Mol. Cell. Proteomics

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe

Use of 18O Labels to Monitor Deamidation during Protein and Peptide Sample Processing

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c· + 58 and z − 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)(n−1)+· − 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of 18O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (̃4 h) trypsin digestion process generates no additional detectable peptide deamidations

The interactions of cisplatin and model proteins studied by electrospray ionization mass spectrometry and tandem mass spectrometry

Protein-cisplatin interactions lie at the heart of both the effectiveness of cisplatin as an anticancer drug and side effects associated with cisplatin treatment. A greater understanding of the protein-cisplatin interactions at the molecular level can not only improve our understanding of the action of cisplatin as an anticancer drug but also inform the design of cisplatin-like agents for future use. Therefore, the interactions of cisplatin with three different model proteins were studied, which may provide theoretical basis for predicating mechanistically relevant protein-cisplatin interactions in biological fluids.;Cytochrome c (cyt c) was used as a model protein to develop a mass spectrometric approach to determine the primary binding site of cisplatin on proteins by coupling Fourier transform mass spectrometry (FT-MS) and tandem mass spectrometry (MS/MS and MS3). FT-MS permits identification of unique fragments in the adduct digest, characterized by MS/MS and MS3 to indicate that Met65 is the primary binding site for cisplatin on cyt c.;The interactions of cisplatin and transplatin with myoglobin (Mb) were compared in order to gain insights into similarities and differences between cisplatin and transplatin in their interactions with globular proteins. Prior to this research, the conditions for Mb denaturation were optimized to obtain the Mb digests for MS/MS and MS3.;Cisplatin and transplatin exhibit similar interactions with Mb. Monoadducts and diadducts were the primary adducts observed in both the interactions. MS/MS and MS3 analyses of the observed unique fragments in the digests of both the Mb-cisplatin and Mb-transplatin adducts indicate a common binding site for cisplatin and transplatin on the His116-His119 residues of Mb. This result coupled with a study of the interactions of cisplatin and transplatin with a dipeptide His∼Ser and the three dimensional (3-D) structure of native Mb shows that cisplatin and transplatin coordinate to the His116 and His119 residues on Mb.;The binding sites of cisplatin on native ubiquitin (Ub) and denatured Ub were compared in order to investigate the effect of protein conformation on the cisplatin binding sites on a protein. Results suggest that cisplatin has more binding sites on the native Ub than on the denatured Ub due to conformation effect. Three cisplatin binding sites are determined on the native Ub, in which two threonines are the primary binding site of cisplatin. On the denatured Ub, the Met1 residue is the specific binding site of cisplatin

Epidemiologic observations guiding clinical application of a urinary peptidomic marker of diastolic left ventricular dysfunction

Hypertension, obesity, and old age are major risk factors for left ventricular (LV) diastolic dysfunction (LVDD), but easily applicable screening tools for people at risk are lacking. We investigated whether HF1, a urinary biomarker consisting of 85 peptides, can predict over a 5-year time span mildly impaired diastolic LV function as assessed by echocardiography. In 645 white Flemish (50.5% women; 50.9 years [mean]), we measured HF1 by capillary electrophoresis coupled with mass spectrometry in 2005-2010. We measured early (E) and late (A) peak velocities of the transmitral blood flow and early (e') and late (a') mitral annular peak velocities and their ratios in 2009-2013. In multivariable-adjusted analyses, per 1-standard deviation increment in HF1, e' was -0.193 cm/s lower (95% confidence interval: -0.352 to -0.033; P = .018) and E/e' 0.174 units higher (0.005-0.342; P = .043). Of 645 participants, 179 (27.8%) had LVDD at follow-up, based on impaired relaxation in 69 patients (38.5%) or an elevated filling pressure in the presence of a normal (74 [43.8%]) or low (36 [20.1%]) age-specific E/A ratio. For a 1-standard deviation increment in HF1, the adjusted odds ratio was 1.37 (confidence interval, 1.07-1.76; P = .013). The integrated discrimination (+1.14%) and net reclassification (+31.7%) improvement of the optimized HF1 threshold (-0.350) in discriminating normal from abnormal diastolic LV function at follow-up over and beyond other risk factors was significant (P ≤ .024). In conclusion, HF1 may allow screening for LVDD over a 5-year horizon in asymptomatic people

A metaproteomic approach to study human-microbial ecosystems at the mucosal luminal interface

Aberrant interactions between the host and the intestinal bacteria are thought to contribute to the pathogenesis of many digestive diseases. However, studying the complex ecosystem at the human mucosal-luminal interface (MLI) is challenging and requires an integrative systems biology approach. Therefore, we developed a novel method integrating lavage sampling of the human mucosal surface, high-throughput proteomics, and a unique suite of bioinformatic and statistical analyses. Shotgun proteomic analysis of secreted proteins recovered from the MLI confirmed the presence of both human and bacterial components. To profile the MLI metaproteome, we collected 205 mucosal lavage samples from 38 healthy subjects, and subjected them to high-throughput proteomics. The spectral data were subjected to a rigorous data processing pipeline to optimize suitability for quantitation and analysis, and then were evaluated using a set of biostatistical tools. Compared to the mucosal transcriptome, the MLI metaproteome was enriched for extracellular proteins involved in response to stimulus and immune system processes. Analysis of the metaproteome revealed significant individual-related as well as anatomic region-related (biogeographic) features. Quantitative shotgun proteomics established the identity and confirmed the biogeographic association of 49 proteins (including 3 functional protein networks) demarcating the proximal and distal colon. This robust and integrated proteomic approach is thus effective for identifying functional features of the human mucosal ecosystem, and a fresh understanding of the basic biology and disease processes at the MLI. © 2011 Li et al

N-terminal Imine Derivatization for Enhanced De Novo Peptide Sequencing: A Study of the Fragmentation Pattern Generated from CID of Peptide-Imines

In this work, the fragmentation pattern derived from model peptides derivatized to create N-terminal imines (Schiff bases) were evaluated. Collision-induced dissociation of the protonated peptide-imines ([M+H]+) generally produced complete series of the sequence informative an and bn ions, now undoubtedly characteristic of the peptide ion species. A novel product ion was also observed, denoted the yǂ ion, determined by IRMPD spectroscopy and density functional theory to be generated from the elimination of the N-terminal amino acid residue despite the N-terminal modification. It was concluded the pathway involved a nucleophilic attack by an amide nitrogen and the possible formation of an imidazole-4-one intermediate, which collapses to generate a truncated, protonated peptide-imine with a conserved primary sequence. N-terminal imine-modification was also observed to eliminate sequence scrambling events, presumably by eliminating the macrocyclic b ion mechanism implicated in the sequence rearrangements. Additionally, the CID mass spectra of Ag-cationized imine-modified peptides were obtained. An apparent even-electron, [M-H]+ peptide ion was observed, determined to be generated by the loss of AgH. The hydrogen abstraction was explicitly identified to originate from the imine-carbon of the argentinated modified peptide. CID of the [M–H]+ ions generated sequence ions analogous to those produced from the [M+H]+ species of imine-modified peptides, however less extensively

Anwendung Massenspektrometrie basierter Technologie zur Entdeckung räumlicher Peptidsignaturen in der Krebsforschung

