A novel strategy for the targeted analysis of protein and peptide metabolites

The detection and quantitation of exogenously administered biological macromolecules (e.g. vaccines, peptide and protein therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related compounds. We describe a method that incorporates stable isotope labeling of the compound of interest allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low molecular weight fragment ions produced during MS/MS that distinguish isotopically labelled material from related endogenous compounds. All isotopically labelled substances can be selected using this scanning technique for further analysis whilst other unlabelled and irrelevant substances are ignored. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α-synuclein in serum and the brain

Kinetics of antigen expression and epitope presentation during virus infection

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.NHMRC (National Health and Medical Research Council of Australia

Targeted analysis of antigen presentation using mass spectrometry

© 2012 Dr. Chor Teck TanThe immune system constantly samples the cellular proteome through the process of antigen presentation. T cells specifically recognize cellular peptides bound to the molecules encoded by the major histocompatibility complex (MHC) that are expressed on the surface of virtually all nucleated cells. In type 1 diabetes (T1D), the immune system mistakenly recognises peptides derived from normal cellular (self) proteins, triggering an attack against the insulin secreting pancreatic β cells, leading to β cell loss and insulin deficiency. Recognition of MHC-bound peptides is critical for both the initiation and progression of the disease. Identifying the peptide epitopes targeted during diabetes remains a critical step in defining the molecular basis of the disease. Human patient testing in combination with the use of the Non-Obese Diabetic (NOD) mouse which develops spontaneous autoimmune diabetes has lead to the characterisation of multiple antigens that are recognised by autoreactive lymphocytes. However, the inherent complexities of antigen processing and presentation, the polygenic nature of the disease, the extensive polymorphism of the MHC and the technical hurdles in working with T cells have made epitope discovery and quantitation challenging. This study examined the peptides selected for presentation by MHC class I molecules on the surface of β cells under normal and inflammatory conditions by mass spectrometry and compared these to those peptides found on the surface of primary tissue (spleen and thymus) from NOD mice. Over 2000 MHC-bound peptides, 1100 of these presented by β cells grown under normal conditions or following exposure to IFNγ, were identified. These include sequences from a number of known autoantigens, however, many previously characterised T cell epitopes in the NOD mouse were not observed. This suggested that many of these peptides were present at very low levels. I therefore sought to develop a more sensitive approach to target analysis towards these species. A novel approach was developed to detect known epitopes and directly quantitate their presentation levels. This methodology involved combining the peptide-MHC complex immunoaffinity purification used for the β cell repertoire analysis followed by interrogation of the peptide sample using a mass spectrometry-based technique called multiple reaction monitoring (MRM). This approach incorporates epitope specific information to target the analysis towards the peptide of choice leading to large gains in sensitivity and specificity. When used in combination with an isotopic peptide standard (AQUA peptide) more accurate quantitation can be achieved. I initially tested and optimised the method by quantitating the ovalbumin derived SIINFEKL epitope from cells that express physiological levels of this antigen (EG-7 cells). In vitro quantitation of SIINFEKL (728 copies per cell) from EG-7 cells and in vivo quantitation of cross-presented SIINFEKL in mouse spleen (4 fmol) were successfully demonstrated. The multiplexing ability of the quantitative workflow was also demonstrated by simultaneous monitoring of 9 peptides purified from antigen presenting cells. The robust and absolute quantitative nature of this newly proposed workflow in addition to its ability to multiplex, allowing for high-throughput analysis of multiple epitopes in a single sample, are superior to existing cell based epitope quantitation techniques. The quantitative workflow was applied in the study of an immunodominant H-2Kd-restricted epitope IGRP206-214. Quantitation of IGRP206-214 revealed low level presentation (~25 complexes/cell) on NIT-1 cells following IFNγ treatment compared with the simultaneous presentation of the endogenously processed H-2Kd-restricted peptide JAK-1355-363 (~15000 copies per cell). The potential to perform quantitative studies of disease related epitope presentation in vivo was also further demonstrated using pancreatic lymph nodes and IGRP206-214. These studies foreshadow future large scale epitope presentation and vaccine efficacy studies from live organisms and tissues. Ultimately, the application of modern mass spectrometry-based workflows will progress our efforts and success in immunological therapy of diabetes and other diseases

Thiol-specific silicon-containing conjugating reagent: β-silyl alkynyl carbonyl compounds

Site-specific modification of thiol-containing biomolecules has been recognized as a versatile and powerful strategy for probing our biological systems and discovering novel therapeutics. The addition of lipophilic silicon moiety opens up new avenues for multi-disciplinary research with broad applications in both the medicinal and material sciences. However, adhering to the strict biocompatibility requirements, and achieving the introduction of labile silicon handle and high chemo-selectivity have been formidable. In this paper, we report silicon-based conjugating reagents including β-trialkylsilyl and silyl ether-tethered alkynones that selectively react with thiols under physiological conditions. The pH-neutral, metal-free and additive-free reaction yields stable products with broad substrate compatibility and full retention of silicon handles in most cases. Besides simple aliphatic and aromatic thiols, this approach is applicable in the labeling of thiols present in proteins, sugars and payloads, thereby expanding the toolbox of thiol conjugation.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Nanyang Technological UniversitySubmitted/Accepted versionWe gratefully acknowledge the financial support from Distinguished University Professor grant (Nanyang Technological University), AcRF Tier1 grant from the Ministry of Education of Singapore (RT14/20) and the Agency for Science, Technology and Research (A*STAR) under its MTC Individual Research Grant (M21K2c0114) and RIE2025 MTC Programmatic Fund (M22K9b0049) for TPL. We also thank “High-end Talents” start up grant (Y0720327K13) from Fujian Normal University for ST

Kinetics of Antigen Expression and Epitope Presentation during Virus Infection

<div><p>Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.</p> </div

Kinetics of VACV antigen presentation.

<p>A) 1×10⁸ DC2.4 cells/time point were infected with 5 pfu of VACV strain WR (or mock treated as time 0) and incubated for 0.5, 3.5, 6.5, 9.5 or 12.5 hours. MHC-peptide complexes were eluted at each time and epitope levels monitored by LC-MRM. Data show the copies of each epitope per cell. B) Epitope data from (A) expressed as the percentage of maximum levels alongside the relative kinetics of each source protein. C) CD8⁺ T cell immunodominance hierarchy of WR infection 7 days after infection as determined brief stimulation with the peptides shown and ICS for IFNγ. Data are the mean and SEM of 3 mice and are representative of multiple experiments.</p

Antigen presentation during virus infection is complex and cannot be described by a single epitope.

<p>DC2.4 cells were infected with VACV strain WR-NP-S-GFP (A) or WR (B) and presentation of MHC class I peptides detected at various times. A) K^b-SIINFEKL presentation levels determined by staining with mAb 25-D1.16 and flow cytometry. Data are representative multiple experiments. B) Presentation of bulk native VACV epitopes determined by incubation with VACV-immune splenocytes and detecting activation by staining for surface CD8⁺ and intracellular IFNγ. Means and SEM of triplicates are shown and the data are representative of two independent experiments.</p

Simultaneous detection of multiple viral epitopes by LC-MRM MS.

<p>A) Demonstration of multiplexed detection of 8 VACV K^b-binding epitopes. A mixture of 100 fmol of each synthetic peptide was loaded and analysed directly by LC-MRM using a method to detect all peptides simultaneously. A single MRM transition per peptide is shown for clarity. B) Schematic of sample workflow. C) DC2.4 cells were pulsed with a 1 µM mixture of each VACV peptide, incubated for 1 hour and washed extensively to remove unbound peptide. Cells were subjected to MHC-peptide elution and each epitope detected by LC-MRM-MS (sum area of all MRM transitions per peptide is shown).</p

Cartoon depiction of the changes in vaccinia viral epitope presentation during infection.

<p>Circles represent infected cells at the indicated times post infection. Size is proportionate to the changing levels of MHC K^b during infection, relative to mock-infected cells.</p

VACV CD8⁺ T cell epitopes.

*<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003129#ppat.1003129-Yang1" target="_blank">[41]</a> Expression time based on cluster analysis of transcript levels during infection. E1.1 and E1.2 are early classes, with E1.1 expressed earlier and at higher levels that E1.2. PR (post replication), typically referred to as the late class.</p>**<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003129#ppat.1003129-Yang2" target="_blank">[43]</a> Earliest promoter associated with gene. E is early, I is intermediate.</p