Identification of the N-termini of NADPH : protochlorophyllide oxidoreductase A and B from barley etioplasts (Hordeum vulgare L.) Matthias Plo ¨ scher 1 , Bernhard Granvogl 1 , Veronika Reisinger 1 and Lutz A. Eichacker 2 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. 1074 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 vulgare L. 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 Ò 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 proteins were then in-gel-digested by a rapid protocol as described in the OMX-S Ò 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 M. Plo ¨ scher et al. N-terminus of protochlorophyllide oxidoreductase FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 1075 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 1.02e 3 TOF MS ES + BPI 728 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). N-terminus of protochlorophyllide oxidoreductase M. Plo ¨ scher et al. 1076 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 residues 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]. M. Plo ¨ scher et al. N-terminus of protochlorophyllide oxidoreductase FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 1077 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 Ò 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). N-terminus of protochlorophyllide oxidoreductase M. Plo ¨ scher et al. 1078 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 72 °C, 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 · 10 8 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 mm 2 ), 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 4 °C 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 72 °C 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-SÒ 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 37 °C for 5 min. Then the orienta- tion of the OMX-SÒ 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 37 °C 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 37 °C 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 lLof50mm disodium tetraborate buffer, pH 8.5, and 2 lL of trypsin at 50 °C 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 37 °C 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 nanoAquityÔ 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. M. Plo ¨ scher et al. 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Proc Natl Acad Sci USA 92, 3254–3258. 9. 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