Manuela Simonato (1) - Sonia Giambelluca (2) - Luca Vedovelli (1) - Lorenza Dalla Massara (3) - Carlo Ori (3)
- Virgilio Paolo Carnielli (4) - Paola Cogo (5)
(1)
Fondazione Istituto di Ricerca Pediatrica Città della Speranza, PCare Laboratory, Padova - (2) Padova
University Hospital, Department of Women’s and Children’s Health, Padova - (3)
Padova University Hospital, Department of Medicine, Section of Anesthesiology and Intensive Care, Padova - (4) Polytechnic University of Marche and Azienda Ospedaliero-Universitaria Ospedali Riuniti, Division of Neonatology, Department of Clinical Sciences, Ancona - (5)University of Udine, Department of Medicine, Udine
Keywords: acute respiratory distress syndrome; albumin; isotope ratio mass spectrometry;
stable isotope
Introduction
Acute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by an increased inflammatory burden and changes in the alveolar-capillary membrane permeability with extravasation of fluid and protein into the alveoli[1].Despite the clear association between plasmatic hypoalbuminemia and poor outcome, there are no data on albumin metabolism in ARDS patients.
Methods
113C-Leucine, as albumin precursor, was administered to 13 ARDS adults and to 8 patients without lung disease (controls). Albumin was isolated from serial plasma and tracheal aspirate (TA) samples, and its kinetic parameters were derived from the 13C enrichment curve obtained by isotope ratio mass spectrometry[2]. As index of alveolar-capillary permeability the TA/plasma albumin ratio was measured.
Results
In patients with ARDS the fractional synthetic rate was significantly higher than in control patients [15.1 (12.3-22.7) vs 11.9 (8.9-12.9) %/day; p=0.04].
Median albumin secretion time and half-life were 0.7 (0.3-1.9) vs 0.6 (0.3-1.0) h, and 43.3 (35.7- 64.9) vs 42.7 (33.3-76.0) h in ARDS and in control patients respectively, no significant differences were observed.
In patients with ARDS the TA to plasma albumin ratio measured in the early phase after the diagnosis of acute lung injury was 7-fold times higher the level found in control patients (p<0.001). In the ARDS group the TA/plasma albumin ratio was significantly correlated with the PaO2/FiO2, as index of lung dysfunction (r=-0.732, p=0.016).
Conclusions
In this study, stable isotopes have been used to better understand albumin metabolism during a disease state like ARDS. Albumin synthesis rate in ARDS patients was higher than in control adults. Furthermore, the comparison between TA/plasma albumin ratio of ARDS and control patients revealed a dramatic increase in alveolar-capillary membrane permeability during the genesis of ARDS.
Novel Aspect
This study will give new insight into albumin turnover and compartmental distribution in patients with ARDS.
References
1. Ware L.B., Matthay M.A., The New England Journal of Medicine, 342, 1334 (2000).
2. Simonato M., Baritussio A., Pioselli B., Ori C., Catinella S., Carnielli V.P., Cogo P., Analitical and Bioanalitical Chemistry, 406, 6225 (2014).
MOr.06-5 МASS-SPECTROMETRY APPROACH FOR RAPID INTRAOPERATIVE IDENTIFICATION OF BRAIN TUMOR TISSUES - A WAY FOR ONLINE ANALYSIS
Stanislav Pekov (1) - Vasily Eliferov (1) - Evgeny Zhvansky (1) - Alexander Vorobyev (1) - Vsevolod
Shurkhay (2) - Anatoly Sorokin(1) - Igor Popov (1) - Alexander Potapov (2) - Evgeny Nikolaev (3)
(1)
Moscow Institute of Physics and Technology, Dolgoprudnyj - (2) N. N. Burdenko Scientific Research Neurosurgery Institute, Moscow - (3) Skolkovo institute of science and technology, Skolkovo
Keywords: ambient mass-spectrometry, clinical application, brain tumor, neurosurgical support,
microextraction.
Introduction
It is known that the lipid metabolism reprogramming is one of the hallmarks of cancer so mass- spectrometry-based method of lipid profiling is a promising approach for precise intraoperative tumor identification. In this research we propose the online method of tissue profiling based on
express and simple capsular extraction followed by ESI with an identification algorithms based on a search through a reference database containing mass spectra of different tumors.
Methods
The online microextraction system consists of disposable capsules compatible with any standard ESI source. Tissues, dissected by the surgeon were placed into the capsules which are then connected to the ESI interface for analysis. Features were extracted from mass-spectrometry data and aligned for further analysis. Significant features were detected by shrinkage discriminant analysis and classifiers were developed with lasso-regression.
Results
It was demonstrated that it is possible to prepare large amount (up to 5 per minute) of capsules with samples immediately during the operation because of simplicity of placing sample into the preassembled capsules so almost all the delay between receiving the tissue from surgeon and analysis depends only from transportation time between operational suite and laboratory room. The minimum amount of sample is very small (less than 1 cubic mm) so proposed method could be used during neurosurgery intervention where amount of resection could be very small. The data obtained with proposed microextraction method are in good correlation with our previous results obtained with spray-from-tissue ionization source. The rapid identification of the tumor type is allowed by the search inside the collected reference database containing mass spectra of samples that are also characterized by histopathological methods.
