Retention time prediction using neural networks increases identifications in crosslinking mass spectrometry

Crosslinking mass spectrometry has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the mass spectra of crosslinked peptides limits the numbers of protein–protein interactions that can be confidently identified. Here, we leverage chromatographic retention time information to aid the identification of crosslinked peptides from mass spectra. Our Siamese machine learning model xiRT achieves highly accurate retention time predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E. coli lysate. Importantly, supplementing the search engine score with retention time features leads to a substantial increase in protein–protein interactions without affecting confidence. This approach is not limited to cell lysates and multi-dimensional separation but also improves considerably the analysis of crosslinked multiprotein complexes with a single chromatographic dimension. Retention times are a powerful complement to mass spectrometric information to increase the sensitivity of crosslinking mass spectrometry analyses

Leveraging parameter dependencies in high-field asymmetric waveform ion-mobility spectrometry and size exclusion chromatography for proteome-wide cross-linking mass spectrometry

[Image: see text] Ion-mobility spectrometry shows great promise to tackle analytically challenging research questions by adding another separation dimension to liquid chromatography–mass spectrometry. The understanding of how analyte properties influence ion mobility has increased through recent studies, but no clear rationale for the design of customized experimental settings has emerged. Here, we leverage machine learning to deepen our understanding of field asymmetric waveform ion-mobility spectrometry for the analysis of cross-linked peptides. Knowing that predominantly m/z and then the size and charge state of an analyte influence the separation, we found ideal compensation voltages correlating with the size exclusion chromatography fraction number. The effect of this relationship on the analytical depth can be substantial as exploiting it allowed us to almost double unique residue pair detections in a proteome-wide cross-linking experiment. Other applications involving liquid- and gas-phase separation may also benefit from considering such parameter dependencies

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

Viruses have evolved means to manipulate the host's ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins

Oxonium ion scanning mass spectrometry for large-scale plasma glycoproteomics

Protein glycosylation, a complex and heterogeneous post-translational modification that is frequently dysregulated in disease, has been difficult to analyse at scale. Here we report a data-independent acquisition technique for the large-scale mass-spectrometric quantification of glycopeptides in plasma samples. The technique, which we named ‘OxoScan-MS’, identifies oxonium ions as glycopeptide fragments and exploits a sliding-quadrupole dimension to generate comprehensive and untargeted oxonium ion maps of precursor masses assigned to fragment ions from non-enriched plasma samples. By applying OxoScan-MS to quantify 1,002 glycopeptide features in the plasma glycoproteomes from patients with COVID-19 and healthy controls, we found that severe COVID-19 induces differential glycosylation in IgA, haptoglobin, transferrin and other disease-relevant plasma glycoproteins. OxoScan-MS may allow for the quantitative mapping of glycoproteomes at the scale of hundreds to thousands of samples

The predictive value of early behavioural assessments in pet dogs: a longitudinal study from neonates to adults

Studies on behavioural development in domestic dogs are of relevance for matching puppies with the right families, identifying predispositions for behavioural problems at an early stage, and predicting suitability for service dog work, police or military service. The literature is, however, inconsistent regarding the predictive value of tests performed during the socialisation period. Additionally, some practitioners use tests with neonates to complement later assessments for selecting puppies as working dogs, but these have not been validated. We here present longitudinal data on a cohort of Border collies, followed up from neonate age until adulthood. A neonate test was conducted with 99 Border collie puppies aged 2–10 days to assess activity, vocalisations when isolated and sucking force. At the age of 40–50 days, 134 puppies (including 93 tested as neonates) were tested in a puppy test at their breeders' homes. All dogs were adopted as pet dogs and 50 of them participated in a behavioural test at the age of 1.5 to 2 years with their owners. Linear mixed models found little correspondence between individuals' behaviour in the neonate, puppy and adult test. Exploratory activity was the only behaviour that was significantly correlated between the puppy and the adult test. We conclude that the predictive validity of early tests for predicting specific behavioural traits in adult pet dogs is limited