skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Mass spectrometry-based technologies for probing the 3D world of plant proteins
Abstract Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions—which are more difficult to study—dynamically regulate the proteome’s 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based “structural” proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function.  more » « less
Award ID(s):
2010789
PAR ID:
10423512
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Plant Physiology
Volume:
189
Issue:
1
ISSN:
0032-0889
Page Range / eLocation ID:
12 to 22
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; (, Methods in molecular biology)
    Jacob, S. (Ed.)
    Protein–protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni²⁺ columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungus Magnaporthe oryzae and might benefit gene function and proteomics studies in plants and other research fields. 
    more » « less
  2. ; ; ; ; ; (, Angewandte Chemie International Edition)
    Abstract Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid‐phase separations. Native proteomics should provide the most accurate bird's‐eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well‐purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra‐high mass range (UHMR) Orbitrap mass spectrometer. The nCZE‐MS technique enabled the measurement of a 115‐kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE‐MS analysis of anE.colicell lysate detected 72 proteoforms or protein complexes in a mass range of 30–400 kDa in a single run while consuming only 50‐ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; et al (, Cell Reports Methods)
    We present an efficient, effective, and economical approach, named E3technology, for proteomics sample preparation. By immobilizing silica microparticles into the polytetrafluoroethylene matrix, we develop a robust membrane medium, which could serve as a reliable platform to generate proteomics-friendly samples in a rapid and low-cost fashion. We benchmark its performance using different formats and demonstrate them with a variety of sample types of varied complexity, quantity, and volume. Our data suggest that E3technology provides proteome-wide identification and quantitation performance equivalent or superior to many existing methods. We further propose an enhanced single-vessel approach, named E4technology, which performs on-filter in-cell digestion with minimal sample loss and high sensitivity, enabling low-input and low-cell proteomics. Lastly, we utilized the above technologies to investigate RNA-binding proteins and profile the intact bacterial cell proteome. 
    more » « less
  4. ; ; ; ; ; ; ; (, Plant Physiology)
    Abstract SPINDLY (SPY) is a novel nucleocytoplasmic protein O-fucosyltransferase that regulates target protein activity or stability via O-fucosylation of specific Ser/Thr residues. Previous genetic studies indicate that AtSPY regulates plant development during vegetative and reproductive growth by modulating gibberellin and cytokinin responses. AtSPY also regulates the circadian clock and plant responses to biotic and abiotic stresses. The pleiotropic phenotypes of spy mutants point to the likely role of AtSPY in regulating key proteins functioning in diverse cellular pathways. However, very few AtSPY targets are known. Here, we identified 88 SPY targets from Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana via the purification of O-fucosylated peptides using Aleuria aurantia lectin followed by electron transfer dissociation-MS/MS analysis. Most AtSPY targets were nuclear proteins that function in DNA repair, transcription, RNA splicing, and nucleocytoplasmic transport. Cytoplasmic AtSPY targets were involved in microtubule-mediated cell division/growth and protein folding. A comparison with the published O-linked-N-acetylglucosamine (O-GlcNAc) proteome revealed that 30% of AtSPY targets were also O-GlcNAcylated, indicating that these distinct glycosylations could co-regulate many protein functions. This study unveiled the roles of O-fucosylation in modulating many key nuclear and cytoplasmic proteins and provided a valuable resource for elucidating the regulatory mechanisms involved. 
    more » « less
  5. ; ; ; ; ; ; ; ; (, Rapid Communications in Mass Spectrometry)
    RationaleTandem‐ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem‐ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes. MethodsHere, we describe the coupling of the separation capabilities of tandem‐trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis. ResultsWe establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2–3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical‐based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here (“UVnoD2”), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility‐separating fragment ions produced from UVPD. ConclusionsThe data demonstrate that UVPD carried out at elevated pressures of 2–3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post‐UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email