Conclusions
Application of microextraction capsules significantly simplifies the manipulations with samples by excluding any special sample preparation steps. Rapidity of method is also very important during the surgery as the minimization of surgery duration improves patient’s postoperative prognosis. Developed data processing algorithms and classifiers provide the reliable differentiation of extracted brain tissue by their lipid profiles.
Novel Aspect
The online mass-spectrometry profiling of brain tissues could be used as the intraoperative technology for characterization and identification of removed tissues.
MOr.07 - Quantitative proteomics
Chairs: Michael Linscheid, Nathalie Norais
MOr.07-1 Keynote: DRUG EFFECTS ON PROTEIN HOMEOSTASIS
Marcus Bantscheff
GlaxoSmithKline, GSK Cellzome GmbH, Heidelberg
Keywords: protein turnover, drug effects, protein homeostasis, targeted degraders Introduction
Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking.
Methods
mPDP enables the sensitive and comprehensive relative quantification of cellular mature and nascent protein pools across a range of different treatment conditions in biological duplicates and in a single mass spectrometric experiment by combining dynamic SILAC with chemical labeling using neutron-encoded tandem mass tags (TMT).
Results
We developed ‘multiplexed proteome dynamics profiling’, mPDP [1], a mass spectrometry-based approach combining dynamic-SILAC labelling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. When applied combination with other quantitative proteomics approaches such as chemoproteomics and thermal protein profiling, mPDP provides unique insights in drug mechanism-of-action. The method was applied in three proof-of-concept studies, and uncovered different responses induced by the bromodomain inhibitor JQ1 versus a JQ1-proteolysis targeting chimera. Further, we elucidated distinct modes-of-action of estrogen receptor modulators, and we comprehensively classified HSP90 clients based on their requirement for HSP90 constitutively or during synthesis demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, higher affinity for the chaperone, vary between cell types and change upon external stimuli.
Conclusions
We have established multiplexed proteome dynamics profiling a powerful means to elucidate the mechanism of action of bioactive molecules, thus complementing current proteome-wide target identification strategies. mPDP identifies targets of protein degraders and more generally enables the discovery of regulatory degradation mechanisms in biological systems.
Novel Aspect
PROTAC target identification and detection of regulated degradation events.
References
Savitski MM et al., Cell 173, 260-274 (2018)
MOr.07-2 MEASURING NASCENT PROTEOMICS IN SIGNALING AND CELLULAR DIFFERENTIATION
Alma Burlingame (1) - Nancy Phillips (2) - Craig Forester (3)
(1)
UCSF, Pharmaceutical Chemistry, San Francisco - (2) UCSF, Pharmaceutical Chemistry, San Francisco -
(3)
UCSF, Urology, San Francisco
Keywords: O-propargylpuromycin, nascent proteome, erythropoiesis, mass spectrometry Introduction
Regulation of gene expression is controlled by mRNA translation and thus defines the functional proteome. Experimental methodologies are needed that can rapidly capture and reveal the actual nascent proteomes. In addition, such methods must be able to measure changes in nascent protein abundances quantitatively in response to pharmacologic or environmental cues.
Methods
We have developed a protocol using an analog of puromycin, a tyrosine-tRNA mimetic, called O- propargylpuromycin (OPP), to identify nascent proteomes rapidly. OPP is cell permeable and catalytically incorporated into nascent polypeptide chains as a specific covalent label during elongation resulting in premature termination. These polypeptides are biotinylated using click chemistry, then isolated and measured by tandem mass spectrometry[1].
Results
We have chosen the erythropoiesis paradigm known to be dependent upon an intricate balance involving maintainence of erythroid precursors and their rapid differentiation to accommodate hematopoietic stresses. In this study, we employed extensively self-renewing erythroblast (ESRE) cells to define the earliest proteomic program unique to the decision between expanding erythroid precursors and terminal maturation. Using OPP-ID, we discovered a network of genes involved in splicing, nucleic acid secondary structure unwinding, and cell cycle regulation which are rapidly repressed in the absence of dexamethasone. While we observed genes previously known to be associated with early hematopoietic precursor states (such as Ddx18, Stag2 and Pold1), we also identified multiple genes such as Prpf6, Sart1, Psen2, and Mycbp2 which have not been previously implicated in erythropoiesis.
Conclusions
This OPP-ID strategy provides rapid experimental view of changes in gene expression at the nascent protein level. This opens a new window for the determination of the actual proteomic changes occurring in response to a variety of cues that direct cellular fates such as proliferation or differentiation.
Novel aspect
This OPP-ID strategy has the potential to ask innumerable questions on the composition and rapid dynamics of proteomic networks at the organismal level.
References
1. Forester C., et al., Proc. Natl. Acad.Sci., 2018, doi: 10.1073/pnas.1707514115
MOr.07-3 A CHEMICAL PROBE FOR UNDERSTANDING HOW PROTEOME FOLDEDNESS CHANGES UNDER