title: Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation.
abstract: Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to today's bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14-261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (-17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N(6)-(retinylidene) modification (266.20 Da) between residues 225-248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b(6)f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane beta-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some transmembrane alpha-helical domains was efficient, other regions were refractory such that their primary structure could only be inferred from the coincidence of genomic translation with precursor and product ions that spanned them.
authors.affiliation: The Pasarow Mass Spectrometry Laboratory, The Neuropsychiatric Institute (NPI)-Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90024, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Ryan
authors.forename: Puneet
authors.initials: P
authors.lastname: Souda
authors.forename: Sara
authors.initials: S
authors.lastname: Bassilian
authors.forename: Rachna
authors.initials: R
authors.lastname: Ujwal
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Jeff
authors.initials: J
authors.lastname: Abramson
authors.forename: Peipei
authors.initials: P
authors.lastname: Ping
authors.forename: Armando
authors.initials: A
authors.lastname: Durazo
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
authors.forename: S Saif
authors.initials: SS
authors.lastname: Hasan
authors.forename: Danas
authors.initials: D
authors.lastname: Baniulis
authors.forename: William A
authors.initials: WA
authors.lastname: Cramer
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D045346:Cytochrome b6f Complex / Q000737:chemistry / Q000378:metabolism; D005583:Fourier Analysis; D019617:Halobacterium salinarum / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D046937:Nostoc / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D021122:Protein Subunits; D001436:Bacteriorhodopsins; D045346:Cytochrome b6f Complex; D050995:Voltage-Dependent Anion Channel 1