Cell Lysis and protein preparation

Axenic cultures of T. pseudonana clone CCMP1335 were grown in enriched artificial seawater (ESAW) medium [16] at 18 °C, at 40 µmol photons m^-2s^-1 on a 12/12 photoperiod and harvested in exponential growth phase. Cells were pelleted (3000 g, 10 min, 4°C), washed twice with lysis buffer (50 mM HEPES, 20 mM KCl, 1 mM EDTA, 0.2 mM DTT, 150 mM sorbitol, pH 7.5) supplemented with PMSF and Complete protease inhibitor mixture (Roche), then broken with 0.3 mm glass beads in a mini-bead-beater (Omni International). The beads were removed by centrifugation (1,000 g, 5 min and the supernatant divided into crude soluble and membrane protein fractions by centrifugation at 16,000 g for 15 min. The supernatant fraction was filtered through a 0.22 µm filter, then concentrated and buffer exchanged to 50 mM HEPES (pH 7.5) using a spin filter device (3 kDa MW cutoff, Millipore). Aliquots of the membrane protein fraction were further purified by sucrose gradient centrifugation, diluted 10 times with 50 mM HEPES (pH 7.5) supplemented with complete protease inhibitor cocktail, sedimented at 45,000 g for 1 h and resuspended in 50 mM HEPES buffer pH 7.5 containing 1% deoxycholate. After heating for 1 min at 95 °C, the non-solubilized materials were pelleted at 14,000 g and the supernatants used for further analysis. All protein fractions were independently used for enrichment of N- terminal peptides.

Enrichment of protein N-terminal peptides

Protein N-terminal peptides were enriched by a polymer-based negative selection method [17]. In short, 500 µg to 1 mg protein from each preparation were denatured, reduced, alkylated, followed by reductive dimethylation of the primary N-terminal α-amines and the ε-amines of Lys side chains using formaldehyde and sodium cyanoborohydrate (ALD coupling solution, Sterogene). Modified proteins were precipitated with chloroform/methanol, resuspended at 1 mg/ml and digested with 1 µg trypsin / 100 µg protein (Trypsin Gold, Promega). After digestion, the peptides with internal trypsin-generated primary amines were covalently bound to water-soluble high molecular weight dentritic polyglycerol-aldehyde polymer (HPG-ALD, Flintbox) using sodium cyanoborohydrate at a ratio of 2.5 mg polymer / mg peptides (Figure 2a). The polymer with coupled tryptic peptides was then removed from the unbound N-terminal peptides (which were either naturally amino-modified or chemically modified by the reductive dimethylation step) by filtration with a spin filter device (10 kDa MW cut-off, Millipore), desalted and identified by high resolution LC-MS/MS.

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Figure 2. T. pseudonana protein termini identified by TAILS.

(a) Schematic representation of the TAILS workflow. Proteins with free or naturally modified (black square) N termini are denatured, followed by chemical modification of all primary amines (grey triangle). Specific digestion with trypsin generates peptides amenable to mass spectrometric identification. N-terminal peptides are blocked, whereas internal or C-terminal peptides exhibit a trypsin-generated primary amine at their N terminus that is used to covalenty bind these peptides to an aldehyde-containing polymer which is subsequently removed by filtration. (b) Position of identified N-terminal peptides with respect to curated protein model. N termini matching the protein models at positions 1 and 2 are cytosolic proteins with intact (+M1) or removed (–M1) initiating Met. Black, acetylated N termini; dark grey, protein N termini present in both dimethylated and acetylated forms; light grey, free N termini identified as dimethylated peptides. (c) Sequence logoplot of the first 6 amino acids of 81 N termini of nuclear encoded proteins with intact initiating Met. (d) Sequence logoplot of 231 N termini of nuclear-encoded proteins starting at protein model position 2 because the initiating Met was removed. (e) Combined logoplot of N termini of 22 plastid-encoded proteins starting at position 2 after N-terminal Met excision plus 18 plastid-imported proteins with Met directly preceding the identified peptide.

https://doi.org/10.1371/journal.pone.0074483.g002

LC-MS/MS

Enriched N-terminal peptides were desalted with C18 stop-and-go extraction tips or fractionated and desalted with C18-SCX-C18 stop-and-go extraction tips as described [18] prior to LC-MS/MS analysis with a linear ion trap–fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR, Thermo) or linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap Velos, Thermo). The LTQ-FTICR and LTQ-Orbitrap instruments were coupled on-line to an Agilent 1100 Series and Agilent 1200 Series HPLC instrument, respectively, which separated peptide samples on columns packed with ReproSil Pur C18 beads using a 6–80% gradient of organic phase over 95 min. Buffer A was 0.5% acetic acid and buffer B was 0.5% acetic acid with 80% acetonitrile. The LTQ-FT was set to acquire a full-range scan at 25,000 resolution from 350 to 1,500 m/z in the FT-ICR cell, followed by selected ion monitoring (SIM) of the top three peptide ions in each cycle at resolution 50,000 for accurate mass measurement and MS/MS of these 3 ions in the LTQ (minimum intensity 500 counts). Parent ions were then excluded from MS/MS for the next 180 sec. The LTQ-Orbitrap was set for acquisition of full-range scans from 350 m/z to 1,500 m/z at a resolution of 60,000, followed by fragmentation of the five most intense ions in the LTQ. Raw data files were acquired by Xcalibur v2.0.1 (for LTQ-FTICR, Thermo) and Xcalibur v2.1.0 (for LTQ-Orbitrap Velos, Thermo).

The raw data associated with this manuscript may be downloaded from the ProteomeCommons.org Tranche network using the following hash:

1. 0 eoIDZbMgFjoLK5DiSKJKDcYZcVzOFJNjxiT5l8nIrS3C32mygEYHb4Fx4xlgBeBEl244gJxMWyWe7OAtAHysPFZTFgAAAAAAAACsQ==

The hash may be used to prove exactly what files were published as part of this manuscript’s dataset, and the hash may also be used to check that the data has not changed since publication. The passcode for accessing the data is “ThapsTAILS”.

Data analysis and identification of putative transit peptide sequences

For peptide identification, a T. pseudonana protein database was assembled combining the Joint Genome Institute (JGI) gene catalog protein models (downloaded from genome.jgi-psf.org/Thaps3/Thaps3.home.html on 2011-09-14) and the plastid-encoded protein sequences (downloaded from chloroplast.ocean.washington.edu on 2011-09-14) with appended reverse decoy sequences. For peptide identification with two search engines, X! TANDEM [19] and MASCOT v2.3 (Matrix Science), raw data files were converted to the mzXML format using ReAdW v4.3.1 (Institute for Systems Biology, Seattle) and mgf format using Proteome Discoverer 1.2 (Thermo Scientific). A custom Perl script completed the headers in the *. mgf files (added time and file name). Search parameters included: Precursor mass tolerance, 10 ppm; Fragment ion mass tolerance, 0.4 Da; enzyme, semi-ArgC (due to the inability of trypsin to cut at dimethylated Lys) with up to two missed cleavages, Peptide modifications: Carboxyamidomethylation of cysteine residues (+57.021464 Da), dimethylation of lysine ε-amines (+28.0313 Da) and variable N-terminal modification by acetylation (+42.010565 Da) or dimethylation (+28.0313 Da), Peptide identifications were subjected to secondary validation with PeptideProphet [20] as implemented in the Trans-Proteomic Pipeline v4.4 [21] and accepted with an estimated FDR <5%. Next, peptide lists from the database searches were combined into a non-redundant peptide list using an in-house script that also summed the number of spectra supporting each unique identified sequence across different experiments. Peptides with unmodified N termini carried over from incomplete depletion were removed from the results list, as were peptides not ending with Arg. The start position of each peptide in relation to the protein model was calculated, and protein annotation information retrieved from Uniprot, JGI gene catalog and CpBase supplementary data as available.

The position of the identified N-terminal peptide in relation to the protein sequence derived from the gene model was used for pre-selection of candidate plastid proteins. This was followed by manual examination of all gene models for which identified N-terminal peptides matched between residue positions 16 and 75, unless the available annotation information indicated a non-plastid destination for the protein. Classification as the mature N terminus of a plastid-targeted protein required an N-terminal ER SP sequence, followed by Phe or Tyr in a variation of the known FXP or FXXP motif [9,10]. In a number of cases, the sequences coding for the ER signal peptides required for the first step of plastid import were found only after upstream extension of incomplete gene models.

To find homologs in other algae with red plastids, the draft genomes available at JGI (http://genome.jgi-psf.org/) were searched using AlgaeBlast (http://genome.jgi-psf.org/Algae/Algae.info.html). Related sequences were also obtained from Genbank (http://blast.ncbi.nlm.nih.gov/).

Sequence logos for aligned sequences were generated using the iceLogo webserver [22].

Identification and characterization of protein N-terminal peptides

Diatom cells are surrounded by a rigid silicaceous wall (frustule), which can only be broken by agitation with glass beads or even more drastic measures that also disrupt the fragile plastids. For this reason, total T. pseudonana lysates fractionated into crude soluble and membrane fractions by centrifugation had to be used. These fractions were independently enriched for N-terminal tryptic peptides using TAILS (Figure 2a) [14]. In TAILS, free N-terminal α-amino groups of proteins that are not modified in vivo, e.g. by acetylation, and lysine ε-amino groups are first blocked in whole proteins by reductive dimethylation. Thus, after tryptic digestion, all peptides originating from true protein N termini are either chemically or naturally blocked, whereas trypsin-generated peptides display primary α-amines that are then covalently coupled to a water-soluble aldehyde-functionalized polymer. The high-molecular weight polymer with bound internal peptides is removed by spin filtration, leaving a filtrate highly enriched in N-terminal peptides.

Enriched N-terminal peptides were analyzed by high resolution LC-MS/MS and individual peptide sequences were identified from a database containing all T. pseudonana protein models predicted from the nuclear and plastid genomes using two different search engines (see Methods). Spectrum-to-sequence assignment searches considered only dimethylation or acetylation as N-terminal modifications, since most other naturally occurring N-termini blocking modifications are rare and affect only a low percentage of proteins [23]. The resulting peptide assignment lists from different experiments and searches (Tables S1 to S4) were combined into a non-redundant peptide list for further analysis.

We identified a total of 1,401 distinct N-terminal peptide sequences. Of these, 1,055 peptides were dimethylated, i.e. originating from proteins that had N-termini with free α-amines in vivo, and 438 were acetylated, i.e. originating from proteins with co- or post-translationally acetylated N-termini. In addition, 92 N-terminal peptides were found in both dimethylated and acetylated forms, indicating partial acetylation in vivo. 1,295 of the peptides matched 939 proteins encoded by the nuclear genome (Table S5), and 106 peptides matched 37 chloroplast-encoded proteins (Table S6).

The N-terminal peptides were sorted into bins according to where they mapped on the matching protein model sequence (Figure 2b). N-termini of nuclear-encoded proteins starting at the first or second amino acid of the model were assumed to be primarily cytosolic, i.e. polypeptides without any organellar or secretory targeting presequence. Proteins with mitochondrial or ER signal sequences should fall in the 16 to 30 residue bin, while a small number of N-termini in bin 3-15 result from limited processing, mostly by amino-peptidases. The best candidates for proteins with a bipartite plastid-targeting sequence were expected in the 30-75 residue bins. Peptides mapping more than 75 amino acids from the beginning of the protein model were regarded as products of internal endoproteolytic processing and were not examined further. However, it should be noted these “internal” N termini result from common physiological processes as well as from technical limitations: i) splicing and alternative translation starts; ii) proteolytic processing, a post-translational modification that regulates the function of many proteins, e.g. zymogen activation [15]; iii) unusually long targeting sequences, e.g. in proteins targeted to the thylakoid lumen; iv) naturally occurring degradation intermediates of abundant proteins that, despite their short half-lives, can be present in higher concentrations than low-abundance proteins; v) background proteolysis during sample preparation by proteases resistant to the inhibitor cocktail used; vi) incorrectly predicted protein models. The proportion of endoproteolytic products (38% mapping at positions >75) may appear surprisingly large, but is comparable to the proportion of “internal” N termini observed in other studies, e.g. mouse tissues (44% [24]) and human Jurkat cell lysates (51% [25]) and thus likely reflects the proportion of proteolytic processing and accumulation of degradation intermediates encountered naturally in vivo.

Almost three-quarters of the cytosolic proteins identified at their predicted protein start had their initiating Met removed (bin 2) as part of their co-translational processing in the cell [23]. In agreement with general specificity rules for Met aminopeptidases [26], the initiating Met was retained if the penultimate residue was a charged residue, e.g. Asp, Asn or Glu (Figure 2c), and removed if the penultimate residue had a small gyration radius, e.g. Ala, Gly, Ser, Thr, or Val (Figure 2d). This shows that diatom Met aminopeptidases follow the same rules as those of other eukaryotes [23,27]. Complete or partial N-terminal acetylation was observed for 70% of the cytosolic proteins, irrespective whether the initiating Met was retained (Figure 2B, bin 1) or removed (Figure 2B, bin 2). Thus N-terminal acetylation is more frequent than reported for yeast (60%), in agreement with increasing percentages of N-terminal acetylation towards more complex eukaryotes [28].

Identification of plastid-targeted proteins

Proteins with N-terminal peptides matching between position 30 and 75 of the corresponding gene model and those with annotations suggesting a plastid location were considered candidates for chloroplast-targeted proteins. Each of the protein models was manually inspected unless the available annotation information clearly indicated a non-plastid destination. Classification as a plastid-targeted protein required that the model had a potential N-terminal bipartite targeting sequence, i.e. an ER signal peptide sequence followed by F or Y (Figure 1b). In a number of cases, the ER signal peptide sequence required for the first step of plastid import was only found after re-evaluation and upstream extension of the T. pseudonana gene models. In total, more than 500 sequences were subjected to expert manual evaluation.

As an example, Figure 3a shows the N-terminal peptides derived from PetC, the Rieske iron-sulfur protein of the plastid cytochrome b₆f complex, which is known to be nuclear-encoded and translated on cytoplasmic ribosomes. Two unique peptide sequences, differing by a missed tryptic cleavage site at their C termini, suggest a single unique N terminus for PetC starting at Ser-35. One peptide was found in both acetylated and dimethylated forms, with approximately one-seventh of the spectra corresponding to the acetylated form. Since acetylation occurs only in vivo, this further supports the identification as the true N terminus of mature PetC. When matched to the complete precursor sequence, the sequence between the SP cleavage site and the mature N terminus suggests a TP sequence 17 amino acids in length. A second example is the Chl a/c light-harvesting protein Lhcr2 (Figure 3b), where two different N-terminal peptides were identified. The first started with FAP, indicating that the protein still had an intact TP at the time of sampling. In addition, 16 spectra defined a unique partially acetylated mature N terminus at Ser-35, from which a 14-residue TP sequence was deduced. A third example is the unknown protein 4820, which is homologous to a putative plastid precursor protein in vascular plants (Figure 3c). Here, two well-supported peptides define a unique, partially acetylated mature N terminus at Gly-49 of the protein model, from which a 26-residue TP is deduced. However, an additional dimethylated peptide indicates an alternative start at Met-48.

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Figure 3. Approach for determining diatom transit peptide sequences.

Transit peptides (orange) and transit peptide cleavage sites were deduced by mapping identified N termini (green) to the protein model sequence after removal of the ER signal sequence (blue). (a) Cytochrome b₆f complex iron-sulfur protein subunit (PetC). Three peptides identify a single unique N terminus at position 35 of the protein model. (b) Light harvesting antenna complex protein Lhcr2. An acetylated and a dimethylated peptide identify the mature protein N terminus at protein model position 30, and a dimethylated peptide begins at the canonical SP cleavage site (ASA-FAP) at protein model position 16, indicating that this protein was incompletely processed or in transit when isolated. (c) Unknown protein 4820, homologous to a putative higher plant plastid precursor protein. Two peptides identify a mature, partially acetylated N terminus starting at protein model position 49, while a third peptide has an N-terminal Met starting at position 48. Bold, observed peptides; underlined, conserved ASA-FAP motif; arrow, inferred ER signal peptide cleavage site; arrowhead, observed protein termini.

