Tobias Knaute

Building ProteomeTools based on a complete synthetic human proteome

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography–tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.

Quantification of the Epitope Diversity of HIV-1-Specific Binding Antibodies by Peptide Microarrays for Global HIV-1 Vaccine Development

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. Summary: Memory T cells have a rapid transcriptional program upon re-stimulation. Chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit this transcriptional memory in T cells.

Antibody Responses After Analytic Treatment Interruption in Human Immunodeficiency Virus-1-Infected Individuals on Early Initiated Antiretroviral Therapy

The examination of antibody responses in human immunodeficiency virus (HIV)-1-infected individuals in the setting of antiretroviral treatment (ART) interruption can provide insight into the evolution of antibody responses during viral rebound. In this study, we assessed antibody responses in 20 subjects in AIDS Clinical Trials Group A5187, wherein subjects were treated with antiretroviral therapy during acute/early HIV-1 infection, underwent analytic treatment interruption, and subsequently demonstrated viral rebound. Our data suggest that early initiation of ART arrests the maturation of HIV-1-specific antibody responses, preventing epitope diversification of antibody binding and the development of functional neutralizing capacity. Antibody responses do not appear permanently blunted, however, because viral rebound triggered the resumption of antibody maturation in our study. We also found that antibody responses measured by these assays did not predict imminent viral rebound. These data have important implications for the HIV-1 vaccine and eradication fields.

PROCAL: A Set of 40 Peptide Standards for Retention Time Indexing, Column Performance Monitoring, and Collision Energy Calibration

Beyond specific applications, such as the relative or absolute quantification of peptides in targeted proteomic experiments, synthetic spike‐in peptides are not yet systematically used as internal standards in bottom‐up proteomics. A number of retention time standards have been reported that enable chromatographic aligning of multiple LC–MS/MS experiments. However, only few peptides are typically included in such sets limiting the analytical parameters that can be monitored. Here, we describe PROCAL (ProteomeTools Calibration Standard), a set of 40 synthetic peptides that span the entire hydrophobicity range of tryptic digests, enabling not only accurate determination of retention time indices but also monitoring of chromatographic separation performance over time. The fragmentation characteristics of the peptides can also be used to calibrate and compare collision energies between mass spectrometers. The sequences of all selected peptides do not occur in any natural protein, thus eliminating the need for stable isotope labeling. We anticipate that this set of peptides will be useful for multiple purposes in individual laboratories but also aiding the transfer of data acquisition and analysis methods between laboratories, notably the use of spectral libraries.

High-Throughput Microarray Incubations Using Multi-Well Chambers.

Peptide microarrays are ideal tools for a variety of applications ranging from epitope mapping to immune monitoring. Here we present a method for high-throughput screening of biological samples using only standard microtiter plate equipment. Parallel incubation of a large number of samples with a small library of peptides is enabled by printing multiple identical mini-arrays on one microarray slide and further combining four slides to yield an incubation frame possessing the dimensions of a 96-well microtiter plate. Applying conventional lab equipment such as ELISA washers, hundreds of samples can be processed in 1 day yielding approx. 200 data points in triplicates per sample.

PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation.

Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells.

Peptide libraries for comprehensive coverage of the tumor mutanome in immune monitoring and immunotherapy

Many cancers are associated with functional mutations which now can be readily identified using new DNA sequencing methods. Sequence information is now used for the generation of personalized cancer vaccines with promising clinical benefit [1]. Intelligent ways to select the relevant, tumor-specific protein-coding mutations are applied to filter the large number of identified mutation to a manageable number. Besides, there is an increasing data pool of mutations, both germline and somatic, the latter frequently even with attached additional information on the tissue and histology. Even though the use of actual sequence of an individual patient is the optimal basis for treatment and immune monitoring it is quite expensive relatively slow to obtain this information. As a fast tool with broad applicability across patient cohorts peptide libraries could find an application in cases where the individual sequence is not (yet) available. Pools of antigen peptides are frequently for T cell stimulation in T cell assays and even for immune stimulation for treatment. Such peptide libraries are well suited for presenting sequence diversity. However, libraries used today normally consist of overlapping peptides of a wild-type antigen sequence. The concept was already extended for the presentation of sequence diversity in antigens of pathogens as HIV where sequence diversity between individual viruses poses many challenges. For the ENV protein an increase in sequence coverage from 10 % to 34 % was achieved. Due to the limited number of peptides a complex filtering of sequence data has to be applied to enrich the library with peptides carrying different tumor-specific and ideally functionally relevant mutations. Our aim is to generate peptide libraries for specific antigens which represent a generic cancer mutanome applicable to a large number of individual patients based on available databases as the catalogue of somatic mutations in cancer and epitope information from the immune epitope database and prediction algorithms. One library type can contain the full length sequence of the antigen enriched by peptides representing overlapping scans for relevant mutations. Alternatively, known or predicted epitopes can be selected and the mutated epitopes can be included. However, mutations can lead to the formation of new epitopes and mutated epitopes can escape from MHC-binding. Epitope prediction can help to account for these effects. We have built a pipeline for the efficient generation of such libraries which will be manufactured.

Universal mapping of humoral immune response using a versatile high-content and high-density peptide microarray

Background Humoral immune responses are often the hallmark of efficient vaccines. The recent RV144 vaccine trial has turned attention to the stimulation of humoral immune response as a potential mode of action for HIV vaccines. Therefore, detailed monitoring of antibody reactivities in patient specimens before and after vaccination is crucial. The determination of these reactivities on a sub-protein level provides information on the site of antigen/antibody interaction. In contrast to assays relying on whole antigens such as ELISA, peptide microarrays are efficient tools to deliver such information. Besides, complex peptide libraries can cover HIV sequence diversity, a special challenge provided by this virus.