Thermo Fisher Scientific (Canada)

Post-translational modifications (PTMs) are critical regulators of protein function, yet confidently identifying and localizing PTM sites across proteomes remains a challenging task. Integrating peptide property predictions into spectrum interpretation improves identification performance, but training data enabling zero-shot prediction across diverse PTMs are scarce. Here, we present a major expansion of the ProteomeTools dataset, comprising over 977,000 synthetic peptides, covering 22 PTM-residue combinations. Furthermore, we developed Prosit-PTM, a model with chemically-informed encoding and amino acid substitution-based augmentation trained with our novel ground-truth dataset, that achieves accurate zero-shot predictions. Applied to modified peptides, Prosit-PTM enhances PTM-site localization in phosphoproteomics, increases identification of multiply modified peptides in histones, and enables data-driven rescoring for unseen modifications such as HLA peptides. Furthermore, the learned embeddings of amino acids and modifications capture physicochemical relationships underlying PTM-driven HLA presentation. Prosit-PTM is integrated into multiple open-source tools, enabling PTM-aware rescoring, site localization, spectral library generation, and beyond.

BackgroundHereditary angioedema (HAE) is a rare disease characterized by unpredictable, recurrent subcutaneous or submucosal swelling that negatively impacts patients’ health-related quality of life (HRQoL). Despite treatment goals aimed at achieving complete control of the disease and normalizing patients' lives, the disease remains poorly controlled for some patients.ObjectivesTo describe the demographic, clinical and treatment characteristics, as well as the HRQoL impairment of patients with sub-optimally controlled HAE type I/II per treating physician's judgment, focusing on understanding the factors influencing the burden of illness.MethodsA chart review was conducted at 32 HAE care centers across 18 European countries, Canada, and Israel between April 2022 and January 2023 in 214 patients aged ≥12 years with HAE type I/II sub-optimally controlled with on-demand treatment (ODT) and/or long-term prophylaxis (LTP). Patients receiving lanadelumab were excluded, as it was not yet widely available during the eligibility period. A cross-sectional survey at patient enrollment included the Angioedema Quality of Life (AE-QoL) and EQ-5D-5L questionnaires to assess the impact of HAE on HRQoL.ResultsPatients with uncontrolled HAE had a mean (standard deviation [SD]) of 9.9 (13) attacks per year, with a mean (SD) duration of 1.9 (1) days per attack. During the one-year observation period, 50.5% of patients were on ODT only, 36.0% used LTP and ODT concurrently, 6.5% used LTP without ODT, and 7.0% were untreated. Attenuated androgens (AA; stanozolol and danazol) in LTP were used by 24.7% of patients, while tranexamic acid (TA) and C1-esterase inhibitor (C1–INH) replacement products were used for LTP by 9.8% and 6.5%, respectively. The mean (SD) AE-QoL total score was 44.4 (24.0), indicating a moderate level of impairment, with women experiencing worse HRQoL (total score of 50.9 [SD 24] vs 37.3 [SD 23] in men) [where the minimal clinically important difference is 6 points] [1]. HRQoL worsened with increasing attack rates, from 41.4 (SD 24.0) among patients with one to 5 attacks/year, still indicating moderate impairment in HRQoL, to 73.0 (SD 27.0) for patients with >40 attacks/year.ConclusionsSuboptimal disease control in HAE was associated with the use of ODT only, as well as LTP mainly with AA/TA. It imposes a substantial burden on patients’ HRQoL, more particularly, but not exclusively, for those with frequent attacks and for women. The results suggest a need for improved HAE management.ClinicalTrial.gov study identifier NCT04957641.

Abstract Gaining a complete and unbiased understanding of the non-tryptic peptide repertoire presented by HLA-I complexes by LC-MS/MS is indispensable for therapy design for cancer, autoimmunity and infectious diseases. A serious concern in HLA peptide analysis is that the routinely used, collision-based fragmentation methods (CID/HCD) do not always render sufficiently informative MS2 spectra, whereby gaps in the fragmentation sequence coverage prevent unambiguous assignments. EThcD can be utilized to generate complementary ion series,i.e.b/y ions and c/z ions, resulting in richer, more informative MS2 spectra, thereby filling in the gaps. Here, we present data generated on a novel hybrid Orbitrap mass spectrometer, facilitating fast and efficient hybrid fragmentation due to the implementation of EThcD in the ion routing multipole. We hypothesized that this would enable more comprehensive and less error-prone analysis of immunopeptidomes at minimal costs in duty-cycle. First, we optimized ETD/EThcD methods using an elastase-digested cell lysate, as this contains peptides of similar length and charge distributions to immunopeptides. Next, we compared HCD and EThcD on immunopeptidomes originating from three cell lines with distinct HLA-I complexes that present peptides with varying physicochemical properties. We demonstrate that the new instrument not only enables efficient and fast ETD reactions, but when combined with collision-based supplemental activation,i.e.EThcD, also consistently increases the sequence coverage and identification of peptide sequences, otherwise missed by using solely HCD. We reveal several of the biochemical properties that make HLA peptides preferably identifiable by EThcD, with internal Arg residues being one of the most dominant determinants. Finally, we demonstrate the power of EThcD for the identification and localization of HLA peptides harboring post-translational modifications, focusing here on HLA Arg mono-/di-methylation. We foresee that this new instrument with efficient EThcD capabilities enhances not only immunopeptidomics analysis, but also analysis of peptides harboring post-translational modifications andde novosequencing.

Abstract Rapid and comprehensive analysis of complex proteomes across large sample sets is vital for unlocking the potential of systems biology. We present UFP-MS, an ultra-fast mass spectrometry (MS) proteomics method that integrates narrow-window data-independent acquisition (nDIA) with short-gradient micro-flow chromatography, enabling profiling of >240 samples per day. This optimized MS approach identifies 6,201 and 7,466 human proteins with 1- and 2-min gradients, respectively. Our streamlined sample preparation workflow features high-throughput homogenization, adaptive focused acoustics (AFA)-assisted proteolysis, and Evotip-accelerated desalting, allowing for the processing of up to 96 tissue samples in 5 h. As a practical application, we analyzed 507 samples from 13 mouse tissues treated with the enzyme-drug L-asparaginase (ASNase) or its glutaminase-free Q59L mutant, generating a quantitative profile of 11,472 proteins following drug treatment. The MS results confirmed the impact of ASNase on amino acid metabolism in solid tissues. Further analysis revealed broad suppression of anticoagulants and cholesterol metabolism and uncovered numerous tissue-specific dysregulated pathways. In summary, the UFP-MS method greatly accelerates the generation of biological insights and clinically actionable hypotheses into tissue-specific vulnerabilities targeted by ASNase.