The underappreciated diversity of bile acid modifications.

The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

A region-resolved proteomic map of the human brain enabled by high-throughput proteomics.

Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).

Predicting glycan structure from tandem mass spectrometry via deep learning.

Glycans constitute the most complicated post-translational modification, modulating protein activity in health and disease. However, structural annotation from tandem mass spectrometry (MS/MS) data is a bottleneck in glycomics, preventing high-throughput endeavors and relegating glycomics to a few experts. Trained on a newly curated set of 500,000 annotated MS/MS spectra, here we present CandyCrunch, a dilated residual neural network predicting glycan structure from raw liquid chromatography-MS/MS data in seconds (top-1 accuracy: 90.3%). We developed an open-access Python-based workflow of raw data conversion and prediction, followed by automated curation and fragment annotation, with predictions recapitulating and extending expert annotation. We demonstrate that this can be used for de novo annotation, diagnostic fragment identification and high-throughput glycomics. For maximum impact, this entire pipeline is tightly interlaced with our glycowork platform and can be easily tested at https://colab.research.google.com/github/BojarLab/CandyCrunch/blob/main/CandyCrunch.ipynb . We envision CandyCrunch to democratize structural glycomics and the elucidation of biological roles of glycans.

Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

Quantitative, high-resolution proteomics for data-driven systems biology.

Systems biology requires comprehensive data at all molecular levels. Mass spectrometry (MS)-based proteomics has emerged as a powerful and universal method for the global measurement of proteins. In the most widespread format, it uses liquid chromatography (LC) coupled to high-resolution tandem mass spectrometry (MS/MS) to identify and quantify peptides at a large scale. This peptide intensity information is the basic quantitative proteomic data type. It is used to quantify proteins between different proteome states, including the temporal variation of the proteome, to determine the complete primary structure of proteins including posttranslational modifications, to localize proteins to organelles, and to determine protein interactions. Here, we describe the principles of analysis and the areas of biology where proteomics can make unique contributions. The large-scale nature of proteomics data and its high accuracy pose special opportunities as well as challenges in systems biology that have been largely untapped so far.

BUDDY: molecular formula discovery via bottom-up MS/MS interrogation.

A substantial fraction of metabolic features remains undetermined in mass spectrometry (MS)-based metabolomics, and molecular formula annotation is the starting point for unraveling their chemical identities. Here we present bottom-up tandem MS (MS/MS) interrogation, a method for de novo formula annotation. Our approach prioritizes MS/MS-explainable formula candidates, implements machine-learned ranking and offers false discovery rate estimation. Compared with the mathematically exhaustive formula enumeration, our approach shrinks the formula candidate space by 42.8% on average. Method benchmarking on annotation accuracy was systematically carried out on reference MS/MS libraries and real metabolomics datasets. Applied on 155,321 recurrent unidentified spectra, our approach confidently annotated >5,000 novel molecular formulae absent from chemical databases. Beyond the level of individual metabolic features, we combined bottom-up MS/MS interrogation with global optimization to refine formula annotations while revealing peak interrelationships. This approach allowed the systematic annotation of 37 fatty acid amide molecules in human fecal data. All bioinformatics pipelines are available in a standalone software, BUDDY ( https://github.com/HuanLab/BUDDY ).

Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

Introducing π-HelixNovo for practical large-scale de novo peptide sequencing.

De novo peptide sequencing is a promising approach for novel peptide discovery, highlighting the performance improvements for the state-of-the-art models. The quality of mass spectra often varies due to unexpected missing of certain ions, presenting a significant challenge in de novo peptide sequencing. Here, we use a novel concept of complementary spectra to enhance ion information of the experimental spectrum and demonstrate it through conceptual and practical analyses. Afterward, we design suitable encoders to encode the experimental spectrum and the corresponding complementary spectrum and propose a de novo sequencing model $\pi$-HelixNovo based on the Transformer architecture. We first demonstrated that $\pi$-HelixNovo outperforms other state-of-the-art models using a series of comparative experiments. Then, we utilized $\pi$-HelixNovo to de novo gut metaproteome peptides for the first time. The results show $\pi$-HelixNovo increases the identification coverage and accuracy of gut metaproteome and enhances the taxonomic resolution of gut metaproteome. We finally trained a powerful $\pi$-HelixNovo utilizing a larger training dataset, and as expected, $\pi$-HelixNovo achieves unprecedented performance, even for peptide-spectrum matches with never-before-seen peptide sequences. We also use the powerful $\pi$-HelixNovo to identify antibody peptides and multi-enzyme cleavage peptides, and $\pi$-HelixNovo is highly robust in these applications. Our results demonstrate the effectivity of the complementary spectrum and take a significant step forward in de novo peptide sequencing.

Ion entropy and accurate entropy-based FDR estimation in metabolomics.

Accurate metabolite annotation and false discovery rate (FDR) control remain challenging in large-scale metabolomics. Recent progress leveraging proteomics experiences and interdisciplinary inspirations has provided valuable insights. While target-decoy strategies have been introduced, generating reliable decoy libraries is difficult due to metabolite complexity. Moreover, continuous bioinformatics innovation is imperative to improve the utilization of expanding spectral resources while reducing false annotations. Here, we introduce the concept of ion entropy for metabolomics and propose two entropy-based decoy generation approaches. Assessment of public databases validates ion entropy as an effective metric to quantify ion information in massive metabolomics datasets. Our entropy-based decoy strategies outperform current representative methods in metabolomics and achieve superior FDR estimation accuracy. Analysis of 46 public datasets provides instructive recommendations for practical application.

Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction.

Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation.

Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Full Mass Range ΦSDM Orbitrap Mass Spectrometry for DIA Proteome Analysis.

Optimizing data-independent acquisition methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here, we describe a real-time full mass range implementation of the phase-constrained spectrum deconvolution method (ΦSDM) for Orbitrap mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals. In a standard 2 h analysis of a 200 ng HeLa digest, this resulted in an increase of 16% in the number of quantified peptides. As the acquisition speed becomes even more important when using fast chromatographic gradients, we further applied ΦSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ΦSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here, the number of identified protein groups and peptides increased by >15% in comparison to enhanced Fourier transformation processing. In conclusion, ΦSDM is an alternative signal processing algorithm for processing Orbitrap data that can improve spectral quality and benefit quantitative accuracy in typical proteomics experiments, especially when using short gradients.

Combining Data Independent Acquisition With Spike-In SILAC (DIA-SiS) Improves Proteome Coverage and Quantification.

Data-independent acquisition (DIA) is increasingly preferred over data-dependent acquisition due to its higher throughput and fewer missing values. Whereas data-dependent acquisition often uses stable isotope labeling to improve quantification, DIA mostly relies on label-free approaches. Efforts to integrate DIA with isotope labeling include chemical methods like mass differential tags for relative and absolute quantification and dimethyl labeling, which, while effective, complicate sample preparation. Stable isotope labeling by amino acids in cell culture (SILAC) achieves high labeling efficiency through the metabolic incorporation of heavy labels into proteins in vivo. However, the need for metabolic incorporation limits the direct use in clinical scenarios and certain high-throughput experiments. Spike-in SILAC (SiS) methods use an externally generated heavy sample as an internal reference, enabling SILAC-based quantification even for samples that cannot be directly labeled. Here, we combine DIA-SiS, leveraging the robust quantification of SILAC without the complexities associated with chemical labeling. We developed DIA-SiS and rigorously assessed its performance with mixed-species benchmark samples on bulk and single cell-like amount level. We demonstrate that DIA-SiS substantially improves proteome coverage and quantification compared to label-free approaches and reduces incorrectly quantified proteins. Additionally, DIA-SiS proves effective in analyzing proteins in low-input formalin-fixed paraffin-embedded tissue sections. DIA-SiS combines the precision of stable isotope-based quantification with the simplicity of label-free sample preparation, facilitating simple, accurate, and comprehensive proteome profiling.

Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down mass spectrometry (MS). Improved sequence coverage, critical for reliable annotation of posttranslational modifications and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as LC/MS of several proteins. Experiments were undertaken on multiple instruments, including quadrupole time-of-flight, Orbitrap, and high-field Fourier-transform ion cyclotron resonance (FT-ICR) across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher energy collision dissociation MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical versus even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD or ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.

Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).

CLIPPER 2.0: Peptide-Level Annotation and Data Analysis for Positional Proteomics.

