Exploring the dynamic landscape of immunopeptidomics: Unravelling posttranslational modifications and navigating bioinformatics terrain.

Immunopeptidomics is becoming an increasingly important field of study. The capability to identify immunopeptides with pivotal roles in the human immune system is essential to shift the current curative medicine towards personalized medicine. Throughout the years, the field has matured, giving insight into the current pitfalls. Nowadays, it is commonly accepted that generalizing shotgun proteomics workflows is malpractice because immunopeptidomics faces numerous challenges. While many of these difficulties have been addressed, the road towards the ideal workflow remains complicated. Although the presence of Posttranslational modifications (PTMs) in the immunopeptidome has been demonstrated, their identification remains highly challenging despite their significance for immunotherapies. The large number of unpredictable modifications in the immunopeptidome plays a pivotal role in the functionality and these challenges. This review provides a comprehensive overview of the current advancements in immunopeptidomics. We delve into the challenges associated with identifying PTMs within the immunopeptidome, aiming to address the current state of the field.

Fragment ion intensity prediction improves the identification rate of non-tryptic peptides in timsTOF.

Immunopeptidomics is crucial for immunotherapy and vaccine development. Because the generation of immunopeptides from their parent proteins does not adhere to clear-cut rules, rather than being able to use known digestion patterns, every possible protein subsequence within human leukocyte antigen (HLA) class-specific length restrictions needs to be considered during sequence database searching. This leads to an inflation of the search space and results in lower spectrum annotation rates. Peptide-spectrum match (PSM) rescoring is a powerful enhancement of standard searching that boosts the spectrum annotation performance. We analyze 302,105 unique synthesized non-tryptic peptides from the ProteomeTools project on a timsTOF-Pro to generate a ground-truth dataset containing 93,227 MS/MS spectra of 74,847 unique peptides, that is used to fine-tune the deep learning-based fragment ion intensity prediction model Prosit. We demonstrate up to 3-fold improvement in the identification of immunopeptides, as well as increased detection of immunopeptides from low input samples.

Machine learning-based peptide-spectrum match rescoring opens up the immunopeptidome.

Immunopeptidomics is a key technology in the discovery of targets for immunotherapy and vaccine development. However, identifying immunopeptides remains challenging due to their non-tryptic nature, which results in distinct spectral characteristics. Moreover, the absence of strict digestion rules leads to extensive search spaces, further amplified by the incorporation of somatic mutations, pathogen genomes, unannotated open reading frames, and post-translational modifications. This inflation in search space leads to an increase in random high-scoring matches, resulting in fewer identifications at a given false discovery rate. Peptide-spectrum match rescoring has emerged as a machine learning-based solution to address challenges in mass spectrometry-based immunopeptidomics data analysis. It involves post-processing unfiltered spectrum annotations to better distinguish between correct and incorrect peptide-spectrum matches. Recently, features based on predicted peptidoform properties, including fragment ion intensities, retention time, and collisional cross section, have been used to improve the accuracy and sensitivity of immunopeptide identification. In this review, we describe the diverse bioinformatics pipelines that are currently available for peptide-spectrum match rescoring and discuss how they can be used for the analysis of immunopeptidomics data. Finally, we provide insights into current and future machine learning solutions to boost immunopeptide identification.

Proteomic comparison between non-purified cerebrospinal fluid and cerebrospinal fluid-derived extracellular vesicles from patients with Alzheimer's, Parkinson's and Lewy body dementia.

Dementia is a leading cause of death worldwide, with increasing prevalence as global life expectancy increases. The most common neurodegenerative disorders are Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD). With this study, we took an in-depth look at the proteome of the (non-purified) cerebrospinal fluid (CSF) and the CSF-derived extracellular vesicles (EVs) of AD, PD, PD-MCI (Parkinson's disease with mild cognitive impairment), PDD and DLB patients analysed by label-free mass spectrometry. This has led to the discovery of differentially expressed proteins that may be helpful for differential diagnosis. We observed a greater number of differentially expressed proteins in CSF-derived EV samples (N = 276) compared to non-purified CSF (N = 169), with minimal overlap between both datasets. This finding suggests that CSF-derived EV samples may be more suitable for the discovery phase of a biomarker study, due to the removal of more abundant proteins, resulting in a narrower dynamic range. As disease-specific markers, we selected a total of 39 biomarker candidates identified in non-purified CSF, and 37 biomarker candidates across the different diseases under investigation in the CSF-derived EV data. After further exploration and validation of these proteins, they can be used to further differentiate between the included dementias and may offer new avenues for research into more disease-specific pharmacological therapeutics.