https://doi.org/10.1371/journal.pone.0074483.g003

This approach led to the identification of 63 precursor proteins with a predicted SP cleavage site followed by Phe or Tyr, and an experimentally determined mature N terminus 13 to 42 amino acids further downstream (Table S7). The proteins were mostly those typical of a plant or green algal chloroplast proteome [29,30]. They included 14 members of the Chl a/c (LHC) protein family, 7 enzymes of heme or Chl biosynthesis, various enzymes of glycolysis, Calvin-Benson cycle, amino acid biosynthesis, carotenoid biosynthesis, lipid biosynthesis and sulfate reduction, 2 elongation factors (EF-Ts, EF-G), the protease subunit ClpB and a putative bicarbonate transporter (Table S7). The list also included a putative plastid-targeted N-acetyl transferase, which may be responsible for the observed N-terminal acetylation of imported and plastid-encoded proteins. Finally, we identified 14 proteins with unknown function that contained a SP followed by a canonical FAP or FXXP. Of these, 12 had homologs in at least one other diatom in the JGI algae databases.

Identification of the transit peptide cleavage motif and subsequent Met removal

The residues bracketing the deduced TP cleavage sites were plotted as an iceLogo (Figure 4a), which shows residues that are significantly over- or underrepresented at each position compared to the natural abundance of each amino acid in the T. pseudonana proteome [22]. The strongest preference for any particular amino acid was not directly at the cleavage site but was a Leu at either the -2 or -3 position. The occurrence of Leu at the -3 position strongly correlated with Met at the -1 position, whereas none of the 63 identified N-terminal peptide sequences started with Met. Since removal of N-terminal Met is ubiquitous in both prokaryotes and eukaryotes [23,26], we hypothesized that in these cases the initial TP cleavage occurred just before a Met that was subsequently removed by a Met-specific aminopeptidase residing in the plastid.

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Figure 4. Amino acid occurrences at the transit peptide cleavage site.

IceLogos of sequences surrounding the putative transit peptide cleavage site (dotted line) of 63 plastid-imported proteins (a) based on alignment of the identified N-terminal sequences and the C-terminus of the deduced transit peptides. (b) based on an alignment of the same 63 sequences, with the 18 sequences that showed a Met at -1 shifted by one position to the right on the assumption that N-terminal Met was removed by a plastid Met-aminopeptidase after import and transit peptide cleavage. Note that iceLogos show the difference between the observed amino acid frequency among the 63 identified sequences and the natural amino acid abundance in T. pseudonana proteins, i.e. overrepresented amino acids are indicated above the line and underrepresented amino acids below the line. Only differences with a p-value of 0.05 or smaller are shown.

https://doi.org/10.1371/journal.pone.0074483.g004

Our data for plastid-encoded proteins synthesized on plastid ribosomes supported this interpretation. We identified N-terminal peptides from 37 out of the 118 plastid-encoded proteins [31], all of which would have been synthesized starting with Met (Table S6). Of those peptides mapping close to the beginning of the gene model, 5 retained their N-terminal Met while 22 started with the second amino acid. A sequence logo plot based on these 22 N termini from Met-processed plastid-synthesized proteins plus the 18 N-terminal peptides with a preceding Met from imported proteins showed that the next amino acid after an excised Met was usually one favored by Met aminopeptidases [26], i.e. Ser, Ala, Val, Thr and Gly (Figure 2e).

When the iceLogo for the TP cleavage site was replotted with the assumption that N-terminal Met were removed after TP cleavage by the stromal processing peptidase (SPP), there was a clear, very strong preference for Leu at position -2 (Figure 4b). In addition, there was a strong preference for hydroxylated residues at positions -3 to -6. However, a wider range of amino acids was observed just before the SPP cleavage site (position -1). There appeared to be a weak preference for small or amidated amino acids, while large aliphatic residues were not found. On the other side of the cleavage site, the first residue after the cleavage site was most frequently Met, Ala or Ser, and the next 5-8 residues of the mature proteins were enriched in negatively charged side-chains.

The final deduced TP sequences ranged from 12 to 42 amino acids in length with an average of 20 (Table S7). Like plant TPs [7], they were highly enriched in Ser and Thr residues (18% and 14%, respectively) and almost completely depleted in acidic residues (Asp and Glu, 0.3% and 0.6%, respectively), resulting in a net positive charge in all but two TPs, with an average of +2.6. The positively charged residues (Arg, Lys) tended to be in the middle of the TP, but the hydroxylated residues were found throughout. Leu and Ile are notably absent or under-represented in positions -1 and -3 to -6 (Figure 4b).

Generality of the transit peptide cleavage motif

Our data suggest that diatom TPs are cleaved at a consensus site with Ala or Ser at -3, Leu at -2, and a charged or small amino acid at -1. The mature N terminus generated by cleavage is usually Ala, Ser or a subsequently removed Met and the next 5-6 amino acids have a negative net charge. In order to examine whether these "rules" for TP cleavage sites are similar in other algae with secondary plastids, databases were searched for homologous gene models encoding a SP followed by the canonical (F/Y) XXP motif. Almost all the proteins in our study (including unknown proteins) had a homolog in at least one other diatom, and many had homologs in other algae. Unfortunately, gene models are often incomplete at the 5'-end or have introns in the targeting sequence, so cannot be aligned in the region of the predicted cleavage site and mature N-terminus. Three representative examples of proteins where it was possible to examine the cleavage site motif across several species are shown in Figure 5.

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Figure 5. Conserved processing sites in related proteins from other organisms with complex plastids.

Processing sites for related proteins of other heterokonts were predicted based on alignments of mature protein sequences. (a) Schematic structure of a nuclear encoded plastid-targeted diatom protein. Proteolytic processing steps and proposed cleavage site consensus sequences are indicated. (b) Alignment of glutamate-1-semialdehyde 2,1-aminomutase from four diatom species and a brown alga (c) Alignment of glutamine synthetase from three diatom and two brown algal species. (d) Alignment of glyceraldehyde-3-phosphate dehydrogenase from five diatoms and two brown algae. Bold letters, mature T. pseudonana N terminal sequences identified in this study. Light blue, SP; orange, TP; yellow, a Met that may be removed from the N terminus after SPP processing; green, mature plastid stroma-targeted protein. Thaps, Thalassiosira pseudonana; Phaeo, Phaeodactylum tricornutum; Fracy, Fragilariopsis cylindrus; Psemu, Pseudo-nitzschia multiseries; Skeco, Skeletonema costatum; Odosi, Odontella sinensis; Ascno, Ascophyllum nodosum; Ectsi, Ectocarpus siliculosis.

https://doi.org/10.1371/journal.pone.0074483.g005

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Figure 6. Comparison of transit peptide cleavage sites in higher plants and diatoms.

(a) Sequence logo based on 47 acetylated N-termini of chloroplast imported A. thaliana proteins identified by Zybailov et al. as semi-tryptic peptides within 10 amino acids from the predicted cleavage site [29]. (b) Sequence logo based on 30 acetylated N-termini of plastid-targeted T. pseudonana proteins identified in this study. Only acetylated N-termini were used to exclude a potential bias from comparing acetylated sequences from A. thaliana with non-acetylated ones from T. pseudonana.

https://doi.org/10.1371/journal.pone.0074483.g006

For glutamate-1-semialdehyde 2,1-aminomutase, the sequences from three additional diatoms and the brown alga Ectocarpus siliculosis (Ectsi) aligned very well with the identified T. pseudonana N terminus. All four diatom TPs contained a Leu at the -2 position of the predicted TP, while the E. siliculosis TP has Val at this position (Figure 5B). For glutamine synthetase, alignment of five diatom sequences predicted that two of the five (T. pseudonana and Skeletonema costatum) would have an Met preceding the mature N terminus. If subsequent plastid Met processing after TP cleavage is taken into account, all five sequences would have a Leu at the -2 position, preceded by several hydroxylated residues (Figure 5C). In the case of GAPDH, the mature N termini of five diatoms and two brown algae (E. siliculosis and Ascophyllum nodosum) would all be preceded by a Met. If Met processing after TP cleavage is taken into account, all five diatoms would have another Met at the -2 position of the inferred TP cleavage site and lack a negative net charge at the protein N terminus, while the two aligned brown algal sequences would have the canonical Leu at -2 and a net negative charge in the following 6 amino acids (Figure 5C). It therefore appears that there is a conserved transit peptide cleavage site motif, at least for diatoms and brown algae.