Positional proteomics methodologies have transformed protease research, and have brought mass spectrometry (MS)-based degradomics studies to the forefront of protease characterization and system-wide interrogation of protease signaling. Considerable advancements in both sensitivity and throughput of liquid chromatography (LC)-MS/MS instrumentation enable the generation of enormous positional proteomics datasets of natural and protein termini and neo-termini of cleaved protease substrates. However, concomitant progress has not been observed to the same extent in data analysis and post-processing steps, arguably constituting the largest bottleneck in positional proteomics workflows. Here, we present a computational tool, CLIPPER 2.0, that builds on prior algorithms developed for MS-based protein termini analysis, facilitating peptide-level annotation and data analysis. CLIPPER 2.0 can be used with several sample preparation workflows and proteomics search algorithms and enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets. We demonstrate the applicability of our tool by analyzing GluC and MMP9 cleavages in HeLa lysates. CLIPPER 2.0 is available at https://github.com/UadKLab/CLIPPER-2.0.

Universal Identification of Pathogenic Viruses by Liquid Chromatography Coupled with Tandem Mass Spectrometry Proteotyping.

Accurate and rapid identification of viruses is crucial for an effective medical diagnosis when dealing with infections. Conventional methods, including DNA amplification techniques or lateral-flow assays, are constrained to a specific set of targets to search for. In this study, we introduce a novel tandem mass spectrometry proteotyping-based method that offers a universal approach for the identification of pathogenic viruses and other components, eliminating the need for a priori knowledge of the sample composition. Our protocol relies on a time and cost-efficient peptide sample preparation, followed by an analysis with liquid chromatography coupled to high-resolution tandem mass spectrometry. As a proof of concept, we first assessed our method on publicly available shotgun proteomics datasets obtained from virus preparations and fecal samples of infected individuals. Successful virus identification was achieved with 53 public datasets, spanning 23 distinct viral species. Furthermore, we illustrated the method's capability to discriminate closely related viruses within the same sample, using alphaviruses as an example. The clinical applicability of our method was demonstrated by the accurate detection of the vaccinia virus in spiked saliva, a matrix of paramount clinical significance due to its non-invasive and easily obtainable nature. This innovative approach represents a significant advancement in pathogen detection and paves the way for enhanced diagnostic capabilities.

GRable Version 1.0: A Software Tool for Site-Specific Glycoform Analysis With Improved MS1-Based Glycopeptide Detection With Parallel Clustering and Confidence Evaluation With MS2 Information.

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).

Enhanced In Situ Spatial Proteomics by Effective Combination of MALDI Imaging and LC-MS/MS.

Highly specialized cells are fundamental for the proper functioning of complex organs. Variations in cell-type-specific gene expression and protein composition have been linked to a variety of diseases. Investigation of the distinctive molecular makeup of these cells within tissues is therefore critical in biomedical research. Although several technologies have emerged as valuable tools to address this cellular heterogeneity, most workflows lack sufficient in situ resolution and are associated with high costs and extremely long analysis times. Here, we present a combination of experimental and computational approaches that allows a more comprehensive investigation of molecular heterogeneity within tissues than by either shotgun LC-MS/MS or MALDI imaging alone. We applied our pipeline to the mouse brain, which contains a wide variety of cell types that not only perform unique functions but also exhibit varying sensitivities to insults. We explored the distinct neuronal populations within the hippocampus, a brain region crucial for learning and memory that is involved in various neurological disorders. As an example, we identified the groups of proteins distinguishing the neuronal populations of the dentate gyrus (DG) and the cornu ammonis (CA) in the same brain section. Most of the annotated proteins matched the regional enrichment of their transcripts, thereby validating the method. As the method is highly reproducible, the identification of individual masses through the combination of MALDI-IMS and LC-MS/MS methods can be used for the much faster and more precise interpretation of MALDI-IMS measurements only. This greatly speeds up spatial proteomic analyses and allows the detection of local protein variations within the same population of cells. The method's general applicability has the potential to be used to investigate different biological conditions and tissues and a much higher throughput than other techniques making it a promising approach for clinical routine applications.

Quantitative proteomics reveals CLR interactome in primary human cells.

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine, and cancer. To date, the network of proteins interacting with CLR ("CLR interactome") in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation utilizing anti-human CLR antibody with label-free quantitative nano LC-MS/MS and quantitative in situ proximity ligation assay. By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ proximity ligation assay). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.