A short dasatinib and quercetin treatment is sufficient to reinstate potent adult neuroregenesis in the aged killifish.

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Open Modification Searching of SARS-CoV-2-Human Protein Interaction Data Reveals Novel Viral Modification Sites.

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus-host protein-protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification-mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus-host protein-protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus-host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.

A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics.

In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design. Therefore, we acquired a hybrid proteome mixture on different instrument platforms and in all currently available families of data acquisition. Here, we present a comprehensive Data-Dependent and Data-Independent Acquisition (DDA/DIA) dataset acquired using several of the most commonly used current day instrumental platforms. The dataset consists of over 700 LC-MS runs, including adequate replicates allowing robust statistics and covering over nearly 10 different data formats, including scanning quadrupole and ion mobility enabled acquisitions. Datasets are available via ProteomeXchange (PXD028735).

Characterising extracellular vesicles from individual low volume cerebrospinal fluid samples, isolated by SmartSEC.

Extracellular vesicles (EVs) are suggested to have a role in the progression of neurodegeneration, and are able to transmit pathological proteins from one cell to another. One of the biofluids from which EVs can be isolated is cerebrospinal fluid (CSF). However, so far, few studies have been performed on small volumes of CSF. Since pooling of patient samples possibly leads to the loss of essential individual patient information, and CSF samples are precious, it is important to have efficient techniques for the isolation of EVs from smaller volumes. In this study, the SmartSEC HT isolation kit from System Biosciences has been evaluated for this purpose. The SmartSEC HT isolation kit was used for isolation of EVs from 500 µL starting volumes of CSF, resulting in two possible EV fractions of 500 µL. Both fractions were characterised and compared to one another using a whole range of characterisation techniques. Results indicated the presence of EVs in both fractions, albeit fraction 1 showed more reproducible results over the different characterisation methods. For example, CMG (CellMask Green membrane stain) fluorescence nanotracking analysis (NTA), ExoView, and the particles/µg ratio demonstrated a clear difference between fraction 1 and 2, where fraction 1 came out as the one where most EVs were eluted with the least contamination. In the other methods, this difference was less noticeable. We successfully performed complementary characterisation tests using only 500 µL of CSF starting volume, and, conclude that fraction 1 consisted of sufficiently pure EVs for further biomarker studies. This means that future EV extractions may be based upon smaller CSF quantities, such as from individual patients. In that way, patient samples do not have to be pooled and individual patient information can be included in forthcoming studies, potentially linking EV content, size and distribution to individualised neurological diagnoses.

Identification of Non-Canonical Translation Products in C. elegans Using Tandem Mass Spectrometry.

Transcriptome and ribosome sequencing have revealed the existence of many non-canonical transcripts, mainly containing splice variants, ncRNA, sORFs and altORFs. However, identification and characterization of products that may be translated out of these remains a challenge. Addressing this, we here report on 552 non-canonical proteins and splice variants in the model organism C. elegans using tandem mass spectrometry. Aided by sequencing-based prediction, we generated a custom proteome database tailored to search for non-canonical translation products of C. elegans. Using this database, we mined available mass spectrometric resources of C. elegans, from which 51 novel, non-canonical proteins could be identified. Furthermore, we utilized diverse proteomic and peptidomic strategies to detect 40 novel non-canonical proteins in C. elegans by LC-TIMS-MS/MS, of which 6 were common with our meta-analysis of existing resources. Together, this permits us to provide a resource with detailed annotation of 467 splice variants and 85 novel proteins mapped onto UTRs, non-coding regions and alternative open reading frames of the C. elegans genome.

Ion Mobility Coupled to a Time-of-Flight Mass Analyzer Combined With Fragment Intensity Predictions Improves Identification of Classical Bioactive Peptides and Small Open Reading Frame-Encoded Peptides.

Bioactive peptides exhibit key roles in a wide variety of complex processes, such as regulation of body weight, learning, aging, and innate immune response. Next to the classical bioactive peptides, emerging from larger precursor proteins by specific proteolytic processing, a new class of peptides originating from small open reading frames (sORFs) have been recognized as important biological regulators. But their intrinsic properties, specific expression pattern and location on presumed non-coding regions have hindered the full characterization of the repertoire of bioactive peptides, despite their predominant role in various pathways. Although the development of peptidomics has offered the opportunity to study these peptides in vivo, it remains challenging to identify the full peptidome as the lack of cleavage enzyme specification and large search space complicates conventional database search approaches. In this study, we introduce a proteogenomics methodology using a new type of mass spectrometry instrument and the implementation of machine learning tools toward improved identification of potential bioactive peptides in the mouse brain. The application of trapped ion mobility spectrometry (tims) coupled to a time-of-flight mass analyzer (TOF) offers improved sensitivity, an enhanced peptide coverage, reduction in chemical noise and the reduced occurrence of chimeric spectra. Subsequent machine learning tools MS2PIP, predicting fragment ion intensities and DeepLC, predicting retention times, improve the database searching based on a large and comprehensive custom database containing both sORFs and alternative ORFs. Finally, the identification of peptides is further enhanced by applying the post-processing semi-supervised learning tool Percolator. Applying this workflow, the first peptidomics workflow combined with spectral intensity and retention time predictions, we identified a total of 167 predicted sORF-encoded peptides, of which 48 originating from presumed non-coding locations, next to 401 peptides from known neuropeptide precursors, linked to 66 annotated bioactive neuropeptides from within 22 different families. Additional PEAKS analysis expanded the pool of SEPs on presumed non-coding locations to 84, while an additional 204 peptides completed the list of peptides from neuropeptide precursors. Altogether, this study provides insights into a new robust pipeline that fuses technological advancements from different fields ensuring an improved coverage of the neuropeptidome in the mouse brain.

PRiSM: A prototype for exhaustive, restriction-free database searching for mass spectrometry-based identification.

RATIONALE: The current methods for identifying peptides in mass spectral product ion data still struggle to do so for the majority of spectra. Based on the experimental setup and other assumptions, such methods restrict the search space to speed up computations, but at the cost of creating blind spots. The proteomics community would greatly benefit from a method that is capable of covering the entire search space without using any restrictions, thus establishing a baseline for identification. METHODS: We conceived the "mass pattern paradigm" (MPP) that enables the creation of such an identification method, and we implemented it into a prototype database search engine "PRiSM" (PRotein-Spectrum Matching). We then assessed its operational characteristics by applying it to publicly available high-precision mass spectra of low and high identification difficulty. We used those characteristics to gain theoretical insights into trade-offs between sensitivity and speed when trying to establish a baseline for identification. RESULTS: Of 100 low difficulty spectra, PRiSM and SEQUEST agree on 84 identifications (of which 75 are statistically significant). Of 15 of 100 spectra not identified in a previous study (using SEQUEST), 13 are considered reliable after visual inspection and represent 3 proteins (out of 9 in total) not detected previously. CONCLUSIONS: Despite leaving noise intact, the simple PRiSM prototype can make statistically reliable identifications, while controlling the false discovery rate by fitting a null distribution. It also identifies some spectra previously unidentifiable in an "extremely open" SEQUEST search, paving the way to establishing a baseline for identification in proteomics.

Urinary Protein Biomarker Panel for the Diagnosis of Antibody-Mediated Rejection in Kidney Transplant Recipients.

INTRODUCTION: Antibody-mediated rejection (ABMR) impacts kidney allograft outcome. The diagnosis is made based on findings from invasive kidney transplant biopsy specimens. The aim of this study was to identify a noninvasive urinary protein biomarker for ABMR after kidney transplantation. METHODS: We performed a multicenter case-control study to identify a urinary biomarker for ABMR (training cohort, n = 249) and an independent, prospective multicenter cohort study for validation (n = 391). We used concomitant biopsies to classify the samples according to the Banff classification. After untargeted protein identification and quantification, we used a support vector machine to train the model in the training cohort. The primary endpoint was the diagnostic accuracy of the urinary biomarker for ABMR in the validation cohort. RESULTS: We identified a set of 10 urinary proteins that accurately discriminated patients with (n = 60) and without (n = 189) ABMR in the training cohort with an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.96-1.00). The diagnostic accuracy was maintained in the validation cohort (AUC, 0.88; 95% CI, 0.8-0.93) for discriminating the presence (n = 43) from the absence (n = 348) of ABMR. The negative predictive value of the 10-protein marker set for exclusion of ABMR was 0.99, and the positive predictive value was 0.33. The diagnostic accuracy was independent of the reason for performing the biopsy, time after transplantation, and better than the accuracy of gross proteinuria (AUC, 0.76). CONCLUSIONS: We identified and validated a urinary protein biomarker set that can be used to exclude ABMR.

Implementation of MALDI Mass Spectrometry Imaging in Cancer Proteomics Research: Applications and Challenges.

Studying the proteome-the entire set of proteins in cells, tissues, organs and body fluids-is of great relevance in cancer research, as differential forms of proteins are expressed in response to specific intrinsic and extrinsic signals. Discovering protein signatures/pathways responsible for cancer transformation may lead to a better understanding of tumor biology and to a more effective diagnosis, prognosis, recurrence and response to therapy. Moreover, proteins can act as a biomarker or potential drug targets. Hence, it is of major importance to implement proteomic, particularly mass spectrometric, approaches in cancer research, to provide new crucial insights into tumor biology. Recently, mass spectrometry imaging (MSI) approaches were implemented in cancer research, to provide individual molecular characteristics of each individual tumor while retaining molecular spatial distribution, essential in the context of personalized disease management and medicine.

Determination of variability due to biological and technical variation in urinary extracellular vesicles as a crucial step in biomarker discovery studies.

Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. A crucial step in biomarker discovery studies is the determination of the variation parameters to perform a sample size calculation. In this way, a biomarker discovery study with sufficient statistical power can be performed to obtain biologically significant biomarkers. Here, a variation study was performed on both the protein and lipid content of urinary EVs of healthy individuals, aged between 52 and 69 years. Ultrafiltration (UF) in combination with size exclusion chromatography (SEC) was used to isolate the EVs from urine. Different experimental variation set-ups were used in this variation study. The calculated standard deviations (SDs) of the 90% least variable peptides and lipids did not exceed 2 and 1.2, respectively. These parameters can be used in a sample size calculation for a well-designed biomarker discovery study at the cargo of EVs.

Beyond Genes: Re-Identifiability of Proteomic Data and Its Implications for Personalized Medicine.

The increasing availability of high throughput proteomics data provides us with opportunities as well as posing new ethical challenges regarding data privacy and re-identifiability of participants. Moreover, the fact that proteomics represents a level between the genotype and the phenotype further exacerbates the situation, introducing dilemmas related to publicly available data, anonymization, ownership of information and incidental findings. In this paper, we try to differentiate proteomics from genomics data and cover the ethical challenges related to proteomics data sharing. Finally, we give an overview of the proposed solutions and the outlook for future studies.

Multiple solvent elution, a method to counter the effects of coelution and ion suppression in LC-MS analysis in bottom up proteomics.

On average a human cell type expresses around 10,000 different protein coding genes synthesizing all the different molecular forms of the protein product (proteoforms) found in a cell. In a typical shotgun bottom up proteomic approach, the proteins are enzymatically cleaved, producing several 100,000 s of different peptides that are analyzed with liquid chromatography-tandem mass spectrometry (LC-MSMS). One of the major consequences of this high sample complexity is that coelution of peptides cannot be avoided. Moreover, low abundant peptides are difficult to identify as they have a lower chance of being selected for fragmentation due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomic analysis where peptides are selected for fragmentation analysis one-by-one as they elute from the column. In the current study we explore a simple novel approach that has the potential to counter some of the effect of coelution of peptides and improves the number of peptide identifications in a bottom-up proteomic analysis. In this method, peptides from a HeLa cell digest were eluted from the reverse phase column using three different elution solvents (acetonitrile, methanol and acetone) in three replicate reversed phase LC-MS/MS shotgun proteomic analysis. Results were compared with three technical replicates using the same solvent, which is common practice in proteomic analysis. In total, we see an increase of up to 10% in unique protein and up to 30% in unique peptide identifications from the combined analysis using different elution solvents when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common in all three LC-MS analyses in our approach is only 23% compared to 50% in the replicates using the same solvent. The method presented here thus provides an easy to implement method to significantly reduce the effects of coelution and ion suppression of peptides and improve protein coverage in shotgun proteomics. Data are available via ProteomeXchange with identifier PXD011908.

MALDI Mass Spectrometry Imaging Linked with Top-Down Proteomics as a Tool to Study the Non-Small-Cell Lung Cancer Tumor Microenvironment.

Advanced non-small-cell lung cancer (NSCLC) is generally linked with a poor prognosis and is one of the leading causes of cancer-related deaths worldwide. Since only a minority of the patients respond well to chemotherapy and/or targeted therapies, immunotherapy might be a valid alternative in the lung cancer treatment field, as immunotherapy attempts to strengthen the body's own immune response to recognize and eliminate malignant tumor cells. However, positive response patterns to immunotherapy remain unclear. In this study, we demonstrate how immune-related factors could be visualized from single NSCLC tissue sections (Biobank@UZA) while retaining their spatial information by using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), in order to unravel the molecular profile of NSCLC patients. In this way, different regions in lung cancerous tissues could be discriminated based on the molecular composition. In addition, we linked visualization (MALDI MSI) and identification (based on liquid chromatography higher resolution mass spectrometry) of the molecules of interest for the correct biological interpretation of the observed molecular differences within the area in which these molecules are detected. This is of major importance to fully understand the underlying molecular profile of the NSCLC tumor microenvironment.

QCQuan: A Web Tool for the Automated Assessment of Protein Expression and Data Quality of Labeled Mass Spectrometry Experiments.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on mixed models can use large amounts of (computing) time. MS-based proteomics laboratories are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at qcquan.net ) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.

Identification of fukinolic acid from Cimicifuga heracleifolia and its derivatives as novel antiviral compounds against enterovirus A71 infection.

Human enterovirus 71 (EV-A71) infections cause a wide array of diseases ranging from diarrhoea and rashes to hand-foot-and-mouth disease and, in rare cases, severe neurological disorders. No specific antiviral drug therapy is currently available. Extracts from 75 Chinese medicinal plants selected for antiviral activity based on the Chinese pharmacopeia and advice from traditional Chinese medicine clinicians were tested for activity against EV-A71. The aqueous extract of the rhizome of Cimicifuga heracleifolia (Sheng Ma) and Arnebia euchroma (Zi Cao) showed potent antiviral activity. The active fractions were isolated by bioassay-guided purification, and identified by a combination of high-resolution mass spectrometry and nuclear magnetic resonance. Fukinolic acid and cimicifugic acid A and J, were identified as active anti-EV-A71 compounds for C. heracleifolia, whereas for A. euchroma, two caffeic acid derivatives were tentatively deduced. Commercially available fukinolic acid analogues such as L-chicoric acid and D-chicoric also showed in vitro micromolar activity against EV-A71 lab-strain and clinical isolates.

The challenges of peptidomics in complementing proteomics in a clinical context.

Naturally occurring peptides, including growth factors, hormones, and neurotransmitters, represent an important class of biomolecules and have crucial roles in human physiology. The study of these peptides in clinical samples is therefore as relevant as ever. Compared to more routine proteomics applications in clinical research, peptidomics research questions are more challenging and have special requirements with regard to sample handling, experimental design, and bioinformatics. In this review, we describe the issues that confront peptidomics in a clinical context. After these hurdles are (partially) overcome, peptidomics will be ready for a successful translation into medical practice.