Impairment of lipid homeostasis causes lysosomal accumulation of endogenous protein aggregates through ESCRT disruption.

Protein aggregation increases during aging and is a pathological hallmark of many age-related diseases. Protein homeostasis (proteostasis) depends on a core network of factors directly influencing protein production, folding, trafficking, and degradation. Cellular proteostasis also depends on the overall composition of the proteome and numerous environmental variables.

Targeting the aminopeptidase ERAP enhances antitumor immunity by disrupting the NKG2A-HLA-E inhibitory checkpoint.

The aminopeptidase, endoplasmic reticulum aminopeptidase 1 (ERAP1), trims peptides for loading into major histocompatibility complex class I (MHC class I), and loss of this activity has broad effects on the MHC class I peptidome. Here, we investigated the impact of targeting ERAP1 in immune checkpoint blockade (ICB), as MHC class I interactions mediate both activating and inhibitory functions in antitumor immunity. Loss of ERAP sensitized mouse tumor models to ICB, and this sensitivity depended on CD8 T cells and natural killer (NK) cells.

Plasma and Kidney Proteome Profiling Combined with Laser Capture Microdissection Reveal Large Increases in Immunoglobulins with Age.

One of the main hallmarks of aging is aging-associated inflammation, also known as inflammaging. In this study, by comparing plasma and kidney proteome profiling of young and old mice using LC-MS profiling, we discovered that immunoglobulins are the proteins that exhibit the highest increase with age. This observation seems to have been disregarded because conventional proteome profiling experiments typically overlook the expression of high-abundance proteins or employ depletion methods to remove them before LC-MS analysis.

A data analysis framework for combining multiple batches increases the power of isobaric proteomics experiments.

We present a framework for the analysis of multiplexed mass spectrometry proteomics data that reduces estimation error when combining multiple isobaric batches. Variations in the number and quality of observations have long complicated the analysis of isobaric proteomics data. Here we show that the power to detect statistical associations is substantially improved by utilizing models that directly account for known sources of variation in the number and quality of observations that occur across batches.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types.

Triple-threat quantitative multiplexed plasma proteomics analysis on immune complex disease MRL-lpr mice.

Systemic lupus erythematosus is a common autoimmune inflammatory disease which is associated with increases in autoantibodies and immune complexes that deposit in the kidney. The MRL-lpr mouse is a common mouse model used for the study of lupus and immune complex glomerulonephritis but very little is known about the plasma proteome changes in this model. We performed in-depth quantitative proteome profiling on MRL-lpr and control (strain MpJ) mice to investigate the changes in the proteome, immunoglobulins and their glycoproteome as well as protein and immune complexes.

Tuning DO:DM Ratios Modulates MHC Class II Immunopeptidomes.

Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen-presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described.

Exercise plasma boosts memory and dampens brain inflammation via clusterin.

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus, yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation.

An Integrated Genomic, Proteomic, and Immunopeptidomic Approach to Discover Treatment-Induced Neoantigens.

All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires - particularly novel MHC presentation of mutation-bearing peptides (neoantigens) - can be potent targets of anti-tumor immune responses.

Automated 16-Plex Plasma Proteomics with Real-Time Search and Ion Mobility Mass Spectrometry Enables Large-Scale Profiling in Naked Mole-Rats and Mice.

Performing large-scale plasma proteome profiling is challenging due to limitations imposed by lengthy preparation and instrument time. We present a fully automated multiplexed proteome profiling platform (AutoMP3) using the Hamilton Vantage liquid handling robot capable of preparing hundreds to thousands of samples. To maximize protein depth in single-shot runs, we combined 16-plex Tandem Mass Tags (TMTpro) with high-field asymmetric waveform ion mobility spectrometry (FAIMS Pro) and real-time search (RTS).

Physiological blood-brain transport is impaired with age by a shift in transcytosis.

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport.

Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins.

Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1's N-terminal domain need not release from the rest of the protein for efficient cholesterol export.

A novel antibody-cell conjugation method to enhance and characterize cytokine-induced killer cells.

Background: Cytokine-induced killer (CIK) cells are an ex vivo-expanded cellular therapy product with potent anti-tumor activity in a subset of patients with solid and hematologic malignancies. We hypothesize that directing CIK cells to a specific tumor antigen will enhance CIK cell anti-tumor cytotoxicity.

Methods: We present a newly developed method for affixing antibodies directly to cell surface proteins.

Predicting HLA class II antigen presentation through integrated deep learning.

Accurate prediction of antigen presentation by human leukocyte antigen (HLA) class II molecules would be valuable for vaccine development and cancer immunotherapies. Current computational methods trained on in vitro binding data are limited by insufficient training data and algorithmic constraints. Here we describe MARIA (major histocompatibility complex analysis with recurrent integrated architecture; https://maria.

Acute myeloid leukemia immunopeptidome reveals HLA presentation of mutated nucleophosmin.

Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapeutic approaches.

TagGraph reveals vast protein modification landscapes from large tandem mass spectrometry datasets.

Although mass spectrometry is well suited to identifying thousands of potential protein post-translational modifications (PTMs), it has historically been biased towards just a few. To measure the entire set of PTMs across diverse proteomes, software must overcome the dual challenges of covering enormous search spaces and distinguishing correct from incorrect spectrum interpretations. Here, we describe TagGraph, a computational tool that overcomes both challenges with an unrestricted string-based search method that is as much as 350-fold faster than existing approaches, and a probabilistic validation model that we optimized for PTM assignments.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr) and a phenylalanyl ( MmPhe) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization.

T-Cell Immunopeptidomes Reveal Cell Subtype Surface Markers Derived From Intracellular Proteins.

Immunopeptidomes promise novel surface markers as ideal immunotherapy targets, but their characterization by mass spectrometry (MS) remains challenging. Until recently, cell numbers exceeding 10 were needed to survey thousands of HLA ligands. Such limited analytical sensitivity has historically constrained the types of clinical specimens that can be evaluated to cell cultures or bulk tissues.

Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens.

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules.

AFFIRM--a multiplexed immunoaffinity platform that combines recombinant antibody fragments and LC-SRM analysis.

Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single-plexed and multiplexed experiments in a concentration range of 5-1000 ng/mL.

Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support.

The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the dock'n'flash technology. In more detail, we have successfully explored the possibility to expand the genetic code of E.

Protein expression profiling of formalin-fixed paraffin-embedded tissue using recombinant antibody microarrays.

Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures.

Grading breast cancer tissues using molecular portraits.

Tumor progression and prognosis in breast cancer patients are difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessments of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, determined using a novel affinity proteomics approach.

Epitope-specificity of recombinant antibodies reveals promiscuous peptide-binding properties.

Protein-peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein-peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody-peptide interaction characteristics, by combining large-scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes.