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In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of ‘missing proteins’ (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.

The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass‐spectrometry‐based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein‐coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease., The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. This data covers 65% and 74% of the predicted and identified human proteome, respectively.

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with “high-mannose” N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

SummaryMalignant melanoma (MM) develops from the melanocytes and in its advanced stage is the most aggressive type of skin cancer. Here we report a comprehensive analysis on a prospective cohort study, including non-tumor, primary and metastasis tissues (n=77) with the corresponding plasma samples (n=56) from patients with malignant melanoma. The tumors and surrounding tissues were characterized with a combination of high-throughput analyses including quantitative proteomics, phosphoproteomics, acetylomics, and whole exome sequencing (WES) combined with in-depth histopathology analysis. Melanoma cell proliferation highly correlates with dysregulation at the proteome, at the posttranslational- and at the transcriptome level. Some of the changes were also verified in the plasma proteome. The metabolic reprogramming in melanoma includes upregulation of the glycolysis and the oxidative phosphorylation, and an increase in glutamine consumption, while downregulated proteins involved in the degradation of amino acids, fatty acids, and the extracellular matrix (ECM) receptor interaction. The pathways most dysregulated in MM including the MAP kinases-, the PI3K-AKT signaling, and the calcium homeostasis, are among the most affected by mutations, thus, dysregulation in these pathways can be manifested as drivers in melanoma development and progression.The phosphoproteome analysis combined with target-based prediction mapped 75% of the human kinome. Melanoma cell proliferation was driven by two key factors: i) metabolic reprogramming leading to upregulation of the glycolysis and oxidative phosphorylation, supported by HIF-1 signaling pathway and mitochondrial translation; and ii) a dysregulation of the immune system response, which was mirrored by immune system processes in the plasma proteome. Regulation of the melanoma acetylome and expression of deacetylase enzymes discriminated between groups based on tissue origin and proliferation, indicating a way to guide the successful use of HDAC inhibitors in melanoma. The disease progression toward metastasis is driven by the downregulation of the immune system response, including MHC class I and II, which allows tumors to evade immune surveillance. Altogether, new evidence is provided at different molecular levels to allow improved understanding of the melanoma progression, ultimately contributing to better treatment strategies.TOC figure

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients., The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and sub‐cellular localization was annotated within both primary and metastatic melanoma.

Background: there are no published national studies that contribute statistical data related to the adaptation of the therapeutic effort in the care units for seriously ill patients of the country.Objective: to characterize the adaptation of the therapeutic effort in the clinical framework of the daily practice in the care units for seriously ill adults of the "Manuel Ascunce Domenech" General Teaching Hospital of Camagüey, from January 1 to December 31, 2017.Methods: a descriptive, explanatory and correlational study was carried out at the aforementioned institution and during the period herein declared. The universe was made up of the 1233 patients of 19 years of age or older who were admitted to the intensive care unit. The sample included 502 patients with indication of adaptation of the therapeutic effort, and unanimous consensus among the healthcare team, the patient, the family or their guardian, for the adaptation of the therapeutic effort. Pregnant and postpartum women were excluded.Results: patients with terminal oncological and chronic diseases were the ones who received the most therapeutic adaptation. Not being admitted to intensive care was the main form of adaptation, followed by non-cardiopulmonary cerebral resuscitation, with the predominance of patients of advanced years, of both sexes. The terminality criterion prevailed. Most of the patients had more than one therapeutic adaptation. 23,9 % of the patients survived.Conclusions: the adaptation of the therapeutic effort in the hospital was characterized. In most of the cases more than one therapeutic adjustment was performed. Not all the patients died.

