GLOBAL AND TARGETED PROFILING OF GTP-BINDING PROTEINS IN BIOLOGICAL SAMPLES BY MASS SPECTROMETRY

Abstract

GTP-binding proteins are among the most important enzyme families that are involved in a plethora of biological processes. However, owing to the enormous diversity of the nucleotide-binding protein family, comprehensive analyses of the expression level, structure, activity, and regulatory mechanisms of GTP-binding proteins remain challenging with the use of conventional approaches. The many advances in mass spectrometry (MS) instrumentation and data acquisition methods, together with a variety of enrichment approaches in sample preparation, render MS a powerful tool for the comprehensive characterizations of the activities and expression levels of various GTP-binding proteins. We review herein the recent developments in the application of MS-based techniques, together with general and widely used affinity enrichment approaches, for the proteome-wide and targeted capture, identification, and quantification of GTP-binding proteins. The working principles, advantages, and limitations of various strategies for profiling the expression level, activity, posttranslational modifications, and interactome of GTP-binding proteins are discussed. It can be envisaged that future applications of MS-based proteomics will lead to a better understanding about the roles of GTP-binding proteins in different biological processes and human diseases. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Keywords: GTP-binding proteins; activity-based protein profiling; posttranslational modifications; shotgun proteomics; small GTPases; targeted proteomics.

Figure 1.. Phylogenetic relationships and gene structure of the Homo Sapiens small GTPase genes.

The unrooted tree was generated using the MEGA v7.0 software with the full-length amino acid sequences of the Homo Sapiens small GTPase proteins using a Neighbor-Joining (NJ) method, including 1,000 boot-strap replications. All the protein sequences were aligned using ClustalW. The phylogenetic tree was generated using the FigTree v1.4.4 software (http://tree.bio.ed.ac.uk/software/figtree/). The five sub-families of small GTPase genes are highlighted with different colored tree branches.

Figure 2.. The chemical structures of the ATP-affinity probes.

(A) Biotin-LC-ATP (B) (+)-biotin-Hex-Acyl-ATP (BHAcATP) (C) (+)-biotin-Hex-Acyl-ADP (BHAcADP)

Figure 3.. The Chemical structures of GTP acyl nucleotide affinity probes and labeling of GTP-binding proteins with these probes.

(A) The chemical structure of the desthiobiotin-GTP probe; (B) A schematic workflow illustrating the conjugation between the desthiobiotin-GTP probe and a GTP-binding protein; (C) The chemical structure of the desthiobiotin-C3-^SGTP structure; (D) Isotope-coded desthiobiotin-C3-GTP probes. ‘H’ and ‘D’ designate hydrogen and deuterium atoms, respectively; (E) A schematic workflow illustrating the conjugation between the desthiobiotin-C3-GTP probe and a GTP-binding protein. Modified from (Xiao, Guo et al. 2013).

Figure 4.. Chemical structures of photoaffinity labeling (PAL)-GTP probes.

(A) The chemical structure and mechanisms of the multi-functional diazirine-based PAL-GTP probe. IMAC: immobilized metal affinity chromatography; (B) The chemical structure of GTP-2′−3′-diol-BP-yne; (C) The chemical structure of GTP-2′−3′-carbonate-BP-yne; (D) Conjugation of biotin tag via Cu(I)-catalyzed click-chemistry reaction. CuAAc: Cu(I)-catalyzed azide/alkyne cycloaddition; (E) BP-yne control. Modified from (Kaneda, Masuda et al. 2007, George Cisar, Nguyen et al. 2013).

Figure 5.. The chemical structures of various GTPase inhibitors and reacting GTP analogues.

Shown are the structures of KRAS inhibitors SML-8-73-1 (A), SML-10-57-1 (B), and SML-10-70-1 (C); the structures of dynamin inhibitor Bis-T (D) and tubulin inhibitor CID 1067700 (E), N²-acryl-GTP (F) and N²-acryl-GppNHp (G). Modified from (Odell, Chau et al. 2009, Lim, Westover et al. 2014, Hong, Guo et al. 2015, Wiegandt, Vieweg et al. 2015)

Figure 6.. Schematic diagrams of AP–MS workflows for interrogating the interactomes of GTP-binding proteins.

(A) Enrichment of GEF proteins from biological samples based on the high-affinity binding of GEFs to nucleotide-free GTPases. Modified from (Koch, Rai et al. 2016); (B) Quantitative GTPase affinity purification (qGAP) assay for the systematic identification of interaction partners of Rho GTPases. Modified from (Paul, Zauber et al. 2017); (C) SILAC-based quantitative AP–MS workflow for the characterizations of the comparative and nucleotide-dependent (KRAS^WT/KRAS^G12D) interactomes of two K-Ras isoforms, KRas4a and KRas4b. Modified from (Zhang, Cao et al. 2018)

Figure 7.. Schematic workflow of BioID–MS in studying Rho GTPase interactomes.

(A) The Rho cycle and the strategy used to define the interactomes of Rho family members by BioID–MS. A total of 20 active, 4 nucleotide-free and 4 WT Rho GTPases were fused with BirA*; (B) Workflow of the BioID–MS approach performed in Flp-In T-Rex HEK293 and HeLa cell lines. Reprinted by permission from Springer Nature Customer Service Centre GmbH: Nat Cell Biol 22: 120–134 (“Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.”, Bagci, H., N. Sriskandarajah, A. Robert, J. Boulais, I. E. Elkholi, V. Tran, Z. Y. Lin, M. P. Thibault, N. Dube, D. Faubert, D. R. Hipfner, A. C. Gingras and J. F. Cote), COPYRIGHT 2019, Springer Nature. (Bagci, Sriskandarajah et al. 2019).

Figure 8.. The Chemical structures of various isoprenoid derivatives used in global proteomic analysis of prenylated proteins and mechanistic studies of PPTases.

Shown are the structures of geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) (A); FPP-azide and GGPP-azide (B); C15Alk-OPP and C20Alk-OPP (C); YnF/YnGG and YnFPP/YnGGPP (D); and biotinylated-geranylpyrophosphate (BGPP) (E). Displayed in (F) is a schematic diagram of BGPP metabolic tagging workflow for LC/MS profiling of prenylome.