A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins

Cadène, Martine; Chait, Brian T.

doi:10.1021/ac000811l

Cited by 159 publications

(162 citation statements)

References 30 publications

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“…2E) on a JEOL JMS-S3000 SpiralTOF mass spectrometer using the ultra-thin-layer sample preparation method 60,61 in which α-cyano-4-hydroxycinnamic acid (Sigma) was used as the matrix. The mass of Qconcat A was internally calibrated with horse myoglobin ([m+H]+ = 16,952.5 Da).…”

Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

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479

894

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Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

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Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

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479

894

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“…Acid-labile surfactant (ALS) and ammonium dodecyl sulfate have been used for preparation of proteins and peptides analyzed by mass spectrometry [22][23][24]. Non-ionic and zwitterionic surfactants have been successfully used to analyze transmembrane proteins and protein digests for improved mass spectral analysis [25,26].…”

mentioning

confidence: 99%

Interactions between sodium dodecyl sulfate micelles and peptides during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of proteolytic digests

Tummala

Green‐Church

Limbach

2005

J. Am. Soc. Mass Spectrom.

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Although sodium dodecyl sulfate (SDS) is routinely used as a denaturing agent for proteins, its presence is highly detrimental on the analysis of peptides and proteins by mass spectrometry. It has been found, however, that when SDS is present in concentrations near to or above its critical micelle concentration (CMC), improvements in the matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) analysis of peptide mixtures or hydrophobic proteins are obtained. To elucidate possible explanations for such improvements, here we have undertaken a study examining the effect of SDS micelles on peptide mixtures. Fluorescently labeled peptides were used as probes to determine whether hydrophobic or hydrophilic peptides interact exclusively with SDS micelles. In addition, four globular proteins were digested with trypsin and then various amounts of SDS were added before MALDI mass spectrometry. To examine the role of mixture complexity on the mass spectral results, the tryptic digest of bovine serum albumin was also fractionated according to hydrophobicity before SDS treatment. Results from these experiments suggest that micelle-peptide interactions increase peptide-matrix cocrystallization irrespective of analyte hydrophobicity. As these studies were performed using the dried-droplet method of sample spotting, the presence of micelles is also hypothesized to reduce Marangoni effects during the crystallization process. (J Am Soc Mass Spectrom 2005, 16, 1438 -1446

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“…The ultra-thin-layer method of sample preparation employed here was adapted from previous work [33]. Both the conditions of the matrix layer preparation as well as the composition of the matrix/sample solution were examined for maximum yield of multiply charged protein ions.…”

Section: Sample Preparationmentioning

confidence: 99%

“…Among them, the thin-layer preparation features higher sensitivity in the detection of peptide and proteins, and has demonstrated tolerance to salts [35]. The ultra-thin-layer method [33], developed from the thin-layer method, facilitates the detection of membrane proteins in the presence of detergent when 1:3:2 FWI is employed as the matrix solvent. The matrix solution composition itself also plays an important role in the incorporation of analyte with matrix.…”

Section: Effect Of Sample Preparationmentioning

confidence: 99%

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Liu

Schey

2008

J. Am. Soc. Mass Spectrom.

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Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach. ragmentation of intact protein gas-phase ions, termed top down proteomics, is becoming more frequently utilized primarily in ion trapping instruments. As was first reported by Loo et al.[1], multiply-charged intact protein ions are formed via electrospray ionization (ESI) and can be collisionally activated to produce sequence-specific fragment ions. Typically, the fragmentation of highly multiply charged intact proteins results in a product ion spectrum displaying a multitude of product ions possessing a range of charge states making spectral interpretation challenging. The high resolving power and mass accuracy of Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has made this instrument platform the method of choice for the interpretation of the complicated product ion spectra produced by fragmentation of multiply charged protein ions. Indeed, automated interpretation algorithms have been constructed based on this approach [2,3]. Using FTMS, proteins over 200 kDa have been successfully fragmented and sequence-specific product ion assignments have been made [4]. Lower mass resolving quadrupole ion trapping instruments have also been successfully utilized for top down proteomics experiments. To meet the challenge of spectral interpretation ion/ion reactions have been employed to reduce the product ion charge by subjecting the product ion population to proton transfer reactions, thereby resulting in the formation of predominantly singly charged product ions and a more readily interpretable product ion spectrum [5]. This strategy removes the product ion charge-state ambiguities arising from dissociation of large multiply charged protein ions and has been successfully demonstrated on ions as large as elastase (25.9 kDa) although it is limited by the m/z range of the instrument [6]. Matrix-assisted laser desorption ionization tandem time-of-flight (MALDI TOF-TOF) mass spectrometry has also been used to produce product i...

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A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins

Cited by 159 publications

References 30 publications

Integrative structure and functional anatomy of a nuclear pore complex

Integrative structure and functional anatomy of a nuclear pore complex

Interactions between sodium dodecyl sulfate micelles and peptides during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of proteolytic digests

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

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