Shotgun Mass Spectrometry Analysis Research Articles

Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three “red complex” bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

Abstract PR03: Immuno-mass spectrometric identification of serum biomarkers of response and toxicity to pembrolizumab

Abstract Immune checkpoint blockade (ICB) is a breakthrough form of cancer immunotherapy that employs antibody targeting of specific inhibitory receptors and ligands, such as cytotoxic T-lymphocyte associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1). The major limitations of ICB are high cost, limited success rate (10-40%), and potential severe toxicity due to immune-related adverse effects (IRAEs), which resemble autoimmune disease. Predictive biomarkers of ICB are not currently widespread in clinical use, despite the growing need for a personalized approach to cancer treatment. Effective immunotherapy causes tumor cell death, which releases tumor-associated antigens (TAAs) into circulation. This results in abnormal presentation of these antigens to immune cells, which leads to B-cell autoantibody production against them. Autoantibodies are effective biomarkers of some autoimmune diseases and may be present before disease onset. We hypothesized that patients who develop immune-related toxicity from immunotherapy will produce specific autoantibodies that are indicative of an autoimmune-like response. Furthermore, we hypothesized that responders to pembrolizumab will develop high titers of serum autoantibodies against TAAs, indicative of a strong humoral immune response to TAAs released during immunotherapy. Likewise, nonresponders will have low levels of these autoantibodies, due to a weaker or nonexistent antitumor and humoral immune response. We used a novel immuno-mass spectrometry method to screen for autoantibodies in the sera of patients with various tumors treated with PD-1 inhibition in the clinical trial called INSPIRE (INvestigator-initiated Phase II Study of Pembrolizumab Immunological Response Evaluation; NCT02644369) at Princess Margaret Cancer Centre. Our methodology involves immunoprecipitation of proteome-wide target antigens of autoantibodies in patient sera with the use of protein G magnetic beads, followed by shotgun mass spectrometry analysis. We analyzed autoantibody responses in the sera before and after immunotherapy initiation in a total of 24 patients, subdivided into 4 patient groups based on their objective response and toxicity status. Candidate autoantibody target antigens, including thyroglobulin, thyroid peroxidase, and ficolin-2, were identified by our pilot study. Validation with additional datasets is planned. Furthermore, we identified PD-1 as an antibody target exclusively in the post-immunotherapy patient sera of all 4 patient groups. This finding confirms the efficacy of our method since pembrolizumab is a humanized antibody targeting PD-1. Predictive biomarkers of cancer immunotherapy will save significant resources, ensure proper patient selection for cancer treatment, and spare certain patients from the toxic effects of immunotherapy. This abstract is also being presented as Poster B05. Citation Format: Milena Music, Marco Iafolla, Antoninus Soosaipillai, Ihor Batruch, Ioannis Prassas, Lillian L. Siu, Eleftherios P. Diamandis. Immuno-mass spectrometric identification of serum biomarkers of response and toxicity to pembrolizumab [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR03.

Characterization of the cellulase-secretome produced by the Antarctic bacterium Flavobacterium sp. AUG42

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.

Quantitative Phosphoproteome Analysis of Clostridioides difficile Toxin B Treated Human Epithelial Cells

The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdBNXN on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO2 followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdBNXN. The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.

Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography

Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.

Algorithm of Protein Sequence Determination by Combination of the Edman Degradation Method and Shotgun Mass Spectrometry

An algorithm combining advantages of the Edman degradation method and de novo mass-spectrometric sequencing was developed. The protein from the “Diaskintest” diagnostic test was used for analysis. The protein was digested with trypsin and 5 steps of Edman degradation were carried out sequentially for the mixture of peptides. At each stage, the resulting mixture was analyzed by shotgun mass spectrometry analysis. The results of mass-spectrometry were analyzed both by the well-known de novo sequencing programs Novor and PepNovo+, and by own program that clustered individual spectra with a C-terminal signature formed by Y-ions. This approach allows us to determine confidently the amino acid sequence of the N-terminal part of the peptides obtained after the protein hydrolysis by trypsin.

Comprehensive Mass Spectrometric Survey of Streptococcus pyogenes Subcellular Proteomes.

Streptococcus pyogenes is a major global health burden causing a wide variety of diseases. Because a vaccine against this bacterium is still lacking, vaccine candidates or antimicrobial therapies are urgently needed. Here we use an invasive and clinically relevant streptococcal M1 serotype to characterize the bacterial proteome in-depth. An elaborate fractionation technique is employed to separate the different cell fractions, followed by shotgun mass-spectrometry analysis, allowing us to confirm the expression of nearly two-thirds (1022) of the 1690 open reading frames predicted for the streptococcal M1 reference proteome. In contrast with other studies, we present the entire isolated membrane proteome, which opens up a whole new source for drug targets. We show both the unique and most prevalent proteins for each cellular fraction and analyze the presence of predicted cell-wall-anchored proteins and lipoproteins. With our approach, we also identify a variety of novel proteins whose presence has not been reported in previous proteome studies. Proteins of interest, potential virulence factors, and drug or vaccine targets are discussed for each cellular fraction. Overall, the results of this work represent the so-far widest proteomic approach to characterize the protein composition and localization in S. pyogenes.

The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

BackgroundThe aminoglycoside antibiotic gentamicin was supposed to induce a crosstalk between the Cpx- and the Arc-two-component systems (TCS). Here, we investigated the physical interaction of the respective TCS components and compared the results with their respective gene expression and protein abundance. The findings were interpreted in relation to the global proteome profile upon gentamicin treatment.ResultsWe observed specific interaction between CpxA and ArcA upon treatment with the aminoglycoside gentamicin using Membrane-Strep-tagged protein interaction experiments (mSPINE). This interaction was neither accompanied by detectable phosphorylation of ArcA nor by activation of the Arc system via CpxA. Furthermore, no changes in absolute amounts of the Cpx- and Arc-TCS could be determined with the sensitive single reaction monitoring (SRM) in presence of gentamicin. Nevertheless, upon applying shotgun mass spectrometry analysis after treatment with gentamicin, we observed a reduction of ArcA ~ P-dependent protein synthesis and a significant Cpx-dependent alteration in the global proteome profile of E. coli.ConclusionsThis study points to the importance of the Cpx-TCS within the complex regulatory network in the E. coli response to aminoglycoside-caused stress.

SIRT2-Mediated Deacetylation and Tetramerization of Pyruvate Kinase Directs Glycolysis and Tumor Growth

Sirtuins participate in sensing nutrient availability and directing metabolic activity to match energy needs with energy production and consumption. However, the pivotal targets for sirtuins in cancer are mainly unknown. In this study, we identify the M2 isoform of pyruvate kinase (PKM2) as a critical target of the sirtuin SIRT2 implicated in cancer. PKM2 directs the synthesis of pyruvate and acetyl-CoA, the latter of which is transported to mitochondria for use in the Krebs cycle to generate ATP. Enabled by a shotgun mass spectrometry analysis founded on tissue culture models, we identified a candidate SIRT2 deacetylation target at PKM2 lysine 305 (K305). Biochemical experiments including site-directed mutants that mimicked constitutive acetylation suggested that acetylation reduced PKM2 activity by preventing tetramerization to the active enzymatic form. Notably, ectopic overexpression of a deacetylated PKM2 mutant in Sirt2-deficient mammary tumor cells altered glucose metabolism and inhibited malignant growth. Taken together, our results argued that loss of SIRT2 function in cancer cells reprograms their glycolytic metabolism via PKM2 regulation, partially explaining the tumor-permissive phenotype of mice lacking Sirt2 Cancer Res; 76(13); 3802-12. ©2016 AACR.

Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis.

The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after "pulse" labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein's half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo. Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide.

Optimized procedure of extraction, purification and proteomic analysis of nuclear proteins from mouse brain

BackgroundThe cell nucleus is a highly dynamic subcellular compartment performing crucial processes for functioning and administration of the cell. Proteomic analysis of isolated nuclear fraction enables in-depth insight into these processes leading to better understanding of physiological and pathological state of the brain. There is no universal method for nuclear proteome investigation and each biological material needs individual optimization. An additional difficulty is the large amount of nucleic acids, which impairs isoelectrofocusing of proteins and shotgun mass spectrometry analysis of complex peptide samples. New methodWe performed the first comprehensive optimization of intact nuclei isolation from mouse brain in combination with nuclear protein purification prior to two-dimensional gel electrophoresis (2DE) and gel-free proteomic analysis. ResultsApplication of sonication, digestion with nuclease and protein precipitation with acetone allowed to obtain high quality 2DE gels. Shotgun mass spectrometry analysis of isolated proteins proved an enrichment in nuclear proteins. The 66.4% of 265 identified proteins had assigned nucleus localization in UniProt database. Gene Ontology analysis using DAVID platform revealed the highest fold enrichment in spliceosome (24.5), nuclear periphery (12.4) and nuclear matrix (11.3). Comparison with existing methodsThe proposed procedure is tailored to mouse brain tissue nuclear subproteome investigation. The quality of isolated nuclei, the effectiveness of the protein purification, efficiency of protein recovery after precipitation and overall method reproducibility was taken into detailed consideration. ConclusionsThe elaborated procedure could be further applied for in-depth proteomic analysis of molecular processes occurring in the mouse brain nucleus.

Isolation of Rhp-PSP, a member of YER057c/YjgF/UK114 protein family with antiviral properties, from the photosynthetic bacterium Rhodopseudomonas palustris strain JSC-3b.