Background: non-invasive mechanical ventilation (NIMV) has been on the increase during the last decades. The specialized literature reveals its undeniable success in medical care of seriously ill patients. However, publications on the use of this method in daily medical practice in Cuba are scarce.Objective: to characterize the patients with criterion of acute respiratory failure who required mechanical ventilation at the intensive care unit of the “Amalia Simoni Argilagos” General Teaching Hospital of Camagüey, from January 2018 to January 2019.Methods: a prospective longitudinal descriptive study was carried out with the patients, institution and period of time stated in the objective. The sample consisted of 52 patients. Two groups were formed: one for those who received non-invasive mechanical ventilation (cases) and another group with patients who were treated invasively according to traditional ventilation criteria (controls). The data were processed according to descriptive statistics.Results: non-invasive ventilation was applied mostly to young patients; pneumonia was the main cause of application of both ventilatory methods, followed by chronic obstructive pulmonary disease; most of those who received treatment with NIMV had a hospital stay of less than 7 days (78,84 %), this group also had a lower mortality.Conclusions: the patients who required mechanical ventilation were characterized. Better results were evidenced when using non-invasive mechanical ventilation.

Dentro de la enorme cantidad de temas reinantes relacionados con el desarrollo científico-tecnológico actual, se encuentra indudablemente el del empleo de la ventilación mecánica no invasiva (VMNI) como medida de soporte vital. Aunque no se trata de una obra especifica que presente un panorama completo y exacto, de todos y cada uno de los problemas relacionados con el empleo de la misma, los autores pretenden con el artículo realizar una breve reflexión del tema desde el enfoque de la ciencia, la tecnología y la sociedad en el momento actual, de manera que constituya un marco útil para el examen de estos temas, su debate, reflexión e intercambio de opiniones en el contexto de los servicios de la salud.

Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426.

Hypertrophic scars (HTS) and keloids are forms of aberrant cutaneous healing with excessive extracellular matrix (ECM) deposition. Current therapies still fall short and cause undesired effects. We aimed to thoroughly evaluate the ability of growth hormone releasing peptide 6 (GHRP6) to both prevent and reverse cutaneous fibrosis and to acquire the earliest proteome data supporting GHRP6's acute impact on aesthetic wound healing. Two independent sets of experiments addressing prevention and reversion effects were conducted on the classic HTS model in rabbits. In the prevention approach, the wounds were assigned to topically receive GHRP6, triamcinolone acetonide (TA), or vehicle (1% sodium carboxy methylcellulose [CMC]) from day 1 to day 30 post‐wounding. The reversion scheme was based on the infiltration of either GHRP6 or sterile saline in mature HTS for 4 consecutive weeks. The incidence and appearance of HTS were systematically monitored. The sub‐epidermal fibrotic core area of HTS was ultrasonographically determined, and the scar elevation index was calculated on haematoxylin/eosin‐stained, microscopic digitised images. Tissue samples were collected for proteomics after 1 hour of HTS induction and treatment with either GHRP6 or vehicle. GHRP6 prevented the onset of HTS without the untoward reactions induced by the first‐line treatment triamcinolone acetonide (TA); however, it failed to significantly reverse mature HTS. The preliminary proteomic study suggests that the anti‐fibrotic preventing effect exerted by GHRP6 depends on different pathways involved in lipid metabolism, cytoskeleton arrangements, epidermal cells’ differentiation, and ECM dynamics. These results enlighten the potential success of GHRP6 as one of the incoming alternatives for HTS prevention.

The protein composition of an Outer Membrane Vesicle (OMV) preparation that constitutes the active pharmaceutical ingredient of VA-MENGOC-BC®, an effective vaccine against Neisseria meningitidis serogroups B, and C is presented. This preparation has a high lipid content and five abundant membrane proteins (FetA, PorA, PorB, RmpM, and Opc), constituting approximately 70% of the total protein mass. The protein composition was determined by combining the use of the Hexapeptide Ligand Library and an orthogonal tandem fractionation of tryptic peptides by reverse-phase chromatography at alkaline and acid pH. This approach equalizes the concentration of tryptic peptides derived from low- and high-abundance proteins as well as considerably simplifying the number of peptides analyzed by LC-MS/MS, enhancing the possibility of identifying low-abundance species. Fifty-one percent of the proteins originally annotated as membrane proteins in the genome of the MC58 strain were identified. One hundred and sixty-eight low-abundance cytosolic proteins presumably occluded within OMV were also identified. Four (NadA, NUbp, GNA2091, and fHbp), out of the five antigens constituting the Bexsero® vaccine, were detected in this OMV preparation. In particular, fHbp is also the active principle of the Trumenba® vaccine developed by Pfizer. The HpuA and HpuB gene products (not annotated in the MC58 genome) were identified in the CU385 strain, a clinical isolate that is used to produce this OMV. Considering the proteins identified here and previous work done by our group, the protein catalogue of this OMV preparation was extended to 266 different protein species.

