Full Subunit Coverage Liquid Chromatography Electrospray Ionization Mass Spectrometry (LCMS+) of an Oligomeric Membrane Protein

Abstract

Highly active cytochrome b 6 f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS؉). Both sizeexclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (؎3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP

Post-translational Modifications of Integral Membrane Proteins Resolved by Top-down Fourier Transform Mass Spectrometry with Collisionally Activated Dissociation

“…The suspension was split into two microcentrifuge tubes (125 l each), and 1 ml of 80% acetone in water (Ϫ20°C stock) was added to each tube prior to Vortex mixing (1 min) and incubation at Ϫ20°C for 1 h. Precipitated protein was recovered by centrifugation (10,000 ϫ g), and the supernatant was removed. Pellets were dried briefly to allow evaporation of residual acetone (5 min, room temperature and pressure) and dissolved in 90% formic acid (total 100 l) for immediate injection into an HPLC system prepared for reverse-phase chromatography (14,18). A column (5 m, 300-Å PLRP/S, 2 ϫ 150 mm; Varian) was previously equilibrated in 95% buffer A (0.1% TFA in water), 5% buffer B (0.05% TFA, 50% acetonitrile, 50% isopropanol) at 100 l/min at 40°C for 30 min prior to sample injection.…”

“…Subsequently, top-down mass spectrometry was performed on small integral subunits of the cytochrome b 6 f complex using quadrupole time-of-flight analyzers (14), and FT-MS was used for the first time on bacteriorhodopsin apoprotein, achieving mass accuracy Ͻ10 ppm (15). Preliminary top-down FT-MS of bacteriorhodopsin holoprotein (16) and a thorough top-down collisionally activated dissociation (CAD) 1 /electron capture dissociation (ECD) FT-MS study of the c-subunit of F 0 of the ATP synthase (17) established the feasibility of performing top-down FT-MS on integral membrane proteins.…”

Multiple Rieske Proteins Enable Short- and Long-term Light Adaptation of Synechocystis sp. PCC 6803

“…The Rieske protein binds a redox-active [2Fe-2S] center close to the p-side of the membranes and carries electrons in the high-potential branch of the bifurcated electron transport chain of the complex (4,5,7,8). The cytochrome b 6 f monomer consist of four large (17.5-32 kDa) core subunits, cytochrome f (PetA), cytochrome b 6 (PetB), the Rieske iron-sulfur protein (PetC), and subunit IV (SU IV, PetD), as well as four additional small (3.3-4.1 kDa) subunits, PetG, PetL, PetM, and PetN (7,33). Except for PetL and PetM all subunits are required for a functional cytochrome b 6 f complex (34,35).…”

Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography–electrospray ionization–mass spectrometry

“…In subsequent studies, the use of chloroform/methanol/aqueous acid was extended further by Whitelegge and co-workers by employing this mixture (4:4:1 v/v/v) as the mobile phase for the separation of membrane proteins by size-exclusion chromatography (SEC) coupled to ES-MS [31;32;33;34]. The method has been successfully applied to the characterization of intact thylakoid membrane proteins [34] and cytochrome b 6 f complexes [33]. The chloroform/methanol/aqueous acid mixture (4:4:1 v/v/v) employed by Whitelegge and co-workers served as starting point to find suitable mobile phases for the analysis of recombinant NTS1 by SEC-MS. NTS1 precipitated upon addition of the 4:4:1 chloroform/ methanol/aqueous formic acid mixture to receptor-containing detergent buffer in preparation for SEC.…”