The Methods Employed in Mass Spectrometric Analysis of Posttranslational Modifications (PTMs) and Protein-Protein Interactions (PPIs)

Abstract

Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.PTMs massively increase the complexity of organismal proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. There are an estimated 300 PTMS with a predicted 5% of the eukaryotic genome coding for enzymes involved in protein modification, however we have not yet been able to reliably map PTM proteomes due to limitations in sample preparation, analytical techniques, data analysis, and the substoichiometric and transient nature of some PTMs. Improvements in proteomic and mass spectrometry methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms. Here we focus on previously published global PTM proteome studies in the Apicomplexan parasites T. gondii and P. falciparum which offer numerous insights into the abundance and function of each of the studied PTM in the Apicomplexa. Integration of these datasets provide a more complete picture of the relative importance of PTM and crosstalk between them and how together PTM globally change the cellular biology of the Apicomplexan protozoa. A multitude of techniques used to investigate PTMs, mostly techniques in MS-based proteomics, are discussed for their ability to uncover relevant biological function.

Keywords: Cell cycle; Experimental techniques; In-silico databases; Mass spectrometry; Posttranslational crosstalk; Posttranslational modifications; Protein-protein interactions; Toxoplasma gondii.

Figure 1

Schematic representations of selected PPI assays. (A) Yeast Two Hybrid (Y2H). (B) Membrane Yeast Two Hybrid (MYTH) and Mammalian Membrane Two Hybrid (MaMTH). (C) Luminescence-based Mammalian Interactome Mapping (LUMIER). (D) Mammalian Protein-Protein Interaction Trap (MAPPIT). (E) Kinase Substrate Sensor (KISS). (F) Bimolecular Fluorescence Complementation (BiFC). (G) Bioluminescence/Fluorescence Resonance Energy Transfer (B/FRET). (H) Affinity Purification-MassSpectrometry (AP-MS). (I) Proximity-dependent Biotin Identification Coupled to Mass Spectrometry (BioID-MS). (J) Proximity Ligation Assay (PLA). (K) Ligand-Receptor Capture-Trifunctional Chemoproteomics Reagents (LRC-TRiCEPS). (L) Avidity-based Extracellular Interaction Screen (AVEXIS). Reprinted and adapted with permission under the terms of the Creative Commons Attribution License.

Figure 2

Interactions between PTM proteomes in T. gondii. Lists of proteins detected by proteomics to be modified by different PTMs in T. gondii were compared to one another using a hypergeometric test of enrichment using methods described by Silmon de Monerri et al. (2015). Both published (Braun et al., 2009; Treeck et al., 2011; Jeffers and Sullivan, 2012; Li et al., 2014; Foe et al., 2015; Silmon de Monerri et al., 2015; Yakubu et al., 2017) and unpublished (O-GlcNAc dataset, Silmon de Monerri and Kim, in preparation) PTM datasets were analyzed. Color key is in -log2 (p-value). Reprinted and adapted with permission from John Wiley and Sons.

Figure 3

PTM display preferences for different metabolic pathways in T. gondii. Proteins identified as PTM targets by proteomic studies in T. gondii were compared to sets of genes with different functions (classified by Gene Ontology [GO] terms) using a hypergeometric test of enrichment using methodology described by Silmon de Monerri (Silmon de Monerri et al., 2015). The five most significantly enriched GO terms are shown in a clustered heatmap. Color key is in -log2 (p-value). Reprinted and adapted with permission from John Wiley and Sons.

Figure 4

Proteins modified by PTMs are enriched in cell cycle regulated genes in T. gondii. Cell cycle enrichment analysis of published (Braun et al., 2009; Treeck et al., 2014; Jeffers and Sullivan, 2012; Li et al., 2014; Foe et al., 2015; Silmon de Monerri et al., 2015; Yakubu et al., 2017) and unpublished (O-GlcNAc, Silmon de Monerri and Kim, in preparation) PTM datasets. Proteins identified in proteomic studies of PTM were compared against cell cycle gene sets by a hypergeometric test of enrichment. Gene sets were composed of genes that are transcriptionally upregulated in G1 or S/M subtranscriptomes at time points during the 8 h T. gondii cell cycle, as described by Croken (Croken et al., 2014). (A) Two peaks of enrichment are seen in G1 phase (h 4.5–5.5; h 6.5–8) and (B) one peak of enrichment in S/M phase (h 3–4). Adjusted p-values (-(log2)- transformed) are plotted. Reprinted and adapted with permission from John Wiley and Sons.