La práctica de la adecuación del esfuerzo terapéutico, constituye un tema de marcado interés social, de reflexión médica vigente, en el que se aborda el dilema del límite entre lo que es técnicamente posible y lo que es éticamente aceptable El trabajo tiene como objetivo fundamental realizar una breve reflexión teórica, desde un análisis ético, acerca de la orden de no reanimación cardiopulmonar y la práctica de la adecuación del esfuerzo terapéutico al final de la vida de los pacientes en situación terminal, específicamente en las unidades de atención al paciente grave.

Son múltiples y variados los aspectos éticos que se derivan de la práctica clínica de la adecuación del esfuerzo terapéutico al final de la vida. La diversidad y complejidad que estos dilemas adquieren en la actualidad exige de los profesionales de la salud no solo constante actualización científico-técnica, sino también la necesidad de replantear las dimensiones éticas de cada una de sus acciones, para lograr una muerte digna y humanizada, donde decidir es todo un reto. Se presenta una breve reflexión ética con el objetivo de lograr una aproximación de los problemas éticos al final de la vida en relación con la adecuación del esfuerzo terapéutico y la muerte digna. Se realizó una revisión bibliográfica en los sitios web de reconocido prestigio, incluyendo revistas nacionales y extranjeras con certificación CITMA, de las cuales se seleccionó la bibliografía suficiente que abordara el tema de la adecuación del esfuerzo terapéutico al final de la vida y la muerte digna en los pacientes terminales, se seleccionaron fundamentalmente aquellos artículos publicados en los últimos seis años y también se incluyó algunos documentos éticos y legales de permanente vigencia en el país. Se exponen los criterios de diferentes autores sobre el tema. Se concluye que el hombre tiene derecho a morir con dignidad, no a una muerte deshumanizada.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas do clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression for the mutated protein is associated with a more aggressive tumor progression. Our study design that is comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis may enable the eventual delineation of patient responders/non-responders and the subsequent therapy of malignant melanoma.

Thoracic empyema is defined by the presence of pus in the pleural space. It is a critical condition which has a worse prognosis in nosocomial infections. The most frequent cause is a history of pneumonia. Associated risk factors include advanced age, type 2 diabetes mellitus, malnutrition, poor oral hygiene, cancer, among others. We present the case of a 45-year-old white female patient, with an apparently healthy past medical history. She presented a clinical productive cough accompanied by mucopurulent sputum, with pleuritic pain on the side, fever of 39 - 40 ºC, night sweats, asthenia and anorexia. When arriving at the emergency room she presented clinical evidence of respiratory failure and elements of sepsis. The clinical findings, thoracentesis, cultures and Gram staining, as well as the imaging studies confirmed the diagnosis of pneumococcal pulmonary empyema. Noninvasive mechanical ventilation and antibiotic therapy were applied. The patient progressed favorably.

