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.

Infrared multiphoton dissociation (IRMPD) has been used in mass spectrometry to fragment peptides and proteins, providing fragments mostly similar to collisional activation. Using the 10.6 μm wavelength of a CO2 laser, IRMPD suffers from the relative low absorption cross-section of peptides and small proteins. Focusing on top-down analysis, we investigate different means to tackle this issue. We first reassess efficient sorting of phosphopeptides from nonphosphopeptides based on IR-absorption cross-sectional enhancement by phosphate moieties. We subsequently demonstrate that a myo-inositol hexakisphosphate (IP6) noncovalent adduct can substantially enhance IRMPD for nonphosphopeptides and that this strategy can be extended to proteins. As a natural next step, we show that native phospho-proteoforms of proteins display a distinct and enhanced fragmentation, compared to their unmodified counterparts, facilitating phospho-group site localization. We then evaluate the impact of size on the IRMPD of proteins and their complexes. When applied to protein complexes ranging from a 365 kDa CRISPR–Cas Csy ribonucleoprotein hetero-decamer, a 800 kDa GroEL homo-tetradecamer in its apo-form or loaded with its ATP cofactor, to a 1 MDa capsid-like homo-hexacontamer, we conclude that while phosphate moieties present in crRNA and ATP molecules enhance IRMPD, an increase in the IR cross-section with the size of the protein assembly also favorably accrues dissociation yields. Overall, our work showcases the versatility of IRMPD in the top-down analysis of peptides, phosphopeptides, proteins, phosphoproteins, ribonucleoprotein assemblies, and large protein complexes.

Mass spectrometry‐based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post‐translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C‐terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline‐directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications.