Top-down proteomics.

Proteoforms, which arise from post-translational modifications, genetic polymorphisms and RNA splice variants, play a pivotal role as drivers in biology. Understanding proteoforms is essential to unravel the intricacies of biological systems and bridge the gap between genotypes and phenotypes. By analysing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and can decipher protein function, uncover disease mechanisms and advance precision medicine. This Primer explores TDP, including the underlying principles, recent advances and an outlook on the future. The experimental section discusses instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques and data collection. The results section looks at how to decipher raw data, visualize intact protein spectra and unravel data analysis. Additionally, proteoform identification, characterization and quantification are summarized, alongside approaches for statistical analysis. Various applications are described, including the human proteoform project and biomedical, biopharmaceutical and clinical sciences. These are complemented by discussions on measurement reproducibility, limitations and a forward-looking perspective that outlines areas where the field can advance, including potential future applications.

Quantitative proteome-wide O-glycoproteomics analysis with FragPipe.

Identification of O-glycopeptides from tandem mass spectrometry data is complicated by the near complete dissociation of O-glycans from the peptide during collisional activation and by the combinatorial explosion of possible glycoforms when glycans are retained intact in electron-based activation. The recent O-Pair search method provides an elegant solution to these problems, using a collisional activation scan to identify the peptide sequence and total glycan mass, and a follow-up electron-based activation scan to localize the glycosite(s) using a graph-based algorithm in a reduced search space. Our previous O-glycoproteomics methods with MSFragger-Glyco allowed for extremely fast and sensitive identification of O-glycopeptides from collisional activation data but had limited support for site localization of glycans and quantification of glycopeptides. Here, we report an improved pipeline for O-glycoproteomics analysis that provides proteome-wide, site-specific, quantitative results by incorporating the O-Pair method as a module within FragPipe. In addition to improved search speed and sensitivity, we add flexible options for oxonium ion-based filtering of glycans and support for a variety of MS acquisition methods and provide a comparison between all software tools currently capable of O-glycosite localization in proteome-wide searches.

IsoBayes: a Bayesian approach for single-isoform proteomics inference.

Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry proteomics, which return peptide identifications, that are indirectly used to infer the presence of protein isoforms. However, the detection and quantification processes are noisy; in particular, peptides may be erroneously detected, and most peptides, known as shared peptides, are associated to multiple protein isoforms. As a consequence, studying individual protein isoforms is challenging, and inferred protein results are often abstracted to the gene-level or to groups of protein isoforms. Here, we introduce IsoBayes, a novel statistical method to perform inference at the isoform level. Our method enhances the information available, by integrating mass spectrometry proteomics and transcriptomics data in a Bayesian probabilistic framework. To account for the uncertainty in the measurement process, we propose a two-layer latent variable approach: first, we sample if a peptide has been correctly detected (or, alternatively filter peptides); second, we allocate the abundance of such selected peptides across the protein(s) they are compatible with. This enables us, starting from peptide-level data, to recover protein-level data; in particular, we: i) infer the presence/absence of each protein isoform (via a posterior probability), ii) estimate its abundance (and credible interval), and iii) target isoforms where transcript and protein relative abundances significantly differ. We benchmarked our approach in simulations, and in two multi-protease real datasets: our method displays good sensitivity and specificity when detecting protein isoforms, its estimated abundances highly correlate with the ground truth, and can detect changes between protein and transcript relative abundances. IsoBayes is freely distributed as a Bioconductor R package, and is accompanied by an example usage vignette.

Five-year outcomes of fractionated stereotactic body radiotherapy for oligometastatic prostate cancer from the TRANSFORM phase II trial.

Metastasis-directed therapy (MDT) for oligometastatic prostate cancer (PCa), including stereotactic body radiotherapy (SBRT), has shown promise but is still considered investigational. This is the 5-year analysis of the TRANSFORM trial, the largest prospective cohort of men with oligometastatic PCa treated with SBRT-based MDT. The primary endpoint was 5-year treatment escalation-free survival (TE-FS), defined as freedom from any new cancer therapy other than further SBRT. In total, 199 men received SBRT; 76.4% were hormone-naïve at baseline. The rate of 5-year TE-FS was 21.7% (95% confidence interval [CI]: 15.7%-28.7%) overall and 25.4% (95% CI: 18.1%-33.9%) in the hormone-naïve subgroup. The subgroups with International Society of Urological Pathology Grade Groups 4-5 disease (hazard ratio [HR] = 1.48, 95% CI: 1.05-2.01, p = .026), a higher baseline prostate-specific antigen (PSA) (HR = 1.06, 95% CI: 1.03-1.09, p < .001) and those who received prior androgen deprivation therapy (ADT) (HR = 2.13, 95% CI: 1.40-3.26, p < .001), were at greater risk of treatment escalation. Outcomes for participants with four or five initial lesions were comparable to those with one to three lesions. At last follow-up, 18.9% (95% CI: 13.2%-25.7%) of participants were free from treatment escalation (median follow-up of 67.9 months) and two participants had an undetectable PSA level. No treatment-related grade three or higher adverse events were reported. The findings of this study demonstrate that SBRT-based MDT is an effective option for delaying systemic treatment escalation in the context of oligometastatic PCa. Future randomised trials comparing SBRT-based MDT to standard-of-care ADT-based approaches are required to evaluate the impact of delaying ADT on survival.

Disparities Between Teleretinal Imaging Findings and Patient-Reported Diabetic Retinopathy Status and Follow-up Eye Care Interval: A 10-Year Prospective Study.

OBJECTIVE: To assess self-reported awareness of diabetic retinopathy (DR) and concordance of eye examination follow-up compared with findings from concurrent retinal images. RESEARCH DESIGN AND METHODS: We conducted a prospective observational 10-year study of 26,876 consecutive patients with diabetes who underwent retinal imaging during an endocrinology visit. Awareness and concordance were evaluated using questionnaires and retinal imaging. RESULTS: Awareness information and gradable images were available in 25,360 patients (94.3%). Severity of DR by imaging was as follows: no DR (n = 14,317; 56.5%), mild DR (n = 6,805; 26.8%), or vision-threatening DR (vtDR; n = 4,238; 16.7%). In the no, mild, and vtDR groups, 96.7%, 88.5%, and 54.9% of patients, respectively, reported being unaware of any prior DR. When DR was present, reporting no prior DR was associated with shorter diabetes duration, milder DR, last eye examination >1 year before, no dilation, no scheduled appointment, and less specialized provider (all P < 0.001). Among patients with vtDR, 41.2%, 58.1%, and 64.2% did not report being aware of any DR and follow-up was concordant with current DR severity in 66.7%, 41.3%, and 25.4% (P < 0.001) of patients when prior examination was performed by a retinal specialist, nonretinal ophthalmologist, or optometrist (P < 0.001), respectively. CONCLUSIONS: Substantial discrepancies exist between DR presence, patient awareness, and concordance of follow-up across all DR severity levels. These discrepancies are present across all eye care provider types, with the magnitude influenced by provider type. Therefore, patient self-report should not be relied upon to reflect DR status. Modification of medical care and education models may be necessary to enhance retention of ophthalmic knowledge in patients with diabetes and ensure accurate communication between all health care providers.

Spectral averaging with outlier rejection algorithms to increase identifications in top-down proteomics.

The identification of proteoforms by top-down proteomics requires both high quality fragmentation spectra and the neutral mass of the proteoform from which the fragments derive. Intact proteoform spectra can be highly complex and may include multiple overlapping proteoforms, as well as many isotopic peaks and charge states. The resulting lower signal-to-noise ratios for intact proteins complicates downstream analyses such as deconvolution. Averaging multiple scans is a common way to improve signal-to-noise, but mass spectrometry data contains artifacts unique to it that can degrade the quality of an averaged spectra. To overcome these limitations and increase signal-to-noise, we have implemented outlier rejection algorithms to remove outlier measurements efficiently and robustly in a set of MS1 scans prior to averaging. We have implemented averaging with rejection algorithms in the open-source, freely available, proteomics search engine MetaMorpheus. Herein, we report the application of the averaging with rejection algorithms to direct injection and online liquid chromatography mass spectrometry data. Averaging with rejection algorithms demonstrated a 45% increase in the number of proteoforms detected in Jurkat T cell lysate. We show that the increase is due to improved spectral quality, particularly in regions surrounding isotopic envelopes.

Efficacy of 38% silver diamine fluoride in reducing gingival inflammation and plaque accumulation in older adults living in retirement-homes: A randomized controlled pilot trial.

OBJECTIVES: Emerging from earlier case reports the potential benefits of 38 % silver diamine fluoride (SDF) in addressing pathogenic biofilms and mitigating gingival inflammation and enlargement have sparked interest. Our study aimed to evaluate the efficacy of 38 % SDF in reducing gingival inflammation and plaque accumulation in older adults living in retirement-homes. METHODS: This 7-week randomized, controlled, double-blinded pilot trial employed a parallel assignment design. The study enrolled older adults (aged ≥65) residing in retirement homes in Dallas County, ultimately comprising a cohort of 40 participants who were evenly divided into two arms. The experimental group received SDF treatment, whereas the comparator group received a placebo. Over three consecutive weeks, both groups had solutions applied to the facial surfaces of all their teeth once per week. The primary outcomes measured the change in Löe-Silness Gingival Index (GI) and Silness-Löe Plaque Index (PI) at 7 weeks following baseline treatment. Repeated measures ANOVA was utilized to assess changes over time within each group (n = 15 each). Post-hoc paired t-tests were conducted to compare changes between week 1 and each subsequent follow-up time point (weeks 3, 5, 7), supplemented with 95 % confidence intervals for change from week 1. RESULTS: In the SDF group, within-group comparisons demonstrated significant reductions (adjusted p < .05) in GI scores within 3 weeks (-.93±.37), as opposed to week 1 (1.90±.39). Between-group comparisons unveiled reductions in both mean GI (p < .05) and PI (p < .05), indicating less gingival inflammation and plaque accumulation in the SDF group at all time points, commencing at week 3. CONCLUSIONS: This study showed that 38 % SDF was effective in reducing gingival inflammation and plaque accumulation in older adults living in retirement-homes. CLINICAL SIGNIFICANCE: Oral health in older adults is a public health concern, especially for the medically compromised or those without traditional care. Our findings offer hope for enhancing oral health quality of life by introducing a cost-effective, compliance-free, noninvasive, and accessible therapeutic. TRIAL REGISTRATION: NCT03445286.(clinicaltrials.gov).

Erratum: Proteomic pathways to metabolic disease and type 2 diabetes in the pancreatic islet.

[This corrects the article DOI: 10.1016/j.isci.2021.103099.].

Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

How the other half screens: A model for partnerships between student-run free clinics and genetic counseling programs to address disparities in hereditary cancer evaluation.

Genetic medicine is considered a major part of the future of preventative care, offering evidence-based, effective interventions to improve health outcomes and reduce morbidity and mortality, especially regarding hereditary cancer screening. Identification of individuals who would benefit from screening is key to improving their cancer-related healthcare outcomes. However, patients without insurance, of historically underserved races, of lower socioeconomic status, and in rural communities have lower access to such care. Barriers to access lead to populations having higher rates of undetected hereditary cancer, and consequently more severe forms of cancer. With an already-established reach, student-run free clinics can work with genetic counseling training programs to incorporate genetic medicine into their workflow. Such partnerships will (1) make genetic care more accessible with goals of improving patient morbidity, mortality, and health outcomes, (2) offer robust educational experiences for genetic counseling learners, particularly in understanding social determinants of health and barriers to care, and (3) actively combat the growing racial and geographic gaps in genetic care. Our study presents how one student-run free clinic implemented genetic counseling into its primary care workflow to improve access to genetics services. We present two examples of how genetic counseling improved patients' medical care. We also identify obstacles encountered during this program's development, as well as solutions-those we incorporated and possible considerations for other clinics. With the hope that other clinics can use this paper to design similar partnerships, we aim to lessen the gap between sickness and screening.

Cysteine Counting via Isotopic Chemical Labeling for Intact Mass Proteoform Identifications in Tissue.

Top-down proteomics, the tandem mass spectrometric analysis of intact proteoforms, is the dominant method for proteoform characterization in complex mixtures. While this strategy produces detailed molecular information, it also requires extensive instrument time per mass spectrum obtained and thus compromises the depth of proteoform coverage that is accessible on liquid chromatography time scales. Such a top-down analysis is necessary for making original proteoform identifications, but once a proteoform has been confidently identified, the extensive characterization it provides may no longer be required for a subsequent identification of the same proteoform. We present a strategy to identify proteoforms in tissue samples on the basis of the combination of an intact mass determination with a measured count of the number of cysteine residues present in each proteoform. We developed and characterized a cysteine tagging chemistry suitable for the efficient and specific labeling of cysteine residues within intact proteoforms and for providing a count of the cysteine amino acids present. On simple protein mixtures, the tagging chemistry yields greater than 98% labeling of all cysteine residues, with a labeling specificity of greater than 95%. Similar results are observed on more complex samples. In a proof-of-principle study, proteoforms present in a human prostate tumor biopsy were characterized. Observed proteoforms, each characterized by an intact mass and a cysteine count, were grouped into proteoform families (groups of proteoforms originating from the same gene). We observed 2190 unique experimental proteoforms, 703 of which were grouped into 275 proteoform families.

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Newly synthesized glycoprotein profiling to identify molecular signatures of warm ischemic injury in donor lungs.

Despite recent technological advances such as ex vivo lung perfusion (EVLP), the outcome of lung transplantation remains unsatisfactory with ischemic injury being a common cause for primary graft dysfunction. New therapeutic developments are hampered by limited understanding of pathogenic mediators of ischemic injury to donor lung grafts. Here, to identify novel proteomic effectors underlying the development of lung graft dysfunction, using bioorthogonal protein engineering, we selectively captured and identified newly synthesized glycoproteins (NewS-glycoproteins) produced during EVLP with unprecedented temporal resolution of 4 h. Comparing the NewS-glycoproteomes in lungs with and without warm ischemic injury, we discovered highly specific proteomic signatures with altered synthesis in ischemic lungs, which exhibited close association to hypoxia response pathways. Inspired by the discovered protein signatures, pharmacological modulation of the calcineurin pathway during EVLP of ischemic lungs offered graft protection and improved posttransplantation outcome. In summary, the described EVLP-NewS-glycoproteomics strategy delivers an effective new means to reveal molecular mediators of donor lung pathophysiology and offers the potential to guide future therapeutic development.NEW & NOTEWORTHY This study developed and implemented a bioorthogonal strategy to chemoselectively label, enrich, and characterize newly synthesized (NewS-)glycoproteins during 4-h ex vivo lung perfusion (EVLP). Through this approach, the investigators uncovered specific proteomic signatures associated with warm ischemic injury in donor lung grafts. These signatures exhibit high biological relevance to ischemia-reperfusion injury, validating the robustness of the presented approach.

Continuous Epidural Analgesia (CEA) via Single Catheter Providing Profound Analgesia for Pediatric Patients Following Posterior Spinal Fusion (PSF) in Adolescent Idiopathic Scoliosis (AIS).

INTRODUCTION: Posterior spinal fusion (PSF) is a commonly performed orthopedic procedure to correct scoliosis in children. Continuous epidural analgesia (CEA) is a proposed means of providing analgesia following PSF. Whether a single epidural catheter with the tip in the upper thorax can provide adequate analgesia for PSF, which often spans the upper thoracic to lower lumbar regions, is unresolved in the literature. METHOD: In this single-center, retrospective study, we reviewed 69 consecutive patients undergoing PSF for adolescent idiopathic scoliosis (AIS) with CEA at our institution from October 1, 2020 to May 26, 2022. Data for the entire cohort was divided into two time intervals before and after epidural removal, group epidural (Epi) and group no epidural (No Epi). Daily intravenous and oral opioid morphine equivalents per kilogram (OME/kg) plus mean and maximal visual analogue pain scores (VAS 0-10) were recorded from post-anesthesia care unit (PACU) discharge to the end of postoperative day (POD) three.  Results: 57 patients were included in the study. Opioid usage was 4.5 times greater in the 19 hours following removal of the epidural catheter when compared to the entire period (mean 65 hours) the epidural was in place (Group Epi 0.154 OME/kg vs Group No Epi 0.690 OME/kg, p<0.001). 51% (29/57) of patients did not require opioids (intravenous or oral) while the epidural was in place, all patients required opioids after epidural removal. Mean opioid usage while the epidural was in place was 9.3 OME, equivalent to approximately 6 mg of oxycodone. Mean and maximum pain scores increased significantly after removal of the epidural on POD 3 (mean pain score: Epi 3.4 (1.8) vs No Epi 4.1 (1.7); p<0.001) (max pain score: Epi 4.9 (2.5) vs No Epi 6.3 (2.1); p<0.001).  Conclusions: This is the first study we are aware of to report pain scores and cumulative opioid requirements for PSF patients receiving CEA with a single epidural catheter before and after epidural removal. Opioid usage increased over four times in the 19 hours after epidural removal compared to the total opioid requirements while the epidural was infusing. Mean and maximum pain scores increased significantly after removal of the epidural on POD 3. This study firmly establishes that CEA with a single epidural catheter can provide profound analgesia for patients having PSF for AIS.

Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

Elucidating the RNA-Protein Interactomes of Target RNAs in Tissue.

RNA-protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA-protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA-protein complexes.

Enhanced Proteomic Data Analysis with MetaMorpheus.

MetaMorpheus is a free and open-source software program dedicated to the comprehensive analysis of proteomic data. In bottom-up proteomics, protein samples are digested into peptides prior to chromatographic separation and tandem mass spectrometric analysis. The resulting fragmentation spectra are subsequently analyzed with search software programs to obtain peptide identifications and infer the presence of proteins in the samples. MetaMorpheus seeks to maximize the information gleaned from proteomic data through the use of (a) mass calibration, (b) post-translational modification discovery, (c) multiple search algorithms, which aid in the analysis of data from traditional, crosslinking, and glycoproteomic experiments, (d) isotope-based or label-free quantification, (e) multi-protease protein inference, and (f) spectral annotation and data visualization capabilities. This protocol provides detailed descriptions of how use MetaMorpheus and how to customize data analysis workflows using MetaMorpheus tasks to meet the specific needs of the user.

Fast, Free, and Flexible Peptide and Protein Quantification with FlashLFQ.

The rapid and accurate quantification of peptides is a critical element of modern proteomics that has become increasingly challenging as proteomic data sets grow in size and complexity. We present here FlashLFQ, a computer program for high-speed label-free quantification of peptides and proteins following a search of bottom-up mass spectrometry data. FlashLFQ is approximately an order of magnitude faster than established label-free quantification methods and can quantify data-dependent analysis (DDA) search results from any proteomics search program. It is available as a graphical user interface program, a command line tool, a Docker image, and integrated into the MetaMorpheus search software.

Overview and considerations in bottom-up proteomics.

Proteins are the key biological actors within cells, driving many biological processes integral to both healthy and diseased states. Understanding the depth of complexity represented within the proteome is crucial to our scientific understanding of cellular biology and to provide disease specific insights for clinical applications. Mass spectrometry-based proteomics is the premier method for proteome analysis, with the ability to both identify and quantify proteins. Although proteomics continues to grow as a robust field of bioanalytical chemistry, advances are still necessary to enable a more comprehensive view of the proteome. In this review, we provide a broad overview of mass spectrometry-based proteomics in general, and highlight four developing areas of bottom-up proteomics: (1) protein inference, (2) alternative proteases, (3) sample-specific databases and (4) post-translational modification discovery.

A Hybrid Spectral Library and Protein Sequence Database Search Strategy for Bottom-Up and Top-Down Proteomic Data Analysis.

Tandem mass spectrometry (MS/MS) is widely employed for the analysis of complex proteomic samples. While protein sequence database searching and spectral library searching are both well-established peptide identification methods, each has shortcomings. Protein sequence databases lack fragment peak intensity information, which can result in poor discrimination between correct and incorrect spectrum assignments. Spectral libraries usually contain fewer peptides than protein sequence databases, which limits the number of peptides that can be identified. Notably, few post-translationally modified peptides are represented in spectral libraries. This is because few search engines can both identify a broad spectrum of PTMs and create corresponding spectral libraries. Also, programs that generate spectral libraries using deep learning approaches are not yet able to accurately predict spectra for the vast majority of PTMs. Here, we address these limitations through use of a hybrid search strategy that combines protein sequence database and spectral library searches to improve identification success rates and sensitivity. This software uses Global PTM Discovery (G-PTM-D) to produce spectral libraries for a wide variety of different PTMs. These features, along with a new spectrum annotation and visualization tool, have been integrated into the freely available and open-source search engine MetaMorpheus.