Se estima que aproximadamente un cuarto de la población mundial está infectado con Áscaris lumbricoides y muchas fallecen anualmente a consecuencia de ello. La ascariasis presenta una mayor prevalencia en niños de países tropicales y subtropicales, especialmente en las regiones donde abunda la pobreza, el hacinamiento y la mala sanidad ambiental. La gran mayoría de los pacientes resuelven con el tratamiento farmacológico. Sin embargo, en ocasiones se necesita del manejo quirúrgico de urgencia. Se presenta el caso clínico de una paciente blanca, de 19 años de edad, que arriba con cuadro doloroso abdominal difuso a predominio de la región epigástrica, periumbilical y porción inferior del abdomen, con dolor a tipo cólico de intensidad variable y distensión abdominal; con sensación de que algo le caminaba por dentro, acompañado de vómitos de contenido alimenticio y luego bilioso claro, con retortijones. Refiere que llevaba más de 15 días sin defecar y sin expulsión de gases. Se le realiza examen físico que, junto al cuadro clínico y el empleo de medios imagenológicos, ultrasonido abdominal, confirman el diagnóstico de oclusión intestinal por Áscaris lumbricoides, etiología poco usual en Cuba. Se realizó proceder quirúrgico tipo laparotomía exploratoria, que evidenció la parasitemia masiva en el interior de las asas intestinales. Mediante maniobras de ordeñamiento de las asas se corroboró la salida en grandes cantidades de los parásitos en su estadio adulto. La paciente evolucionó de forma favorable.

To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

Large cohorts of carefully collected clinical tissue materials play a central role in acquiring sufficient depth and statistical power to discover disease-related mechanisms and biomarkers of clinical significance. Manual preparation of such large sample cohorts requires experienced laboratory personnel. This carries other possible downsides such as low throughput, high risk of errors, and low reproducibility. In this work, three automated technologies for high-throughput proteomics of frozen sectioned tissues were compared. The instruments evaluated included the Bioruptor for tissue disruption and protein extraction; the Barocycler, which is able to disrupt tissues and digest the proteins; and the AssayMAP Bravo, a microchromatography platform for protein digestion, peptide desalting, and fractionation. Wide varieties of tissue samples from rat spleen, malignant melanoma, and pancreatic tumors were used for the assessment. The three instruments displayed reproducible and consistent results, as was proven by high correlations and low coefficients of variation between technical replicates and even more importantly, between replicates that were processed in different batches or at different time points. The results from this study allowed us to integrate these technologies into an automated sample preparation workflow for large-scale proteomic studies that are currently ongoing. Data are available via ProteomeXchange with identifiers PXD010296 and PXD011295.

There is an increasing global interest to support research areas that can assist in understanding disease and improving patient care. The National Cancer Institute (NIH) has identified precision medicine-based approaches as key research strategies to expedite advances in cancer research. The Cancer Moonshot program (https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative) is the largest cancer program of all time, and has been launched to accelerate cancer research that aims to increase the availability of therapies to more patients and, ultimately, to eradicate cancer. Mass spectrometry-based proteomics has been extensively used to study the molecular mechanisms of cancer, to define molecular subtypes of tumors, to map cancer-associated protein interaction networks and post-translational modifications, and to aid in the development of new therapeutics and new diagnostic and prognostic tests. To establish the basis for our melanoma studies, we have established the Southern Sweden Malignant Melanoma Biobank. Tissues collected over many years have been accurately characterized with respect to the tumor and patient information. The extreme variability displayed in the protein profiles and the detection of missense mutations has confirmed the complexity and heterogeneity of the disease. It is envisaged that the combined analysis of clinical, histological, and proteomic data will provide patients with a more personalized medical treatment. With respect to disease presentation, targeted treatment and medical mass spectrometry analysis and imaging, this overview report will outline and summarize the current achievements and status within malignant melanoma. We present data generated by our cancer research center in Lund, Sweden, where we have built extensive capabilities in biobanking, proteogenomics, and patient treatments over an extensive time period. © 2018, The Author(s)., Funding information This study was supported by a joint grant from Berta Kamprad Foundation, and grants from the National Research Foundation of Korea, funded by the government of Republic of Korea (MSIP; 2012M3A9D1054520, 2015K1A1A2028365, 2015M3A9C4076 321), European Union’s Horizon 2020, Interreg. Program: NYPS 20200407, BReproUnion^, and European Union Regional Development Fund, Interreg. Oresund, BMAX4ESSFUN, LU-062^.

Hypoxemia represents a drastic consequence of numerous diseases. It is imperative that the physician count on all the tools needed to assess it effectively and early. The PaO2/FiO2 ratio, also called Kirby index, is merely a quotient that indirectly reflects changes in the ventilation-perfusion ratio or presence of pulmonary shunts. Many studies are in favor of its application as a predictor of mortality, as well as a success predictor of non-invasive mechanical ventilation, besides being useful in the follow-up of a seriously ill patient, from a hemogasometric viewpoint, who is receiving this type of ventilation. This the reason why a bibliographic review article was carried out using the services available in Infomed with the objective to identify relevant research works that deal with the importance of the Kirby index as a success and follow-up predictor of the non-invasive mechanical ventilation.

The scientific and technological development has an impact in all the spheres of society. The adaptation of the therapeutic effort is example of this. The author’s motive to carry out this bibliographic review article laid on the technological abuse as a result of the indiscriminate and excessive use of the vital support measures, on the growing potentials offered by science and technology to medical professionals, and on the need to present the foundations which help these professionals develop the abilities to accept the values that regulate and limit the use of these modern technologies. The main aim of the article is to get a theoretical approach taking into consideration the ethical thought about this present-day problem. It is necessary that science and humanness, as well as the scientific knowledge and the moral and ethical values governing the medical practice, get together in only one process.

Background: the predictive factors of success of non-invasive mechanical ventilation in patients with acute respiratory failure are important to define their progress.Objective: to determine the effectiveness of non-invasive mechanical ventilation through successful predictors as heart rate, respiratory rate, mean arterial pressure, PaCO2, PaO2, PaO2 / FiO2 and the Glasgow Scale, in patients of the intensive care wards of the "Manuel Ascunce Domenech" Hospital, Camagüey, from March 21, 2016 to March 21, 2017.Methods: a quasi-experimental study was carried out, taking patients with criterion of acute respiratory failure to whom non-invasive mechanical ventilation was performed and were compared to those who required invasive ventilation. The sample consisted of 51 patients divided into two groups: one of 38 patients who underwent non-invasive mechanical ventilation and another group of 13 patients who had not resolved and were treated invasively.Results: all the used successful predictors showed the effectiveness of non-invasive mechanical ventilation in the clinical diagnoses, with significance for the outcome in chronic obstructive pulmonary disease (76,47 %), bronchial asthma (81,81 %) and post-extubation acute respiratory failure (75 %). The physiological parameters decreased at the end of the assessment, with a statistically significant increase for the Glasgow Scale of 16,7±1,2. Average incremental changes were estimated for pH (7,40), PaCO2 (97,6), SaO2 (100,5) and the PaO2 / FiO2 ratio (286,3).Conclusions: the successful predictors made it possible to assess the satisfactory progress of the patients with non-invasive mechanical ventilation.

To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.

A random hexapeptide library (one-bead-one-compound), containing sixteen amino acids (16(6) different sequences) was synthesized on a Tentagel resin previously modified with a dipeptide linker (Asp-Pro). This peptide bond is highly susceptible to cleavage under mild acidic conditions in a salt-free solution prepared with H(2)(16)O/H(2)(18)O (60/40% v/v). In the hydrolysis, hexapeptides are released with an additional Asp residue partially labeled with (18)O at the C-terminus. These conditions are fully compatible with ESI-MS analysis and facilitate sequencing by MS, as N- and C-terminal ions can be easily differentiated in MS/MS spectra. The peptides were sequenced manually and also with de novo sequencing programs, and identifying them in a database containing all possible heptapeptide sequences or in a filtered database. The proposed strategy is also compatible with stepwise Edman degradation using either intact beads or the released free peptides.

Here we describe an integrated approach for the selective separation of peptides from complex mixtures using strong cation-exchange chromatography. The procedure exploits the charge differences produced by reversible modification of primary amino groups in peptides, enabling their separation into three major fractions: 1) neutral peptides 2) peptides with one positive charge and 3) peptides with 2 or more positive charges. The procedure demonstrated an excellent selectivity which allowed restricted MS/MS ion searches with peptide-centric databases.

