Dataset Information

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

ABSTRACT: Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).

SUBMITTER: Debelyy MO

PROVIDER: S-EPMC5658093 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Debelyy Mykhaylo O MO Waridel Patrice P Quadroni Manfredo M Schneiter Roger R Conzelmann Andreas A

PloS one 20171026 10

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimi ...[more]

PMID: 29073188

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Project description: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.

Dataset Information

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Publications

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

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