Cancer is one of the leading causes of death worldwide, within the molecular and structure complexity of tumors are causal factors for disease progression and treatment standards. With the development of molecular biological techniques, physicians could use genetic variation or protein and metabolic expression profile besides histo-morphologicial evaluation to classify more accurate risk assessment and to guide treatment decisions. The biomarker-driven personalized therapies might improve clinical care, avoid unnecessary treatments and reduce the duration and costs for hospital stay. Therefore, there is a strong demand for more reliable molecular biomarker profiles. In this dissertation, a novel technique called imaging mass spectrometry (MADLI-MSI) is used to investigate the potential of spatially resolved peptide signatures (directly from tumor tissue; in situ) for (i) discrimination of subtypes of serous ovarian cancer (HGSOC) and (ii) risk assessment of neuroblastoma. Univariate and multivariate static methods were used to determine associated peptide signatures. Using complementary methods, liquid chromatography-based mass spectrometry the corresponding proteins to the peptides were identified and verified by immunohistology. Consequently, peptide signatures were identified to predict disease recurrence in early-stage HGSOC patients and to distinguish high-risk neuroblastoma patients from other risk groups. These results suggest that the MALDI-MSI technique is a promising analytical method that facilitates diagnosis and treatment decision-making. It has also provided new biological insights into tumor heterogeneity, that could benefit the development of molecular biomarker profiles. The data of this dissertation have been really published in Journal “Cancers (MDPI)” 2020 and 2021.Onkologische Erkrankungen (Krebs) sind weltweit eine der häufigsten Todesursachen. Die molekulare und strukturelle Komplexität von Tumoren sind ursächlich für die Krankheitsprogression und Therapieanspruch. Mit der Entwicklung von neuen molekularbiologischen Verfahren könnten Ärzte neben der histo-morphologischen Bewertung auch genetische Variationen oder Protein- und Metabolit-Expressionsprofile nutzen, um eine genauere Risikobewertung vorzunehmen und die Behandlungsentscheidung zu treffen. Die personalisierten Therapien können die klinische Versorgung verbessern durch Vermeidung unnötiger Behandlungen und verringerte Dauer und Kosten des Krankenhausaufenthalts. Daher besteht ein starker Bedarf an zuverlässigeren molekularen Biomarker Profilen. In dieser Dissertation wird ein neuartiges Verfahren, die sogenannten bildgebenden Massenspektrometrie (MADLI-MSI) eingesetzte um das Potential von räumlich aufgelösten Peptide-Signaturen (direkt aus dem Tumorgewebe; in situ) für (i) die Diskriminierung von Subtypen des serösen Ovarialkarzinom (HGSOC) zu untersuchen und (ii) die Risikoabschätzung des Neuroblastomes. Dabei wurden univariate und multivariate statischer Verfahren eingesetzt, um assoziierten Peptide- Signaturen zu bestimmen. Mittels komplementärer Verfahren, Flüssigkeitschromatographie basierte Massenspektrometrie wurden die korrespondierenden Proteine zu den Peptiden identifiziert und Immunhistologisch verifiziert. Folglich wurden Peptidsignaturen zur Vorhersage des Wiederauftretens der Krankheit bei HGSOC-Patienten im Frühstadium und zur Unterscheidung von Hochrisiko-Neuroblastom Patienten von anderen Risikogruppen identifiziert. Diese Ergebnisse deuten darauf hin, dass die MALDI-MSI-Technik eine vielversprechende Analysemethode ist, die die Diagnose und die Entscheidung über die Behandlung erleichtert. Außerdem hat sie neue biologische Erkenntnisse über die Heterogenität des Tumors geliefert, die der Entwicklung von molekularen Biomarker-Profilen zu Gute kommen könnten. Die Daten dieser Dissertation wurden in der Zeitschrift „Cancers (MDPI)" 2020 und 2021 veröffentlicht

Application of Ion-Molecule Reactions to Structure Characterization and Thermochemical Property Measurements

This thesis explores several novel applications of gas-phase ion-molecule reactions to solve analytical problems. In all cases, the application of an ion-molecule reaction to solve a specific challenge is based on a thorough understanding of the fundamental aspects of the reaction, including kinetics, production distributions and most notably, its reaction mechanism.Ion-molecule reactions of the atomic oxygen radical anion, O *- , with selected ketones are investigated to explore a strategy to synthesize 1,3-distonic radical anions, which are precursors to 1,3-diyls. The O *- /cyclopentanone reaction is examined in detail, under the well-defined thermal conditions uniquely available in the flowing afterglow, to ascertain if cyclopentan-2-one-1,3-diyl radical anion is formed. To further the understanding of this key reaction, a series of related ketones are also examined. Rate coefficients for each ketone reaction are measured, products are identified, and the branching ratios are determined. A strategy is developed to differentiate the 1,1- and 1,3-H 2 *+ isobaric abstraction products. A total yield of 48% [M-2H] *- is obtained for the O *- /cyclopentanone reaction, wherein ∼15% is the absolute yield of the 1,1- and ∼33% is the absolute yield of the 1,3-H 2 *+ abstraction product.A chemical reagent that specifically cleaves the peptide backbone will greatly simplify peptide sequencing as compared to nonselective energetic collisions with inert gases. In the search for peptide cleavage reagents, translationally-driven, endothermic ion-molecule reactions between peptide ions and potential cleavage reagents are investigated in a custom-built, electrospray ionization, triple quadrupole mass spectrometer. Strategies are adopted to minimize nonselective energetic fragmentation processes and to favor amide bond cleavage.The kinetic method is used to derive the relative and absolute proton affinities of two neuropeptides, leucine-enkephalin and methionine-enkephalin. Based on analyses of the collision induced fragmentation of the proton-bound heterodimer of leucine-enkephalin and methionine-enkephalin, leucine-enkephalin is established to be 0.1 kcal/mol lower in proton affinity than methionine-enkephalin. Based on analyses of the collision induced fragmentation of the proton-bound heterodimer of leucine-enkephalin or methionine-enkephalin and triethylamine, tripropylamine or tributylamine, the absolute proton affinities of leucine-enkephalin and methionine-enkephalin are established to be 238.5 and 238.6 (±5.0) kcal/mol