Rhodopseudomonas palustris strain JSC-3b isolated from a water canal adjacent to a vegetable field produces a protein that was purified by bioactivity-guided fractionation based on ammonium sulfate precipitation, ion-exchange absorption and size exclusion. The protein was further identified as an endoribonuclease L-PSP (Liver-Perchloric acid-soluble protein) by shotgun mass spectrometry analysis and gene identification, and it is member of YER057c/YjgF/UK114 protein family. Herein, this protein is designated Rhp-PSP. Rhp-PSP exhibited significant inhibitory activities against tobacco mosaic virus (TMV) in vivo and in vitro. To our knowledge, this represents the first report on the antiviral activity of a protein of the YER057c/YjgF/UK114 family and also the first antiviral protein isolated from R. palustris. Our research provides insight into the potential of photosynthetic bacterial resources in biological control of plant virus diseases and sustainable agriculture.

Phospholipidomics reveals differences in glycerophosphoserine profiles of hypothermically stored red blood cells and microvesicles

During their normal in vivo life cycle erythrocytes (red blood cells, RBCs) undergo biochemical changes leading to membrane microvesiculation and shedding. RBC microvesiculation also occurs in vitro under conditions of blood bank storage, so microvesicles (MVs) accumulate in the storage (preservation) medium over storage time. Considerable effort has been put into gaining a mechanistic understanding of the RBC microvesiculation process, as this is crucial to better understand RBC biology in disease and in health. Additionally, MVs accumulated in stored RBCs have been implicated in transfusion adverse inflammatory reactions, with chloroform extractable compounds, thus lipophilic, known to trigger the effect. However, because thin layer chromatography resolution of RBC and MV lipids has always enabled one to conclude high compositional similarities, in depth analysis of MV lipids has not been extensively pursued. Here we present an orbitrap mass spectrometry (MS) approach to compare the phospholipid composition of RBCs and MVs from leukoreduced, hypothermically (2–6°C) stored RBC units. We used shotgun MS analysis and electrospray ionization (ESI) intra-source separation, and demonstrated high similarity of compositional profiles, except for glycerophosphoserines (PS). Contrasting abundances of PS 38:4 and PS 38:1 characterized MV and RBC profiles and suggested that storage-associated microvesiculation possibly involves shedding of specific membrane rafts. This finding indicates that phospholipidomics could likely contribute to a better understanding of the RBC microvesiculation process.

P120 The cytokine mRNA-interactome – An unbiased approach to identify RNA–protein interactions

Introduction The first line of defense against invading pathogens is the innate immune system. Recognition of an intruder results in transcriptional up-regulation of several genes, including cytokines. The production of these potent immune mediators needs to be tightly monitored as aberrant cytokine expression results in tissue damage or development of autoimmune diseases. It becomes increasingly evident that post-transcriptional regulation of gene expression also strongly impacts on cytokine production, especially during the resolution phase of the immune response. One means to post-transcriptionally control cytokine expression is by modulating the stability of their mRNA via adenosine/uridine (AU)-rich elements (ARE), which are found within the 3’untranslated region (UTR) of several cytokine mRNAs. Although several ARE-interacting proteins (ARE-BPs) have been described, it is not clear how they regulate individual AREs of distinct cytokine mRNAs during the immune response. Methods Here we propose to use affinity purification coupled to mass spectrometry to analyze the dynamic interactions of selected cytokine ARE elements with ARE-BPs during an immune response. We chose to study the human interferon-beta (IFN-b) mRNA, which contains two instability determining sequences, an ARE in the 3’UTR and a coding region instability element (CIRD) within the open reading frame. We used 3’biotinylated IFN-b ARE RNA oligomers as affinity reagent to isolate ARE-BPs from total cell extracts of mock-treated or polyinosinic:polycytidylic acid (p(I:C)) stimulated cells. Isolated interacting proteins were analyzed via 1D shot gun mass spectrometry analysis. Results We could identify the human antigen R ( HuR ) as the most prominent IFN-b ARE binding protein. Previous studies have pointed to a role for HuR mRNA binding in antagonizing the rapid decay of some ARE-containing mRNAs, which correlates with reports of IFN-b message stabilization during the immune response. Conclusion The stability of cytokine mRNAs is highly regulated, as deregulation of cytokine production can have detrimental effects. Our work will shed new light on the dynamic interactions of cytokine ARE elements and their ARE-BPs during an immune response.

Depletion of hemoglobin and carbonic anhydrase from erythrocyte cytosolic samples by preparative clear native electrophoresis

Proteomic analysis of red cells is compromised by the presence of high-abundance proteins (hemoglobin and carbonic anhydrase-1), which completely obscure low-abundance species. The depletion method presented here involves performing native gel electrophoresis in a polyacrylamide gel tube using a modified electroelution cell. The electrophoretic run is interrupted intermittently to allow the recovery of at least three different liquid fractions, which can be analyzed by both native PAGE and 2D isoelectric focusing SDS-PAGE, or by shotgun mass spectrometry analysis after trypsin in-solution protein digestion. This low-cost, reproducible technique can be used to process large amounts of sample, and it increases the likelihood of detecting low-abundance proteins, thereby resulting in greater proteome coverage. The separation procedure takes approximately 6-7 h.