title: Improved identification of membrane proteins by MALDI-TOF MS/MS using vacuum sublimated matrix spots on an ultraphobic chip surface.
abstract: Integral membrane proteins are notoriously difficult to identify and analyze by mass spectrometry because of their low abundance and limited number of trypsin cleavage sites. Our strategy to address this problem is based on a novel technology for MALDI-MS peptide sample preparation that increases the success rate of membrane protein identification by increasing the sensitivity of the MALDI-TOF system. For this, we used sample plates with predeposited matrix spots of CHCA crystals prepared by vacuum sublimation onto an extremely low wettable (ultraphobic) surface. In experiments using standard peptides, an up to 10-fold gain of sensitivity was found for on-chip preparations compared with classical dried-droplet preparations on a steel target. In order to assess the performance of the chips with membrane proteins, three model proteins (bacteriorhodopsin, subunit IV(a) of ATP synthase, and the cp47 subunit from photosystem II) were analyzed. To mimic realistic analysis conditions, purified proteins were separated by SDS-PAGE and digested with trypsin. The digest MALDI samples were prepared either by dried-droplet technique on steel plates using CHCA as matrix, or applied directly onto the matrix spots of the chip surface. Significantly higher signal-to-noise ratios were observed for all of the spectra resulting from on-chip preparations of different peptides.In a second series of experiments, the membrane proteome of Rhodococcus jostii RHA1 was investigated by AIEC/SDS-PAGE in combination with MALDI-TOF MS/MS. As in the first experiments, Coomassie-stained SDS-PAGE bands were digested and the two different preparation methods were compared. For preparations on the Mass.Spec.Turbo Chip, 43 of 60 proteins were identified, whereas only 30 proteins were reliably identified after classical sample preparation. Comparison of the obtained Mascot scores, which reflect the confidence level of the protein identifications, revealed that for 70% of the identified proteins, higher scores were obtained by on-chip sample preparation. Typically, this gain was a consequence of higher sequence coverage due to increased sensitivity.
authors.affiliation: Department of Biochemistry of Plants, University of Bochum, Germany.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Poetsch
authors.forename: Daniela
authors.initials: D
authors.lastname: Schlüsener
authors.forename: Christine
authors.initials: C
authors.lastname: Florizone
authors.forename: Lindsay
authors.initials: L
authors.lastname: Eltis
authors.forename: Christoph
authors.initials: C
authors.lastname: Menzel
authors.forename: Matthias
authors.initials: M
authors.lastname: Rögner
authors.forename: Kerstin
authors.initials: K
authors.lastname: Steinert
authors.forename: Udo
authors.initials: U
authors.lastname: Roth
mesh_terms: D001436:Bacteriorhodopsins / Q000302:isolation & purification; D001709:Biotechnology; D025222:Chloroplast Proton-Translocating ATPases / Q000302:isolation & purification; D008565:Membrane Proteins / Q000302:isolation & purification*; D045332:Photosystem II Protein Complex / Q000302:isolation & purification; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D020543:Proteome / Q000302:isolation & purification; D012240:Rhodococcus / Q000737:chemistry; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000706:statistics & numerical data; D013499:Surface Properties; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data
chemical_list: D008565:Membrane Proteins; D045332:Photosystem II Protein Complex; D020543:Proteome; D001436:Bacteriorhodopsins; D025222:Chloroplast Proton-Translocating ATPases