Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.     

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.     

Proteomic analysis of photosystem I components from different plant species

In this study, the photosystem I (PSI) highly hydrophobic proteins present within stroma lamellae of the thylakoid membrane were separated by RP-HPLC and identified either by in-solution trypsin digestion peptide fragment fingerprinting or by the close correspondence between the intact mass measurements (IMMs) and those expected from the DNA sequence. Protein identification performed by MS/MS was as reliable as IMMs. Thus, IMM is an easy and valid method for identifying proteins that have no PTMs. This paper reports the M(r) for all PSI proteins in ten different species, including those whose genes have not yet been cloned. Lhca5 was revealed unequivocally in four species, corroborating that it is indeed a protein belonging to the light-harvesting antenna of PSI. In all species examined, the product of the Lhca6 gene has never been revealed. Concerning core proteins, Psa-O has been revealed in three species; isoforms of Psa-D and Psa-E have been found in both monocots and dicots. Small proteins like Psa-I and Psa-J are well separated and identified. RP-HPLC produces reliable fingerprints and reveals that the relative amounts of PSI proteins appear to be markedly different.     

Isoforms of photosystem II antenna proteins in different plant species revealed by liquid chromatography-electrospray ionization mass spectrometry

The high selectivity offered by reversed-phase high-performance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14-280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.     

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins

In higher plants, both photosystem I (PSI) and II (PSII) consist of membrane-embedded proteins that contain more than one transmembrane alpha helix. PSI is a multiprotein complex consisting of a core complex of thirteen proteins surrounded by four different types of light harvesting antenna proteins. Up to now, the protein components of both photosystems have been characterized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not sufficient to discriminate between individual membrane proteins. This is also complicated by the fact that some proteins, such as the antenna proteins, have almost identical molecular mass and amino acid sequence, making it difficult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins and most of the core components of PSI from spinach, together with the identification of proteins by molecular mass, successfully deduced by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). The proposed RP-HPLC-ESI-MS method holds several advantages over SDS-PAGE, including better protein separation, especially for antenna proteins, mass accuracy, speed, efficiency, and the potential to reveal isomeric forms. Moreover, the molecular masses determined by HPLC-ESI-MS are in good agreement with the molecular masses of the individual components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of post-translational modifications in these proteins. It follows that if the method proposed is useful for these highly hydrophobic proteins, it may be of general use for any membrane proteins, where the presence of detergent for solubilization may compromise their characterization.     

Genome-wide analysis of the family of light-harvesting chlorophyll a/b-binding proteins in arabidopsis and rice

Light-harvesting antenna system possesses an inherent property of photoprotection. The single-helix proteins found in cyanobacteria play role in photoprotection and/or pigment metabolism. The photoprotective functions are also manifested by the two- and four-helix proteins. The photoprotection mechanism evolved earlier to the mechanism of light-harvesting of the antenna complex. Here, the light-harvesting complex genes of photosystems I and II from Arabidopsis are enlisted, and almost similar set of genes are identified in rice. Also, the three-helix early light-inducible proteins (ELIPs), two-helix stress-enhanced proteins (SEPs) and one-helix high light-inducible proteins [one-helix proteins (OHPs)] are identified in rice. Interestingly, two independent genomic loci encoding PsbS protein are also identified with implications on additional mode of non-photochemical quenching (NPQ) mechanism in rice. A few additional LHC-related genes are also identified in rice (LOC_Os09g12540, LOC_Os02g03330). This is the first report of identification of light-harvesting complex genes and light-inducible genes in rice.Key words: Lhca and Lhcb proteins, Lhc proteins evolution, light-inducible proteins, protein alignment, PsbSThe light-harvesting proteins are present in different taxa. The proteins of light-harvesting systems from higher plants, cyano-bacteria, purple bacteria and green sulphur bacteria share no sequence similarity however little structural similarity can be seen.¹ Apparently, the light-harvesting systems in these different taxa might have evolved independently from each other.¹ To enable efficient transfer of excitation energy into the reaction centers, where charge separation takes place, different proteins are recruited in order to coordinate the photosynthetic pigment molecules. The light-harvesting and light dissipation are tightly coupled processes involving the higher plant light-harvesting antenna. Here, genome-wide analysis of the light-harvesting chlorophyll a/b-binding proteins and light-inducible proteins in Arabidopsis thaliana L. and Oryza sativa L. (rice) is conducted. This study wherein genes coding for antenna proteins are identified and named can be used as a nomenclature guide to the light-harvesting complex gene family members and their relatives in rice.     

CP43-like chlorophyll binding proteins: structural and evolutionary implications

CP43, encoded by the psbC gene, is a chlorophyll (Chl)-binding protein of Photosystem II (PSII), the water-splitting and oxygen-evolving enzyme of photosynthesis. CP47, encoded by psbB, a Chl-binding protein of PSII, is closely related to CP43. The Chl-binding six transmembrane helical unit typified by CP43, is also structurally related to the N-terminal domains of the PsaA and PsaB proteins of Photosystem I (PSI) as well as to the family of light-harvesting proteins encoded by cyanobacterial isiA genes and prochlorophyte pcb genes. Here we use recent structural information derived for PSII and PSI to review similarities and differences between the various members of the CP43-like class of light-harvesting proteins, exploring both functional and evolutionary implications.     

Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry

A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.     

A red-shifted antenna protein associated with photosystem II in Physcomitrella patens

Antenna systems of plants and green algae are made up of pigment-protein complexes belonging to the light-harvesting complex (LHC) multigene family. LHCs increase the light-harvesting cross-section of photosystems I and II and catalyze photoprotective reactions that prevent light-induced damage in an oxygenic environment. The genome of the moss Physcomitrella patens contains two genes encoding LHCb9, a new antenna protein that bears an overall sequence similarity to photosystem II antenna proteins but carries a specific motif typical of photosystem I antenna proteins. This consists of the presence of an asparagine residue as a ligand for Chl 603 (A5) chromophore rather than a histidine, the common ligand in all other LHCbs. Asparagine as a Chl 603 (A5) ligand generates red-shifted spectral forms associated with photosystem I rather than with photosystem II, suggesting that in P. patens, the energy landscape of photosystem II might be different with respect to that of most green algae and plants. In this work, we show that the in vitro refolded LHCb9-pigment complexes carry a red-shifted fluorescence emission peak, different from all other known photosystem II antenna proteins. By using a specific antibody, we localized LHCb9 within PSII supercomplexes in the thylakoid membranes. This is the first report of red-shifted spectral forms in a PSII antenna system, suggesting that this biophysical feature might have a special role either in optimization of light use efficiency or in photoprotection in the specific environmental conditions experienced by this moss.     

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b ₆ protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.     

Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.     

Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts

The photosystem II (PSII) light-harvesting antenna in higher plants contains a number of highly conserved gene products whose function is unknown. Arabidopsis thaliana plants depleted of one of these, the CP24 light-harvesting complex, have been analyzed. CP24-deficient plants showed a decrease in light-limited photosynthetic rate and growth, but the pigment and protein content of the thylakoid membranes were otherwise almost unchanged. However, there was a major change in the macroorganization of PSII within these membranes; electron microscopy and image analysis revealed the complete absence of the C(2)S(2)M(2) light-harvesting complex II (LHCII)/PSII supercomplex predominant in wild-type plants. Instead, only C(2)S(2) supercomplexes, which are deficient in the LHCIIb M-trimers, were found. Spectroscopic analysis confirmed the disruption of the wild-type macroorganization of PSII. It was found that the functions of the PSII antenna were disturbed: connectivity between PSII centers was reduced, and maximum photochemical yield was lowered; rapidly reversible nonphotochemical quenching was inhibited; and the state transitions were altered kinetically. CP24 is therefore an important factor in determining the structure and function of the PSII light-harvesting antenna, providing the linker for association of the M-trimer into the PSII complex, allowing a specific macroorganization that is necessary both for maximum quantum efficiency and for photoprotective dissipation of excess excitation energy.     

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo‐adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C‐terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co‐localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light‐induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two‐dimensional gel electrophoresis discriminated, for the first time, the 58‐kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC‐MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Small,acid-soluble proteins as biomarkers in mass spectrometry analysis of Bacillus spores

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor alpha/beta- and gamma-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.     

Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii

Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.     

A simple intact protein analysis by MALDI-MS for characterization of ribosomal proteins of two genome-sequenced lactic acid bacteria and verification of their amino acid sequences

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.