3.1 Chromatographic separation of PSII subunits
The primary reversed phase LC-MS+ separation yielded a retention map that was annotated with protein identities where they are known, and intact masses where they remain unknown (). Mass accuracy on the low-resolution quadrupole instrument was typically 100 ppm (1 Da at 10,000 Da) and the spectra were used to guide targeted top-down high-resolution experiments on selected fractions from the LC-MS+ experiment in order to fully define primary structure and post-translational modifications of PSII subunits. The physical basis of the separation relies upon hydrophobicity, and thus the first proteins to elute are the hydrophilic peripheral PSII subunits PsbO, PsbU, PsbV, Psb28 (PsbW) and Psb27 (‘PsbZ-like’). These were followed by a set of proteins in the 16 kD class, identified as phycobiliproteins (ApcA, ApcB, ApcD, ApcF, CpcA, CpcB, CpcG) and a pair of 55 kD proteins identified as AtpA and AtpB of ATP synthase (data not shown). Association of phycobiliproteins with PSII is predictable, while ATP synthase is probably a contaminant of the preparation. The remainder of the chromatogram was dominated by elution of the small and large integral subunits of PSII spanning a substantial range of hydrophobicity, with the PsbE and PsbF subunits that bind cytochrome b559 eluting around 70 minutes, and PsbZ not eluting until 133 minutes. Several of the small subunits (PsbF, PsbM, PsbT, PsbI, PsbH) had singly oxidized isoforms that eluted a little earlier than the unmodified population. The intensities of different peaks in the total ion chromatogram shown in are dependent on abundance and ionization efficiency and thus stoichiometry should not be inferred. lists intact mass tags (IMTs) that were assigned to PSII subunits based upon coincidence of measured average mass with calculated average mass based upon primary structure and limited post-translational modification. These assignments remain coincidental, with little statistical confidence until a high-resolution top-down analysis is completed (). Out of around 40 detected IMT’s, 20 PSII subunits were assigned with different PTMs. The 3409 Da IMT eluting at 78 minutes is potentially a PSII subunit but so far remains unidentified.
Figure 2 Reversed phase total ion chromatogram elution profile of PSII from the red alga, Galdieria sulphuraria. PSII proteins were precipitated with acetone and dissolved in formic acid for loading onto a reversed phase column as described in the experimental (more ...)
Average masses of PSII subunits from low resolution LC-MS+
Monoisotopic masses of PSII subunits from high resolution Fourier-transform mass spectrometry (FT-MS)
3.2 Larger integral membrane subunits
The four large subunits (PsbA, PsbB, PsbC and PsbD), which account for twenty-two of the total thirty four transmembrane alpha helices in PSII, eluted between 88 and 106 minutes along with other smaller subunits (). The large subunits PsbA, PsbB, PsbC and PsbD measured 38184, 56551, 50927 and 39350 Da respectively, largely consistent with their gene sequences and known N- and C- terminal post-translational modifications (). The experimentally determined average mass of 38184 Da for PsbA was consistent with the removal of the initiating methionine at the N-terminus with acetylation of Thr 2, and removal of 15 amino acids at the C-terminus () in conservation with other species. The difference of 23 Da relative to the calculated average mass (38160.7 Da) could be due to minor DNA sequence differences between the strain (074W) used for PSII preparation and sequencing. In PsbD, the difference between the calculated (39344.1 Da) and experimental (39346 Da) average masses is within experimental measurement error (). For subunit PsbB, the experimentally derived mass (56551 Da) is in good agreement with the calculated mass (56558.8 Da) taking into account the loss of initial methionine and N-terminal acetylation (). In PsbC, if 14 amino acids are removed to form a processed PsbC with a free N-terminus in conservation with higher plants, the calculated mass (50588.3) is lower than measured (50888 Da) by 300 Da, indicating either divergent N-terminal processing or some other DNA sequence discrepancy. Despite minor inconsistencies, the intact mass tags of the larger integral subunits can be assigned with confidence. Since the main focus of this study was to identify and characterize the smaller integral (< 10 kDa) and the peripheral subunits constituting Galdieria PSII further experiments on the large subunits were not performed.
Of the three (PsbO, PsbU, PsbV) OEC stabilizing subunits identified, PsbO and PsbU are encoded by the nucleus and carry an N- terminal chloroplast target peptide. PsbO has a target peptide consisting of the first 21 amino acids so the mature form has 242 amino acids. The experimentally determined monoisotopic mass of 28796.7520 Da could be reconciled (Δ = 2.6387 ppm) with the calculated monoisotopic mass of 28796.67415 Da with inclusion of the formation of a single disulfide bond (−2.01565 Da) between the only two Cys residues, Cys23 and Cys47 ( and ). Assignment of product ions from a CAD experiment of the disulfide linked mature protein yielded 11 y-and 9 b- ion matches within a 10 ppm tolerance and a consequent P score of 1.41E-26 (, ). In the case of PsbU, the first 81 amino acids of the signal peptide are cleaved at the N terminus, forming a mature protein of 93 amino acids, with a calculated monoisotopic mass of 10579.4347 Da (). Top-down CAD analysis of the N-terminally truncated protein confirmed the primary structure of PsbU with a experimentally determined monoisotopic mass of 10579.4648 Da, within 5 ppm of the calculated monoisotopic mass (Δ = 2.8361 ppm), and a CAD experiment yielded 32 b- and 23 y- product ions for a P score of 5.26E-75 (, ). The third extrinsic subunit PsbV was assigned to an intact mass tag of 15704 Da with proposed modifications including removal of 30 amino acid residues from the N-terminus, removal of 10 residues from the C-terminus and attachment of a covalently bound heme group (615.16947 Da) (, ). Analysis of the high-resolution CAD dataset for PsbV gave a mass difference of −2.0602 Da (−131.2860 ppm) with 11 b- and 15 y- ions matched. Further optimization of the match between measured and calculated mass was reached by changing Thr 80 to Val (mature protein numbering) significantly increasing coverage with 19 b- and 24 y- product ions matched and a P score of 3.32E-45 (Δ = 5.1595 ppm) (, ).
Figure 3 Top-down mass spectrometry of oxygen evolving complex subunits. Selected fractions were analyzed by static nanospray on a 7 Tesla LTQ-FT Ultra. Collisionally activated dissociation experiments on isolated precursor ions yielded complex tandem mass spectra (more ...)
Besides the 3 OEC stabilizing subunits, 2 other peripheral subunits were identified, Psb27 and Psb28. In the primary LC-MS+ analysis, a hydrophilic protein eluted at 39 minutes with an intact mass tag of 13247 Da and was assigned as Psb28 with removal of Met1 (, ). This protein is similar to the PsbW identified in Synechocystis and will be referred to as Psb28 hereafter (see discussion). Psb28 was assigned to a monoisotopic mass peak of 13237.7755 Da (), confirming the identity and processing with 4 b- and 17 y- product ions and the precursor matched below 5 ppm (Δ = 4.6250 ppm) for a P score of 9.55E-28 (). The identity of Psb28 was also confirmed by ECD () with 57 c- and 39 z- product ions, and agreement between calculated and measured monoisotopic masses for the precursor ion within 5 ppm (Δ = 4.6250 ppm) for a P score of 1.02E-152 (, ). An IMT of 12820 Da was assigned to Psb27 (psbz-like) using top-down mass spectrometry after attempts to assign it with low-resolution data were unsuccessful. In the assignments for the dissociation experiments performed on an LTQ-Orbitrap (HCD and ETD) are shown. Both experiments yield b- and y-, and c- and z- ions that support complete agreement with the C-terminal sequence over most of the polypeptide chain. However, the data strongly suggests that the sequence reported for Psb27 has some errors towards its N-terminus and we have therefore represented this region as an unknown modification on the Ala residue shown in of mass 1375.65 Da to optimize b- and c- ion matches. Using this strategy we matched the experimental monoisotopic mass of 12811.3742 Da to the calculated value at <1 ppm, and matched 12 b- and 23 y- ions in the CAD (HCD) experiment and 36 c- and 44 z- ions in the ETD experiment. The measured P scores for each experiment (respectively) confirm the accuracy of the identification (). The nature of the N-terminus is under further study.
Figure 4 Top-down mass spectrometry of subunit Psb28 (PsbW). A protein of average mass 13247 Da detected by LC-MS+, was analyzed by top-down FT-MS with ECD. The 14-charge m/z 947 precursor ion was isolated as shown inset left. The product spectrum is shown from (more ...)
Top-down mass spectrometry of subunit Psb27 (PsbZ-like).
Effort was expended finding the PsbQ, PsbQ’ and PsbP proteins, genes, which can be identified in the Galdieria genome. Attempts to match intact mass tags and top-down MS experiments in the 37 – 76 minute retention range to these sequences were unsuccessful so one minute fractions across this range were digested with trypsin and analyzed by nano-liquid chromatography with tandem mass spectrometry. While these experiments successfully identified many peptides from PsbO, PsbU, PsbV, Psb27, Psb28, ApcA, ApcB, ApcD, ApcF, CpcA, CpcB, AtpA, AtpB as well as some of the integral subunits there were no peptides detected for PsbQ, PsbQ’ or PsbP (data not shown).
3.4 Small integral membrane subunits
The analysis confirmed the presence of core subunits PsbE, PsbF and Psb I, and the remaining integral subunits PsbH, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. The results of top-down CAD and, for PsbF and PsbL, ECD, are shown in , with coverage and P scores described in the legend and measured/calculated masses in . P scores ranged from 2.81E-16 for PsbE and 6.95E-88 for PsbF demonstrating the statistical confidence of the assignments achieved using high-resolution FT-MS with CAD. Even higher P scores were achieved for some of the ECD/ETD experiments. The small subunits could be grouped into three categories based upon post-translational processing. PsbI, PsbT, PsbX and PsbZ were unprocessed with initiating formyl-Met residue intact. PsbE, PsbF, PsbH, PsbJ and PsbL had Met1 removed though only PsbF, PsbJ and PsbL were subsequently acetylated. PsbK and PsbM had longer signal peptides removed from their N-termini and were not acetylated.
Figure 6 Top-down mass spectrometry of small integral membrane subunits A. PsbE, B. PsbF, C. PsbH, D. PsbI, E. PsbJ, F. PsbK G. PsbL H. PsbM I. PsbT J. PsbX and K. PsbZ. The ion isolation used for CAD/ECD for each subunit is shown on the left, while the product (more ...)