Assessment and Evaluation of Psychosocial Distress in Outpatients with Cancer at a University Hospital in Germany.

INTRODUCTION: Cancer patients (pts) suffer from a significant amount of psychosocial distress related to tumor disease itself or straining treatments. Despite recommendations on how to screen for and to deal with psychosocial distress in cancer pts, data about implementation of psycho-oncological interventions (poi) in outpatient settings of cancer pts are scarce. The aim of this study was to identify outpatients with cancer in need of poi and to evaluate different assessment instruments. METHODS: N = 200 outpatients with hemat-/oncological malignancies were interviewed between October 2015 and December 2017 at the University Hospital Mannheim using the Basic Documentation for Psycho-Oncology (PO-Bado) and the Hornheider Screening Instrument (HSI) - both clinician-administered assessment tools - followed by descriptive, univariate, and agreement analysis. RESULTS: N = 61 cancer pts (31%) were identified to be in need for poi considering the results of both questionnaires. The number of identified pts in need of poi was lower when analyzing the results of the PO-Bado (n = 42, 21%) and the HSI (n = 39, 20%) separately. The degree of agreement between the results of PO-Bado and HSI was low (kappa = 0.3655). Several factors like gender, age and diagnosis were identified to have significant impact on the need for poi (p ≤ 0.05). CONCLUSION: Our study underlines that different screening instruments for psychosocial distress may identify disparate populations of cancer pts. The study data also revealed significant characteristics that might be associated with elevated levels of psychosocial distress and a clear indication for poi. However, further analyses on larger populations of cancer pts are needed to provide information how to transfer positive screening to poi in clinical routine.

N-glycoproteome of E14.Tg2a mouse embryonic stem cells.

E14.Tg2a mouse embryonic stem (mES) cells are a widely used host in gene trap and gene targeting techniques. Molecular characterization of host cells will provide background information for a better understanding of functions of the knockout genes. Using a highly selective glycopeptide-capture approach but ordinary liquid chromatography coupled mass spectrometry (LC-MS), we characterized the N-glycoproteins of E14.Tg2a cells and analyzed the close relationship between the obtained N-glycoproteome and cell-surface proteomes. Our results provide a global view of cell surface protein molecular properties, in which receptors seem to be much more diverse but lower in abundance than transporters on average. In addition, our results provide a systematic view of the E14.Tg2a N-glycosylation, from which we discovered some striking patterns, including an evolutionarily preserved and maybe functionally selected complementarity between N-glycosylation and the transmembrane structure in protein sequences. We also observed an environmentally influenced N-glycosylation pattern among glycoenzymes and extracellular matrix proteins. We hope that the acquired information enhances our molecular understanding of mES E14.Tg2a as well as the biological roles played by N-glycosylation in cell biology in general.

Analysis of RP-HPLC loading conditions for maximizing peptide identifications in shotgun proteomics.

Substantial energy and resources have been invested in improving mass spectrometry (MS) instrumentation, upstream sample preparation protocols, and database search strategies to maximize peptide and protein identifications. The role of HPLC sample loading methods in maximizing MS identifications has been largely overlooked, and there exists an immense heterogeneity in the methods employed in the proteomics literature. We sought to optimize loading methods by testing multiple loading conditions (buffer composition, resin, initial gradient) using tryptic digests of an 18 protein mixture and whole yeast lysate. The loading buffer acetonitrile (ACN) concentration greatly affected peptide identifications: up to a 26% increase in peptide identifications was observed by decreasing the ACN concentration from 5 to 2% during sample loading. Hydrophilic peptides were the main contributors to the increase in peptide identifications and, at higher ACN concentrations, were washed from the precolumn during desalting. Sampling of the hydrophilic peptides was enhanced by using a shallow initial ACN gradient. The results were found to be resin-specific and not generalizable. Our investigation demonstrates the often unappreciated importance of optimizing sample loading conditions to reflect the aims of the research and the characteristics of the LC configurations employed.

Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N.

Proteomic analysis typically has been performed using proteins digested with trypsin because of the excellent fragmentation patterns they produce in collision induced dissociation (CID). For analyses in which high protein coverage is desirable, such as global monitoring of post-translational modifications, additional sequences can be seen using parallel digestion with a second enzyme. We have benchmarked a relatively obscure basidomycete-derived zinc metalloendopeptidase, Lys-N, that selectively cleaves the amide bond N-terminal of lysine residues. We have found that Lys-N digestion yields peptides with easily assigned CID spectra. Using a mixture of purified proteins as well as a complex yeast lysate, we have shown that Lys-N efficiently digests all proteins at the predicted sites of cleavage. Shotgun proteomics analyses of Lys-N digests of both the standard mixture and yeast lysate yielded peptide and protein identification numbers that were generally comparable to trypsin digestion, whereas the combination data from Lys-N and trypsin digestion substantially enhanced protein coverage. During CID fragmentation, the additional amino terminal basicity enhanced b-ion intensity which was reflected in long b-ion tags that were particularly pronounced during CID in a quadrupole. Finally, immonium ion peaks produced from Lys-N digested peptides originate from the carboxy terminus in contrast to tryptic peptides where immonium ions originate from the amino terminus.

Nonenzymatic biotinylation of histone H2A.

Holocarboxylase synthetase (HCS, eukaryotic enzyme) and BirA (prokaryotic) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases via the reactive intermediate, bio-5'-AMP. In this study, we examined the in vitro mechanism of biotin attachment to histone H2A in the presence of HCS and BirA. The experiment derives from our observations that HCS is found in the nucleus of cells in addition to the cytoplasm, and it has the ability to attach biotin to histones in vitro (Narang et al., Hum Mol Genet 2004; 13:15-23). Using recombinant HCS or BirA, the rate of biotin attachment was considerably slower with histone H2A than with the biotin binding domain of an apocarboxylase. However, on incubation of recombinant H2A with chemically synthesized bio-5'-AMP, H2A was observed to be rapidly labeled with biotin in the absence of enzyme. Nonenzymatic biotinylation of a truncated apocarboxylase (BCCP87) has been previously reported (Streaker and Beckett, Protein Sci 2006; 15:1928-1935), though at a much slower rate than we observe for H2A. The specific attachment sites of nonenzymatically biotinylated recombinant H2A at different time points were identified using mass spectrometry, and were found to consist of a similar pattern of biotin attachment as seen in the presence of HCS, with preference for lysines in the highly basic N-terminal region of the histone. None of the lysine sites within H2A resembles the biotin attachment consensus sequence seen in carboxylases, suggesting a novel mechanism for histone biotinylation.

Staphylococcus aureus elicits marked alterations in the airway proteome during early pneumonia.

Pneumonia caused by Staphylococcus aureus is a growing concern in the health care community. We hypothesized that characterization of the early innate immune response to bacteria in the lungs would provide insight into the mechanisms used by the host to protect itself from infection. An adult mouse model of Staphylococcus aureus pneumonia was utilized to define the early events in the innate immune response and to assess the changes in the airway proteome during the first 6 h of pneumonia. S. aureus actively replicated in the lungs of mice inoculated intranasally under anesthesia to cause significant morbidity and mortality. By 6 h postinoculation, the release of proinflammatory cytokines caused effective recruitment of neutrophils to the airway. Neutrophil influx, loss of alveolar architecture, and consolidated pneumonia were observed histologically 6 h postinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) or S. aureus were depleted of overabundant proteins and subjected to strong cation exchange fractionation followed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the airway. No significant changes in response to PBS inoculation or 30 min following S. aureus inoculation were observed. However, a dramatic increase in extracellular proteins was observed 6 h postinoculation with S. aureus, with the increase dominated by inflammatory and coagulation proteins. The data presented here provide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the host airway during the earliest stages of S. aureus pneumonia.

Quantification of the compositional information provided by immonium ions on a quadrupole-time-of-flight mass spectrometer.

Immonium ions have been largely overlooked during the rapid expansion of mass spectrometry-based proteomics largely due to the dominance of ion trap instruments in the field. However, immonium ions are visible in hybrid quadrupole-time-of-flight (QTOF) mass spectrometers, which are now widely available. We have created the largest database to date of high-confidence sequence assignments to characterize the appearance of immonium ions in CID spectra using a QTOF instrument under "typical" operating conditions. With these data, we are able to demonstrate excellent correlation between immonium ion peak intensity and the likelihood of the appearance of the expected amino acid in the assigned sequence for phenylalanine, tyrosine, tryptophan, proline, histidine, valine, and the indistinguishable leucine and isoleucine residues. In addition, we have clearly demonstrated a positional effect whereby the proximity of the amino acid generating the immonium ion to the amino terminal of the peptide correlates with the strength of the immonium ion peak. This compositional information provided by the immonium ion peaks could substantially improve algorithms used for spectral assignment in mass spectrometry analysis using QTOF platforms.

The standard protein mix database: a diverse data set to assist in the production of improved Peptide and protein identification software tools.

Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.

The anatomy of microbial cell state transitions in response to oxygen.

Adjustment of physiology in response to changes in oxygen availability is critical for the survival of all organisms. However, the chronology of events and the regulatory processes that determine how and when changes in environmental oxygen tension result in an appropriate cellular response is not well understood at a systems level. Therefore, transcriptome, proteome, ATP, and growth changes were analyzed in a halophilic archaeon to generate a temporal model that describes the cellular events that drive the transition between the organism's two opposing cell states of anoxic quiescence and aerobic growth. According to this model, upon oxygen influx, an initial burst of protein synthesis precedes ATP and transcription induction, rapidly driving the cell out of anoxic quiescence, culminating in the resumption of growth. This model also suggests that quiescent cells appear to remain actively poised for energy production from a variety of different sources. Dynamic temporal analysis of relationships between transcription and translation of key genes suggests several important mechanisms for cellular sustenance under anoxia as well as specific instances of post-transcriptional regulation.

Assessing bias in experiment design for large scale mass spectrometry-based quantitative proteomics.

Mass spectrometry-based proteomics holds great promise as a discovery tool for biomarker candidates in the early detection of diseases. Recently much emphasis has been placed upon producing highly reliable data for quantitative profiling for which highly reproducible methodologies are indispensable. The main problems that affect experimental reproducibility stem from variations introduced by sample collection, preparation, and storage protocols and LC-MS settings and conditions. On the basis of a formally precise and quantitative definition of similarity between LC-MS experiments, we have developed Chaorder, a fully automatic software tool that can assess experimental reproducibility of sets of large scale LC-MS experiments. By visualizing the similarity relationships within a set of experiments, this tool can form the basis of systematic quality control and thus help assess the comparability of mass spectrometry data over time, across different laboratories, and between instruments. Applying Chaorder to data from multiple laboratories and a range of instruments, experimental protocols, and sample complexities revealed biases introduced by the sample processing steps, experimental protocols, and instrument choices. Moreover we show that reducing bias by correcting for just a few steps, for example randomizing the run order, does not provide much gain in statistical power for biomarker discovery.

An integrated systems approach for understanding cellular responses to gamma radiation.

Cellular response to stress entails complex mRNA and protein abundance changes, which translate into physiological adjustments to maintain homeostasis as well as to repair and minimize damage to cellular components. We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to (60)Co ionizing gamma radiation in an effort to understand the correlation between genetic information processing and physiological change. The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein-DNA interactions and evolutionarily conserved functional associations. This systems view reveals cooperation among several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady-state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation.