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To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells. ISSN:1535-3893 ISSN:1535-3907

The degree of genomic relatedness influences many social behaviours in humans and animals. Mice, as nocturnal rodents, take advantage of their excellent sense of smell to identify kin, conspecifics and members of other species via body odours or urine scent marks. Selected groups of peptides, including those that are presented by major histocompatibility complex (MHC) proteins, are recognised by mouse chemosensory neurons with very high sensitivity (up to 10^-14 M). MHC peptide ligands have been proposed to influence social interactions in many species, including mice and humans. However, the lack of knowledge about such peptides in natural sources accessible for nasal recognition has been a major barrier for this hypothesis. Here we analyse urinary peptides from selected inbred strains of Mus musculus with respect to genotype-related differences. We discover many abundant peptides with single amino acid variations (SAVs) corresponding to genomic nucleotide variations. The polymorphism of major urinary proteins (MUPs) is reflected by variations in prominent urinary peptides, and further types of genomic variability are predicted to affect the urinary peptidome. Several peptides with MHC binding motifs are readily detectable in urine at concentrations of up to 10^-6 M, but their occurrence is independent of the MHC. On the other hand, by comparing mouse strains expressing chicken ovalbumin together with, or without, functional MHC class I molecules, we provide conclusive evidence that the prototypic H2-Kb ligand SIINFEKL (derived from ovalbumin) exists in urine at a concentration of 4 x 10^-12 M in an MHC-dependent manner. In cooperation with neurobiologists, we show that chemoreceptive neurons in the vomeronasal organ detect and discriminate SAV peptides as well as SIINFEKL. The present thesis fills a critical gap in the hypothesis that MHC peptide ligands may serve as signals of individuality in mice by providing the first evidence for such a ligand occurring in urine in an MHC-dependent manner. However, at the same time, this study profoundly questions the same hypothesis by showing, first, that the respective peptide concentrations in urine are about eight orders of magnitude lower than those previously applied in behavioural experiments and second, by demonstrating abundant MHC-independent MHC motif peptides. I therefore suppose that the copious peptides which reflect genomic individuality independent of MHC genes are far more important in determining social behaviours than the MHC-dependent ones. Collectively, urinary peptides represent a real-time sampling of the expressed genome available for chemosensory assessment by other individuals. Der genetische Verwandtschaftsgrad wirkt sich auf viele soziale Verhaltensweisen von Mensch und Tier aus. Als nachtaktive Nagetiere nutzen Mäuse ihren hervorragenden Geruchsinn, um Familienangehörige, Artgenossen und Vertreter fremder Arten zu identifizieren. Die benötigten Informationen können sie dabei auch aus Urinduftmarken gewinnen. Chemosensorische Nervenzellen der Maus erkennen bestimmte Peptidsorten, einschließlich denjenigen, die von den Proteinen des Haupthistokompatibilitätskomplexes (major histocompatibility complex, MHC) präsentiert werden, mit sehr hohen Sensitivitäten von bis zu 10^-14 M. Es wurde postuliert, dass MHC-Peptidliganden soziale Beziehungen bei vielen verschiedenen Arten, einschließlich Maus und Mensch, beeinflussen. Ein grundlegendes Problem bei dieser Hypothese ist jedoch das fehlende Wissen über solche Peptide in Duftquellen, die natürlicherweise für die Geruchserkennung zur Verfügung stehen. Hier untersuchen wir Urinpeptide von ausgewählten Inzuchtstämmen von Mus musculus in Bezug auf Unterschiede, die mit dem Genotyp zusammenhängen. Dabei entdecken wir viele häufige Peptide mit Einzelaminosäurevariationen (EAVs), welche genomischen Nukleotidvariationen entsprechen. Der Polymorphismus der Major Urinary Proteine (MUPs) spiegelt sich in Unterschieden in prominenten Urinpeptiden wider, und es kann vorhergesagt werden, dass weitere Arten genomischer Vielfalt das Urinpeptidom beeinflussen. Mehrere Peptide mit MHC-Bindemotiven sind im Harn in Konzentrationen von kleiner-gleich 10^-6 M leicht nachzuweisen, treten aber unabhängig vom MHC auf. Andererseits können wir einen schlüssigen Nachweis liefern, dass der prototypische H2-Kb-Ligand SIINFEKL, welcher aus Hühner-Ovalbumin stammt, MHC-abhängig mit einer Konzentration von 4 x 10^-12 M im Harn Ovalbumin-transgener Mäuse vorkommt. Der Nachweis der MHC-Abhängigkeit erfolgt dabei unter Nutzung eines Mausstammes, der Ovalbumin, aber keine funktionellen MHC-Klasse-I-Moleküle exprimiert. In Zusammenarbeit mit Neurobiologen zeigen wir, dass chemosensorische Nervenzellen im Vomeronasalorgan sowohl EAV-Peptide als auch SIINFEKL erkennen und unterscheiden können. Die vorliegende Doktorarbeit schließt eine entscheidende Lücke in der Hypothese, dass MHC-Peptidliganden als Signale der Individualität von Mäusen dienen können, indem sie den ersten Nachweis für einen solchen Liganden erbringt, der MHC-abhängig im Harn auftritt. Gleichzeitig stellt unsere Studie dieselbe Hypothese jedoch grundlegend in Frage, weil sie erstens zeigt, dass die entsprechenden Peptidkonzentrationen im Urin etwa acht Größenordnungen unter denjenigen liegen, welche in vorangegangenen Verhaltensexperimenten verwendet wurden und weil sie zweitens häufige MHC-unabhängige MHC-Motivpeptide nachweist. Ich vermute daher, dass die zahlreichen Peptide, welche die genomische Individualität unabhängig von MHC-Genen widerspiegeln, für das Sozialverhalten weitaus wichtiger sind als die MHC-abhängigen. In ihrer Gesamtheit bilden Urinpeptide Teile des exprimierten Genoms in Echtzeit ab und machen es somit für die chemosensorische Analyse durch andere Individuen zugänglich.

Minimal information about an immuno‐peptidomics experiment (MIAIPE) is an initiative of the members of the Human Immuno‐Peptidome Project (HIPP), an international program organized by the Human Proteome Organization (HUPO). The aim of the MIAIPE guidelines is to deliver technical guidelines representing the minimal information required to sufficiently support the evaluation and interpretation of immunopeptidomics experiments. The MIAIPE document has been designed to report essential information about sample preparation, mass spectrometric measurement, and associated mass spectrometry (MS)‐related bioinformatics aspects that are unique to immunopeptidomics and may not be covered by the general proteomics MIAPE (minimal information about a proteomics experiment) guidelines., Proteomics, 18 (12)

To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells., Journal of Proteome Research, 20 (1), ISSN:1535-3893, ISSN:1535-3907