Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabl...

Significance: Nearly all proteins are posttranslationally modified, a phenomenon known to alter protein function. Recently, multiple posttranslational modifications (PTMs) have been documented to exist on the same proteins, revealing an additional level of complexity (named “PTM crosstalk”) that, due to its dynamic nature, is challenging to predict. Here, we propose a motif for PTM crosstalk between two of the most common PTMs: phosphorylation and O-GlcNAcylation. Through the use of a kinetic-based high-resolution mass spectrometry assay, we highlight specific residues that, when phosphorylated, hamper O-GlcNAcylation at nearby sites. In addition, we show that the Ser/Thr residues in one of the most common kinase motifs, PX(S/T)P, cannot be O-GlcNAcylated, demonstrating that reciprocal PTM crosstalk does not occur with Pro-directed kinases.

A wide variety of protein post‐translational modifications (PTMs) decorate cellular proteins, regulating their structure, interactions and ultimately their function. The density of co‐occurring PTMs on proteins can be very high, where multiple PTMs can positively or negatively influence each other's actions, termed PTM crosstalk. In this review, we highlight recent progress in the area of PTM crosstalk, whereby we focus on crosstalk between protein phosphorylation and O‐GlcNAcylation. These two PTMs largely target identical (i.e., Ser and Thr) amino acids in proteins. Phosphorylation/O‐GlcNAcylation crosstalk comes in many flavors, for instance by competition for the same site/residue (reciprocal crosstalk), as well as by modifications influencing each other in proximity or even distal on the protein sequence. PTM crosstalk is observed on the writers of these modifications (i.e., kinases and O‐GlcNAc transferase), on the erasers (i.e., phosphatases and O‐GlcNAcase), and on the readers and the substrates. We describe examples of all these different flavors of crosstalk, and additionally the methods that are emerging to better investigate in particular phosphorylation/O‐GlcNAcylation crosstalk.

Membrane proteins are notoriously challenging to analyze using mass spectrometry (MS) because of their insolubility in aqueous solution. Current MS methods for studying intact membrane proteins involve solubilization in detergent. However, detergents can destabilize proteins, leading to protein unfolding and aggregation, or resulting in inactive entities. Amphipathic polymers, termed amphipols, can be used as a substitute for detergents and have been shown to enhance the stability of membrane proteins. Here, we show the utility of amphipols for investigating the structural and functional properties of membrane proteins using electrospray ionization mass spectrometry (ESI-MS). The functional properties of two bacterial outer-membrane β-barrel proteins, OmpT and PagP, in complex with the amphipol A8-35 are demonstrated, and their structural integrities are confirmed in the gas phase using ESI-MS coupled with ion mobility spectrometry (IMS). The data illustrate the power of ESI-IMS-MS in separating distinct populations of amphipathic polymers from the amphipol–membrane complex while maintaining a conformationally “nativelike” membrane protein structure in the gas phase. Together, the data indicate the potential importance and utility of amphipols for the analysis of membrane proteins using MS.