Determination of Photosystem II Subunits by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Photosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes.Photosystem II is a pigment-protein complex of the thylakoid membrane of higher plants, eukaryotic algae, and cyanobacteria. It catalyzes light-induced electron transfer from water to plastoquinone, with associated production of molecular oxygen. PSII 1 consists of a large complex with a number of polypeptide components, most of which are integral membrane proteins. A number of extrinsic proteins are also associated with it at the membrane surface. The entire set of electron transfer cofactors, including chlorophyll a, pheophytin a, plastoquinones, and non-heme iron, is associated with the D1/D2 heterodimer. These two proteins, together with the ␣ and ␤ subunits of cytochrome b 559 and the PsbI protein, constitute the so-called reaction center II (RCII), which is the smallest PSII subparticle still able to perform light-induced charge separation (1). Two large integral membrane proteins, CP43 and CP47, each coordinating a number (12-15) of chlorophyll a molecules, and several low molecular mass (Ͻ10 kDa) polypeptides are constituents of the PSII core complex, which is very similar in higher plants and prokaryotic cyanobacteria.During the last decade, considerable progress has been made in our understanding of the organization of polypeptides constituting the reaction center of PSII, but the topology and functional role of the small protein subunits are still largely unknown. Some of them are universal, whereas others are present only in cyanobacteria (PsbU and PsbV) or only in higher plants (e.g. PsbP, PsbQ, PsbS) (2).Investigation of the structural and functional roles of the various PSII subunits requires, as a preliminary step, a suitable method to detect them in the thylakoid membrane or purified subparticle preparations. The detection and study of stimuli-induced modifications of several PSII components have mainly been based on the use of polyclonal antibodies. However, these are often not available and, when available, are time-consuming to use.A different technique, capable of accurately detecting even small amounts of the various PSII subunits in an integrated system, could be of great help in studying the function of these protein...

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I

“…2A). m / z Peaks at 38 070, 39 300, 51 400 and 55 500 Da have recently been attributed to D1, D2, CP43 and CP47, respectively, by comparing the molecular masses obtained by MALDI with the protein masses calculated from the nucleotide sequences of the PSII components [22]. The peak corresponding to CP43 is almost completely absent in the spectra of CP47–RC, whereas the relative intensities of peaks attributed to D1, D2 and CP47 remain basically unaltered.…”

“…The peak corresponding to CP43 is almost completely absent in the spectra of CP47–RC, whereas the relative intensities of peaks attributed to D1, D2 and CP47 remain basically unaltered. The difference in the amplitudes of the peaks assigned to CP47 and CP43 does not reflect a stoichiometry different from 1 : 1 but is rather due to a different cross section for ionization, caused by the more external position of CP43 respect to CP47 in the PSII complex [22]. Figure 2C shows the proteins of the CP47–RC complex in the 2‐ to 20‐kDa range.…”

Isoforms of Photosystem II Antenna Proteins in Different Plant Species Revealed by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

“…Various analytical techniques, including SDS-polyacrylamide gel electrophoresis (PAGE), SDS-isoelectric focusing, high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) (15,16), or matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) (17,18), are applicable to the characterization of minute differences in heterogeneous proteins. Traditionally, the protein components of the PS II major and minor antenna system are resolved by SDS-PAGE into several closely migrating protein bands (19,20).…”

Liquid Extraction-Ultracentrifugation-Liquid Chromatography-Mass Spectrometry: A Potent Tool for Separation and Identification of Thylakoid Membrane Proteins

“…This is particularly true for intrinsic thylakoid membrane proteins, because their solubilization normally requires strong detergent, which can interfere with biochemical or mass spectrometric post-separation identification protocols. Thus the combination of HPLC and MS can facilitate the identification of photosynthetic proteins either by intact molecular mass determinations [27][28][29][30][31], peptide mass fingerprinting, or peptide fragment fingerprinting [32,33]. The applicability of intact molecular mass measurements for the study of the thylakoid membrane PSII subdomains using HPLC-ESI-MS will be discussed later.…”