Edman degradation in the gas phase has been observed by collision activated dissociation of N-terminal phenylthiocarbamoyl (PTC) protonated peptide to yield abundant complementary b₁ and y(n-1) ion pairs. Here, we demonstrated the relation between the observed losses of aniline and/or the entire PTC derivatizing group with the availability of mobile protons using electrospray ionization mass spectrometry. In order to select the peptides with more efficient fragmentation, while simplifying the mixture of peptides, we extend the phenylisotiocyanate (PITC) derivatization of amino groups to the selective isolation of multiply charged peptides (those having the number of arginines and histidines residues higher than one) using a procedure previously developed in our group. Thus, it was possible to identify in the filtered protein database the sequence of the isolated multiply charged peptides derived from a single protein and a complex mixture of proteins extracted from Escherichia coli using only the molecular mass and the N-terminal amino acid information. For this purpose, we developed a novel bioinformatic tool for automatic identification of peptides from liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments, which potentially can be used in high-throughput proteomics.

A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3- and d0-acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N-acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R + H ≤ 1) that are neither retained nor analyzed, whereas the multiply charged peptides (R + H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae.

Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC®) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains.

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.

Recently, we reported a method for the identification of free or blocked N-terminal peptides of proteins. 1 Briefly, the amino groups on lysine side chains and the N-terminal of the gel-entrapped protein are blocked with normal acetic or succinic anhydride and digested with a high-specificity protease. The generated peptides are retreated with an equimolar mixture of normal and deuterated acetic anhydride. In the mass spectrum, all the peptides show a complex isotopic ion distribution due to the artificial incorporation of heavy isotopes, with the exception of the N-terminal peptide that appears as a normal signal, allowing its rapid identification. Two main problems were observed in this method: (1) during electrospray ionization mass spectrometry (ESIMS) analysis, the C-terminal ions of any internal peptide originated by an in-source fragmentation process also have natural isotopic ion distribution and could be missassigned as an N-terminal peptide and (2) since the N-terminal peptide is not isotopically-labeled, their N- and the C-terminal ions can not be differentiated in the tandem mass spectrum (MS/MS) to achieve an easier and more reliable sequencing which is particularly advantageous when proteins derived from an organism with an unknown genome are analyzed. In this sense, a number of methods have been developed to facilitate the interpretation of MS/MS spectra. 2–7 In some of these works, a stable isotope label is introduced at the peptide N-terminus using a 1 : 1 mixture of normal and deuterated acetic anhydride, allowing an easy and unambiguous identification of the N- and C-terminal ion series in the product ion spectrum. 8,9

In this work, a method devised for the selective isolation of multiply-charged peptide applied to a complex protein mixture was evaluated for the first time using a mass spectrometer with low resolution (LTQ). In this procedure, all primary amino groups of tryptic peptides derived from human liver tissue interstitial fluid (TIF) are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply-charged peptides (#R + #H > 1) are retained in the column and separated with high selectivity from singly (#R + #H = 1) and neutral peptides (#R + #H = 0) which are collected together in the flow-through. Using liquid chromatography electrospray ionization tandem mass spectrometry analysis the retained fraction displayed a 95% enrichment of multiply charged peptides while in the flow-through; only 4% of multiply-charged peptides were identified.

Peptide sequence matching algorithms used for peptide identification by tandem mass spectrometry (MS/MS) enumerate theoretical peptides from the database, predict their fragment ions, and match them to the experimental MS/MS spectra. Here, we present an approach for scoring MS/MS identifications based on the high mass accuracy matching of precursor ions, the identification of a high intensity b1 fragment ion, and partial sequence tags from phenylthiocarbamoyl-derivatized peptides. This derivatization process boosts the b1 fragment ion signal, which turns it into a powerful feature for peptide identification. We demonstrate the effectiveness of our scoring system by implementing it on a computational tool called "HI-bone" and by identifying mass spectra of an Escherichia coli sample acquired on an Orbitrap Velos instrument using Higher-energy C-trap dissociation. Following this strategy, we identified 1614 peptide spectrum matches with a peptide false discovery rate (FDR) below 1%. These results were significantly higher than those from Mascot and SEQUEST using a similar FDR.