.5 mm

hydroxylamine and 100 mm NaOH to the reaction chamber

and incubating at 37C for 15 min Thereafter, 12 lL

aceto-nitrile was added to the sample, and the solution was

removed by reverse centrifugation

After in-gel acetylation, in-gel digestion was carried out

utilizing 20 lL of 50 mm disodium tetraborate buffer,

pH 8.5, and 2 lL of trypsin at 50C for 45 min The pep-tide mixture was removed from the gel pieces and split into two equal parts (A and B) The volume of part A was increased to 30 lL using 50 mm disodium tetraborate buffer, pH 8.5, and acidified with 5 lL of 40% formic acid to stop trypsin digestion The peptide mixture in part B was modified with 100 mm TNBS solution in water For this modification, the sample volume was increased to 28 lL with disodium tetraborate buffer,

pH 9.8, resulting in a final pH of the sample of 9.5 Then

2 lL of 100 mm TNBS solution were added, and the mix-ture was incubated at 37C for 1 h Finally, the sample was acidified using 5 lL of 40% formic acid

UPLC separation and mass spectrometry For peptide separation, a Waters nanoAquity 10 000 psi UPLC system (Waters Corporation, Milford, MA, USA) was used, equipped with a BEH130 C18 nanoflow column, particle size 1.7 lm, with an inner diameter of 100 lm and

a length of 100 mm, and a Symmetry C18 trapping column, particle size 5 lm, and dimensions 180 lm· 20 mm Two solvent systems – standard solvents acidified with formic acid and neutral solvents with ammonium formate – were used In the standard approach, solvent A was composed

of 95 : 5 (v⁄ v) water ⁄ acetonitrile and the solvent was acidi-fied by addition of 0.1% formic acid, and solvent B was composed of 99.9 : 0.1 (v⁄ v) acetonitrile ⁄ formic acid In the neutral approach, solvent A comprised 95 : 5 (v⁄ v) water⁄ acetonitrile and 10 mm ammonium formate was added, and solvent B comprised 100% acetonitrile The same solvent gradient was used for both approaches The sample was trapped for 2 min at a flow rate of 15 lLÆ min)1, followed by a linear gradient from 1% to 80% solvent B applied over 16 min with a flow rate of 1.2 lLÆmin)1

The UPLC equipment was connected to a Micromass Q-TOF Premier mass spectrometer (Waters Corporation, Milford, MA, USA) Mass spectra were obtained by auto-mated LC-MS and LC-MS⁄ MS analysis, and peptides were identified using masslynx version 4.1 (Waters Corpora-tion) With standard solvents, a capillary voltage of 3000 V and a cone voltage of 45 V was used With neutral solvents,

a capillary voltage of 3500 V and a cone voltage of 35 V was used The two MS chromatograms from parts A and B

of each sample were aligned using masslynx

Acknowledgements

The work was funded by the Deutsche Forschungs-gemeinschaft and the Sonderforschungsbereich Trans-regio1 (SFB TR1) The antibody against the mature part of POR was donated by Professor Sundquvist, Sweden

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