Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer's Disease.

To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD).

Alzheimer proteopathic tau seeds are biochemically a forme fruste of mature paired helical filaments.

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils.

multiFLEX-LF: A Computational Approach to Quantify the Modification Stoichiometries in Label-Free Proteomics Data Sets.

In liquid-chromatography-tandem-mass-spectrometry-based proteomics, information about the presence and stoichiometry of protein modifications is not readily available. To overcome this problem, we developed multiFLEX-LF, a computational tool that builds upon FLEXIQuant, which detects modified peptide precursors and quantifies their modification extent by monitoring the differences between observed and expected intensities of the unmodified precursors. multiFLEX-LF relies on robust linear regression to calculate the modification extent of a given precursor relative to a within-study reference. multiFLEX-LF can analyze entire label-free discovery proteomics data sets in a precursor-centric manner without preselecting a protein of interest. To analyze modification dynamics and coregulated modifications, we hierarchically clustered the precursors of all proteins based on their computed relative modification scores. We applied multiFLEX-LF to a data-independent-acquisition-based data set acquired using the anaphase-promoting complex/cyclosome (APC/C) isolated at various time points during mitosis. The clustering of the precursors allows for identifying varying modification dynamics and ordering the modification events. Overall, multiFLEX-LF enables the fast identification of potentially differentially modified peptide precursors and the quantification of their differential modification extent in large data sets using a personal computer. Additionally, multiFLEX-LF can drive the large-scale investigation of the modification dynamics of peptide precursors in time-series and case-control studies. multiFLEX-LF is available at https://gitlab.com/SteenOmicsLab/multiflex-lf.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation.

Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C(CDH1)-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.

FLEXITau: Quantifying Post-translational Modifications of Tau Protein in Vitro and in Human Disease.

Tauopathies, including Alzheimer's disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as "hyperphosphorylated". Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies.

FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms.

We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this assay we combined our full-length expressed stable isotope-labeled protein for quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multikinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n × 80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3ß (GSK3ß) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3ß activity and permitting an advanced model for GSK3ß-mediated signaling.

A non-parametric cutout index for robust evaluation of identified proteins.

This paper proposes a novel, automated method for evaluating sets of proteins identified using mass spectrometry. The remaining peptide-spectrum match score distributions of protein sets are compared to an empirical absent peptide-spectrum match score distribution, and a Bayesian non-parametric method reminiscent of the Dirichlet process is presented to accurately perform this comparison. Thus, for a given protein set, the process computes the likelihood that the proteins identified are correctly identified. First, the method is used to evaluate protein sets chosen using different protein-level false discovery rate (FDR) thresholds, assigning each protein set a likelihood. The protein set assigned the highest likelihood is used to choose a non-arbitrary protein-level FDR threshold. Because the method can be used to evaluate any protein identification strategy (and is not limited to mere comparisons of different FDR thresholds), we subsequently use the method to compare and evaluate multiple simple methods for merging peptide evidence over replicate experiments. The general statistical approach can be applied to other types of data (e.g. RNA sequencing) and generalizes to multivariate problems.

SweetSEQer, simple de novo filtering and annotation of glycoconjugate mass spectra.

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.

A classifier based on accurate mass measurements to aid large scale, unbiased glycoproteomics.

Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses.

Diaminothiazoles modify Tau phosphorylation and improve the tauopathy in mouse models.

Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3ß. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3ß in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3ß to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting.

mz5: space- and time-efficient storage of mass spectrometry data sets.

Across a host of MS-driven-omics fields, researchers witness the acquisition of ever increasing amounts of high throughput MS data and face the need for their compact yet efficiently accessible storage. Addressing the need for an open data exchange format, the Proteomics Standards Initiative and the Seattle Proteome Center at the Institute for Systems Biology independently developed the mzData and mzXML formats, respectively. In a subsequent joint effort, they defined an ontology and associated controlled vocabulary that specifies the contents of MS data files, implemented as the newer mzML format. All three formats are based on XML and are thus not particularly efficient in either storage space requirements or read/write speed. This contribution introduces mz5, a complete reimplementation of the mzML ontology that is based on the efficient, industrial strength storage backend HDF5. Compared with the current mzML standard, this strategy yields an average file size reduction to ∼54% and increases linear read and write speeds ∼3-4-fold. The format is implemented as part of the ProteoWizard project and is available under a permissive Apache license. Additional information and download links are available from http://software.steenlab.org/mz5.

Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits.

Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of ß-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.

Quantitative profiling of posttranslational modifications of pathological tau via sarkosyl fractionation and mass spectrometry.

Tau protein aggregation is associated with posttranslational modifications (PTMs) in 75% of all dementia cases. The distribution of tau pathology and the presence of specific tau phosphorylation sites of interest are typically visualized and measured using antibodies. However, previous knowledge of the target epitopes is required. Additionally, antibodies can be used in a semi-quantitative manner but cannot be used to determine the absolute amount of tau or the extent of the modifications at specific sites or domains. Here we present a discovery assay that characterizes the global qualitative and quantitative tau modification landscape of a sample without a priori knowledge. Our workflow uses sarkosyl fractionation to extract the pathological tau species from sample-limited brain specimens, followed by mass spectrometry (MS) to characterize and quantify tau PTMs. The two-step MS-based proteomics approach includes an exploratory tau PTM analysis and a targeted full-length expressed stable isotope-labeled tau assay, which monitors specific unmodified tau peptides using a heavy isotope-labeled internal standard as a reference. This enables the absolute quantification of the respective tau peptides and the total tau amount in the sample, thus providing the modification extent of tau PTMs. This approach provides precise, comprehensive, qualitative and quantitative tau PTM profiling of the sample. It also enables the detailed molecular comparison of tau across multiple experiments, including a comparison between neurodegenerative diseases, stages of the disease, human patient heterogeneity and characterization of animal models. The approach is useful for studying the molecular features of pathological tau in neurodegeneration. The procedure requires 7-8 d and is suitable for users with expertise in targeted and untargeted MS-based protein analysis.

Longitudinal plasma proteomic analysis of 1117 hospitalized patients with COVID-19 identifies features associated with severity and outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by highly heterogeneous manifestations ranging from asymptomatic cases to death for still incompletely understood reasons. As part of the IMmunoPhenotyping Assessment in a COVID-19 Cohort study, we mapped the plasma proteomes of 1117 hospitalized patients with COVID-19 from 15 hospitals across the United States. Up to six samples were collected within ~28 days of hospitalization resulting in one of the largest COVID-19 plasma proteomics cohorts with 2934 samples. Using perchloric acid to deplete the most abundant plasma proteins allowed for detecting 2910 proteins. Our findings show that increased levels of neutrophil extracellular trap and heart damage markers are associated with fatal outcomes. Our analysis also identified prognostic biomarkers for worsening severity and death. Our comprehensive longitudinal plasma proteomics study, involving 1117 participants and 2934 samples, allowed for testing the generalizability of the findings of many previous COVID-19 plasma proteomics studies using much smaller cohorts.

Nonparametric Bayesian evaluation of differential protein quantification.

Arbitrary cutoffs are ubiquitous in quantitative computational proteomics: maximum acceptable MS/MS PSM or peptide q value, minimum ion intensity to calculate a fold change, the minimum number of peptides that must be available to trust the estimated protein fold change (or the minimum number of PSMs that must be available to trust the estimated peptide fold change), and the "significant" fold change cutoff. Here we introduce a novel experimental setup and nonparametric Bayesian algorithm for determining the statistical quality of a proposed differential set of proteins or peptides. By comparing putatively nonchanging case-control evidence to an empirical null distribution derived from a control-control experiment, we successfully avoid some of these common parameters. We then apply our method to evaluating different fold-change rules and find that for our data a 1.2-fold change is the most permissive of the plausible fold-change rules.

A screen for regulators of survival of motor neuron protein levels.

The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK-PI3K-AKT-GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.

Meta-analysis of published cerebrospinal fluid proteomics data identifies and validates metabolic enzyme panel as Alzheimer's disease biomarkers.

To develop therapies for Alzheimer's disease, we need accurate in vivo diagnostics. Multiple proteomic studies mapping biomarker candidates in cerebrospinal fluid (CSF) resulted in little overlap. To overcome this shortcoming, we apply the rarely used concept of proteomics meta-analysis to identify an effective biomarker panel. We combine ten independent datasets for biomarker identification: seven datasets from 150 patients/controls for discovery, one dataset with 20 patients/controls for down-selection, and two datasets with 494 patients/controls for validation. The discovery results in 21 biomarker candidates and down-selection in three, to be validated in the two additional large-scale proteomics datasets with 228 diseased and 266 control samples. This resulting 3-protein biomarker panel differentiates Alzheimer's disease (AD) from controls in the two validation cohorts with areas under the receiver operating characteristic curve (AUROCs) of 0.83 and 0.87, respectively. This study highlights the value of systematically re-analyzing previously published proteomics data and the need for more stringent data deposition.

Common mouse models of tauopathy reflect early but not late human disease.

BACKGROUND: Mouse models that overexpress human mutant Tau (P301S and P301L) are commonly used in preclinical studies of Alzheimer's Disease (AD) and while several drugs showed therapeutic effects in these mice, they were ineffective in humans. This leads to the question to which extent the murine models reflect human Tau pathology on the molecular level. METHODS: We isolated insoluble, aggregated Tau species from two common AD mouse models during different stages of disease and characterized the modification landscape of the aggregated Tau using targeted and untargeted mass spectrometry-based proteomics. The results were compared to human AD and to human patients that suffered from early onset dementia and that carry the P301L Tau mutation. RESULTS: Both mouse models accumulate insoluble Tau species during disease. The Tau aggregation is driven by progressive phosphorylation within the proline rich domain and the C-terminus of the protein. This is reflective of early disease stages of human AD and of the pathology of dementia patients carrying the P301L Tau mutation. However, Tau ubiquitination and acetylation, which are important to late-stage human AD are not represented in the mouse models. CONCLUSION: AD mouse models that overexpress human Tau using risk mutations are a suitable tool for testing drug candidates that aim to intervene in the early formation of insoluble Tau species promoted by increased phosphorylation of Tau.