Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

Crosslinking mass spectrometry (XL-MS) has received considerable interest due to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison to other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by use of bi-functional crosslinking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by mass spectrometry. XL-MS has received the required boost to tackle more complex samples with recent advances in crosslinking chemistry with MS-cleavable or reporter-based crosslinkers and faster, more sensitive and more versatile mass spectrometry platforms. This protocol provides a detailed description of our optimized conditions for a full proteome native protein preparation followed by crosslinking using the gas-phase cleavable crosslinking reagent DSSO. Following crosslinking, we demonstrate extensive sample fractionation and significantly simplified data analysis with XlinkX in Proteome Discoverer and subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within approximately 10 d including structural investigations.

Chemical cross-linking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, substoichiometric reaction efficiencies render routine detection of cross-linked peptides problematic. Here, we present a new trifunctional cross-linking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the cross-linked peptides amenable to the well-established immobilized metal affinity chromatography (IMAC) enrichment. The handle allows for 300× enrichment efficiency and 97% specificity. We exemplify the approach on various model proteins and protein complexes, e.g., resulting in a structural model of the LRP1/RAP complex. Almost completely removing linear peptides allows PhoX, although noncleavable, to be applied to complex lysates. Focusing the database search to the 1400 most abundant proteins, we were able to identify 1156 cross-links in a single 3 h measurement.