A Galaxy of informatics resources for MS-based proteomics.

INTRODUCTION: Continuous advances in mass spectrometry (MS) technologies have enabled deeper and more reproducible proteome characterization and a better understanding of biological systems when integrated with other 'omics data. Bioinformatic resources meeting the analysis requirements of increasingly complex MS-based proteomic data and associated multi-omic data are critically needed. These requirements included availability of software that would span diverse types of analyses, scalability for large-scale, compute-intensive applications, and mechanisms to ease adoption of the software. AREAS COVERED: The Galaxy ecosystem meets these requirements by offering a multitude of open-source tools for MS-based proteomics analyses and applications, all in an adaptable, scalable, and accessible computing environment. A thriving global community maintains these software and associated training resources to empower researcher-driven analyses. EXPERT OPINION: The community-supported Galaxy ecosystem remains a crucial contributor to basic biological and clinical studies using MS-based proteomics. In addition to the current status of Galaxy-based resources, we describe ongoing developments for meeting emerging challenges in MS-based proteomic informatics. We hope this review will catalyze increased use of Galaxy by researchers employing MS-based proteomics and inspire software developers to join the community and implement new tools, workflows, and associated training content that will add further value to this already rich ecosystem.

Prediction of Overall Survival by Thymidine Kinase 1 Combined with Prostate-Specific Antigen in Men with Prostate Cancer.

Thymidine kinase 1 (TK1) is an intracellular enzyme involved in DNA-precursor synthesis. Increased serum TK1 levels are used as a biomarker in various malignancies. We combined serum TK1 with PSA and evaluated its capacity to predict overall survival (OS) in 175 men with prostate cancer (PCa), detected by screening in 1988-1989 (n = 52) and during follow-up (median 22.6 years) (n = 123). TK1 was measured in frozen serum, age was stratified into four groups, and dates of PCa diagnosis and dates of death were obtained from Swedish population-based registries. The median concentration of TK1 and PSA was 0.25 and 3.8 ng/ml. TK1 was an independent variable of OS. In the multivariate analysis, PSA was not statistically significant in combination with age whereas the significance remained for TK1 + PSA. Measured once, TK1 + PSA predicted a difference of up to 10 years (depending on patient subgroup) in OS at a median of 9 years before PCa diagnosis. The TK1 concentration in 193 controls without malignancies did not differ from that of the PCa patients, hence TK1 was likely not released from incidental PCa. Thus, TK1 in the blood circulation may indicate the release of TK1 from sources other than cancers, nonetheless associated with OS.

Serum thymidine kinase 1 concentration as a predictive biomarker in prostate cancer.

BACKGROUND: Thymidine kinase 1 (TK1) recycles DNA before cell division. We do not know if baseline blood concentrations of TK1 predict death in prostate cancer within 30 years. Our objective is to determine if there is an association between baseline levels of TK1 and future prostate cancer-specific mortality. METHODS: With a "proof of concept" approach, we performed a nested case-control study among 1782 individuals screened for prostate cancer between 1988 and 1989. The concentration of TK1 was measured in frozen serum from 330 men, 36 of whom have died of prostate cancer. The primary endpoint was prostate cancer-specific mortality and outcomes after 30 years were analyzed using logistic regression modeling odds ratios (Ors). RESULTS: The estimated OR (adjusted for age) for dying from prostate cancer among the men who had a TK1 value in the upper tertile was 2.39 (95% confidence interval 1.02-5.63). The corresponding OR, regardless of the cause of death, was 2.81 (1.24-6.34). CONCLUSIONS: High levels of TK1 predicts death in prostate cancer within 30 years of follow-up.

Association between one-time prostate-specific antigen (PSA) test with free/total PSA ratio and prostate cancer mortality: A 30-year prospective cohort study.

OBJECTIVES: To explore if there is a long-term association between baseline prostate-specific antigen (PSA), including free/total PSA ratio and long-term (30-year) risk for prostate cancer death. SUBJECTS AND METHODS: In all, 1782 men were screened for prostate cancer through PSA analysis. Some years later, frozen plasma samples were used to calculate the ratio of free to total PSA (f/t PSA). At 30-year follow-up, baseline PSA and f/t PSA were compared with recent data extracts from the Swedish Cause of Death Registry and Swedish Cancer Registry. PSA values and f/t PSA values were treated as continuous variables in a multivariable analysis and also stratified according to their distribution and useful clinical thresholds. RESULTS: Risk of death from prostate cancer after 30 years of follow-up was significantly increased with a higher baseline PSA level, with the hazard ratio being 1.04 (95% confidence interval 1.03-1.09) per increase of one unit of PSA. Adding f/t PSA increased the model's ability to discriminate (concordance index 0.84-0.88). Men with PSA levels <1.0 ng/mL had a very low long-term risk of prostate cancer death (1.2% risk). An f/t PSA ≥ 0.25 extended the low-risk range to PSA < 2.0 ng/mL (1.5% risk). CONCLUSION: Prostate-specific antigen testing can be carried out less frequently or can be discontinued in men aged 55-70 years if their PSA levels are <2.0 ng/mL and the f/t PSA is ≥0.25.

Association between dihydrotestosterone and long-term risk for prostate cancer mortality: A prospective cohort study.

BACKGROUND: The androgen metabolism plays an important role in the progression of prostate cancer. Contradictory to what one might assume given the androgenic potency of dihydrotestosterone (DHT) there are indications that high DHT levels protect from prostate cancer. We want to determine whether there is a long-term association between baseline levels of DHT and death from prostate cancer. METHOD: During the years 1988 and 1989, 1782 men out of 2400 invited were screened for prostate cancer. The invited men were randomly selected from a background population of more than 27 000 men. Serum levels of DHT were analyzed for all 65 men diagnosed in the trial and 130 controls from the same cohort without any signs of prostate cancer. In this study we evaluate outcomes for the whole cohort (n = 195), the men without clinical signs of prostate cancer at beginning of follow up (n = 130) and men with screening detected cancer (n = 65). The cohort was followed up after 30 years and data from the Swedish Cause of Death Registry and the Swedish Cancer Registry were extracted. Hazard ratios (HRs) were calculated using Cox regression models. RESULT: High DHT levels were positively correlated to a lower risk for prostate cancer death in the entire cohort: HR = 0.44 (0.25-0.77 95% confidence interval [CI]). The positive correlation remained significant for the subgroup analysis. HR for the men enrolled in the study without any clinical signs of prostate cancer was 0.25 (0.07-0.88 95% CI) and for the men with a prostate cancer diagnosis at time of inclusion: HR = 0.50 (0.26-0.94 95% CI). CONCLUSION: DHT is negatively associated with long-term prostate cancer death regardless of clinical presentation at time of inclusion.

Secondary Structural Changes in Proteins as a Result of Electroadsorption at Aqueous-Organogel Interfaces.

The electroadsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated antiparallel ß-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI ToF MS measurements, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamic simulations, which modeled the structure of lysozyme at an aqueous-organic interface. On the basis of these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins.

Long-Term Outcome of a Single Intervention Population Based Prostate Cancer Screening Study.

PURPOSE: We evaluated the long-term effect of screening for prostate cancer. MATERIALS AND METHODS: In 1988 we randomly selected 2,400 men from a background population of 27,464 men. The 2,400 men were invited to undergo screening, of whom 1,779 (74%) accepted and were examined with digital rectal examination, ultrasound and prostate specific antigen measurement. Biopsy was performed if there were suspicious findings on ultrasound or digital rectal examination, or prostate specific antigen was greater than 10 ng/ml. The subpopulations have now been reassessed after 20 years. RESULTS: Participants had a decreased overall mortality rate compared to the source population (IRR 0.93, 95% CI 0.86-0.98). Nonparticipants had an increased overall mortality rate (IRR 1.25, 95% CI 1.14-1.37). There was no difference between the groups in prostate cancer specific survival. The incidence of prostate cancer remained higher in the screened population throughout followup. CONCLUSIONS: A single screening intervention in men 50 to 75 years old using prostate specific antigen, digital rectal examination and transrectal ultrasound, and a prostate specific antigen cutoff of 10 ng/ml for biopsy carried a significant risk of prostate cancer detection without a concomitant reduction in prostate cancer specific mortality after 20 years. This intervention should not be considered for public screening. Nonparticipants were at greater risk for death of all causes. In addition to being a single intervention trial, the limitations of this study include an outdated prostate specific antigen cutoff for biopsy. Despite the outdated screening method the source population failed to reach the same level of prostate cancer incidence as the screened population even after 20 years.

Plasma Proteome Association and Catalytic Activity of Stealth Polymer-Grafted Iron Oxide Nanoparticles.

Polyethylene glycol (PEG) is widely used as an antifouling and stealth polymer in surface engineering and nanomedicine. However, recent research has revealed adverse effects of bioaccumulation and immunogenicity following the administration of PEG, prompting this proteomic examination of the plasma protein coronae association with superparamagnetic iron oxide nanoparticles (IONPs) grafted with brushed PEG (bPEG) and an alternative, brushed phosphorylcholine (bPC). Using label-free quantitation by liquid chromatography tandem-mass spectrometry, this study determines protein abundances for the in vitro hard coronae of bare, bPC-, and bPEG-grafted IONPs in human plasma. This study also shows unique protein compositions in the plasma coronae of each IONP, including enrichment of coagulation factors and immunogenic complement proteins with bPEG, and enhanced binding of apolipoproteins with bPC. Functional analysis reveals that plasma protein coronae elevate the horseradish peroxidase-like activities of the bPC- and bPEG-IONPs by approximately twofold, an effect likely mediated by the diverse composition and physicochemical properties of the polymers as well as their associated plasma proteins. Taken together, these observations support the rational design of stealth polymers based on a quantitative understanding of the interplay between IONPs and the plasma proteome, and should prove beneficial for the development of materials for nanomedicine, biosensing, and catalysis.

Transarterial Chemoembolization of Renal Cell Carcinoma: A Prospective Controlled Trial.

PURPOSE: To prospectively assess feasibility, safety, and cytoreductive effect of transarterial chemoembolization on renal cell carcinoma (RCC) using drug-eluting embolic agent (DEE) saturated with doxorubicin compared with transarterial embolization (TAE). MATERIALS AND METHODS: Between 2012 and 2015, 12 patients (male/female = 5/7, age 66 y ± 9.8) with biopsy-verified RCC eligible for nephron-sparing surgery or radical nephrectomy were recruited. Mean tumor size was 3.2 cm ± 0.62. Patients were randomized at 1:1 ratio to receive either DEE transarterial chemoembolization or TAE before planned surgery. A microcatheter was used to inject particles selectively into arteries feeding the tumors. Response was evaluated by CT according to modified Response Evaluation Criteria In Solid Tumors and by microscopy of excised tumors. Complications were scored according to the Society of Interventional Radiology classification. RESULTS: DEE transarterial chemoembolization (n = 6) resulted in a significantly (P = .018) higher degree of necrosis with an average of 88.3% (range, 70%-100%) compared with TAE (n = 5), which resulted in an average of 29.4% (range, 0-77%), as evaluated by CT. Histopathologic evaluation showed similar results (P = .016) with an average necrosis of 87.5% (range, 80%-95%) for DEE transarterial chemoembolization (n = 4) versus 26% (range, 0-70%) for TAE (n = 5). Percentage of necrosis seen on microscopy correlated significantly (P = .0005) with radiologic findings, as 4 tumors in each arm were evaluated by both CT and microscopy. No major complications were observed in either group. CONCLUSIONS: DEE transarterial chemoembolization is safe for treating localized RCC and has a significantly superior cytoreductive effect compared with TAE.

Proteomic developments in the analysis of formalin-fixed tissue.

Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics.

Silver Coating for High-Mass-Accuracy Imaging Mass Spectrometry of Fingerprints on Nanostructured Silicon.

Nanostructure imaging mass spectrometry (NIMS) using porous silicon (pSi) is a key technique for molecular imaging of exogenous and endogenous low molecular weight compounds from fingerprints. However, high-mass-accuracy NIMS can be difficult to achieve as time-of-flight (ToF) mass analyzers, which dominate the field, cannot sufficiently compensate for shifts in measured m/z values. Here, we show internal recalibration using a thin layer of silver (Ag) sputter-coated onto functionalized pSi substrates. NIMS peaks for several previously reported fingerprint components were selected and mass accuracy was compared to theoretical values. Mass accuracy was improved by more than an order of magnitude in several cases. This straightforward method should form part of the standard guidelines for NIMS studies for spatial characterization of small molecules.

MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney.

Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.

Tryptic peptide reference data sets for MALDI imaging mass spectrometry on formalin-fixed ovarian cancer tissues.

MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC-MS/MS. The sample preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This resulted in 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per sample. From this, a total of 840 proteins and, on average, 297 proteins per sample could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several m/z species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer.

Matrix-assisted laser desorption/ionization imaging protocol for in situ characterization of tryptic peptide identity and distribution in formalin-fixed tissue.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the means to map the in situ distribution of tryptic peptides in formalin-fixed clinical tissue samples. The ability to analyze clinical samples is of great importance to further developments in the imaging field. However, there is a requirement in this field of research for additional methods describing the characterization of tryptic peptides by MALDI imaging. METHODS AND RESULTS: This protocol gives highly detailed instructions, with examples, for (1) successfully performing tryptic peptide MALDI imaging on formalin-fixed cancer tissue using a MALDI-TOF/TOF MS instrument, (2) tentatively generating identifications through nLC/MS/MS, and (3) validating these identifications by in situ MS/MS of peptides of interest. CONCLUSIONS: This protocol provides a detailed and straightforward description of the methods required for groups new to MALDI imaging to begin analysis of formalin-fixed clinical samples.

Active and passive short-wave infrared and near-infrared imaging for horizontal and slant paths close to ground.

This paper investigates active and passive short-wave infrared (SWIR) imaging for slant paths close to ground. The main sensor, a gated SWIR camera, was collecting both passive and active images along a 2 km long path over an airfield and also from our rooftop laboratory looking over open fields. For some investigations we also used a gated system working in the near-infrared region and thermal as well as color CCD cameras. The sensor was elevated by a lift in steps from 1.6-13.5 m or placed in a rooftop laboratory 13 m above ground. Targets were resolution charts and man targets. The turbulence was measured along the path with anemometers and scintillometers. The image performance was evaluated by measurement of the image blur and also by performing observer perception tests. The results reveal a strong dependence on the sensor height especially during daytime.

99mTc-Sestamibi SPECT/CT and histopathological features of oncocytic renal neoplasia.

BACKGROUND: 99mTc-Sestamibi Single Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) contributes to the non-invasive differentiation of renal oncocytoma (RO) from renal cell carcinoma (RCC) by characterising renal tumours as Sestamibi positive or Sestamibi negative regarding their 99mTc-Sestamibi uptake compared to the non-tumoral renal parenchyma. PURPOSE: To determine whether 99mTc- Sestamibi uptake in renal tumour and the non-tumoral renal parenchyma measured using Standard Uptake Value (SUV) SPECT, has a beneficial role in differentiating RO from RCC. MATERIAL AND METHODS: Fifty-seven renal tumours from 52 patients were evaluated. In addition to visual evaluation of 99mTc-Sestamibi uptake, SUVmax measurements were performed in the renal tumour and the ipsilateral non-tumoral renal parenchyma. Analysis of the area under the receiver operating characteristic curve identified an optimal cut-off value for detecting RO, based on the relative ratio of 99mTc- Sestamibi uptake. RESULTS: Semiquantitative evaluation of 99mTc-Sestamibi uptake did not improve the performance of 99mTc- Sestamibi SPECT/CT in detecting RO. 99mTc- Sestamibi SPECT/CT identifies a group of mostly indolent Sestamibi-positive tumours with low malignant potential containing RO, Low-Grade Oncocytic Tumours, Hybrid Oncocytic Tumours, and a subset of chromophobe RCCs. CONCLUSION: The imaging limitations for accurate differentiation of Sestamibi-positive renal tumours mirror the recognised diagnostic complexities of the histopathologic evaluation of oncocytic neoplasia. Patients with Sestamibi-positive renal tumours could be better suited for biopsy and follow-up, according to the current active surveillance protocols.

Correlation between circulatory, local prostatic, and intra-prostatic androgen levels.

BACKGROUND: Testosterone is converted to the more potent androgen dihydrotestosterone (DHT) in the prostate. DHT and androgen metabolites are inactivated by uridine diphospho (UDP)-glucuronosyl transferase (UGT) enzymes. Here we have studied the influence of the prostate gland on the systemic levels of DHT. Moreover, genetic variation in androgen metabolizing UGT enzymes and the intra-prostatic levels of glucuronidated DHT metabolites were investigated. METHODS: We collected peripheral serum, serum from the local prostatic veins and prostatic tissue from 25 patients undergoing radical prostatectomy. The serum and intra-tissular level of different androgen metabolites were determined by immunological assays and gas chromatography-mass spectrometry (GCMS), respectively. RESULTS: We found a significant positive correlation between the local prostatic serum DHT levels and (a) prostate weight and (b) circulatory serum levels. There were no correlation between in intra-prostatic hormonal levels and local DHT serum levels. The DHT metabolite 3α-diol-17-glucuronide and 3α-diol-3-glucuronide were significantly associated with UGT2B17 deletion and UGT2B15 Asp85Tyr polymorphisms, respectively. CONCLUSION: These results indicate that local prostatic DHT production has an influence on systemic serum DHT levels. Moreover, our results support the evidence that the prostate is the main DHT producer and that UGTs are important in the intra-prostatic regulation of androgens.

Rate of incisional hernia after minimally invasive and open surgery for renal cell carcinoma: a nationwide population-based study.

OBJECTIVES: To determine the rate of incisional hernia after surgery for renal cell carcinoma, to compare the rate after open vs minimally invasive surgery and radical nephrectomy vs partial nephrectomy and to identify risk factors for incisional hernia. MATERIALS AND METHODS: From the Renal Cell Cancer Database Sweden we identified all patients (n = 9,638) diagnosed with renal cell carcinoma in Sweden between January 2005 and November 2015. Of these, 6,417 were included in the analyses to determine comorbidity and subsequent diagnosis of or surgery for incisional hernia. RESULTS: In all, 6,417 patients underwent surgery for renal cell carcinoma between January 2005 and November 2015, of these 5,216 (81%) underwent open surgery and 1,201 (19%) underwent minimally invasive surgery. Altogether 140 patients were diagnosed with incisional hernia. The cumulative rate of incisional hernia after 5 years was 5.2% (95% confidence interval [CI] = 4.0-6.4%) after open surgery and 2.4% (95% CI = 1.0-3.4%) after minimally invasive surgery (p < 0.05). In Cox proportional hazard analysis, age and left-sided surgery were associated with incisional hernia in the open surgery group (both p < 0.05), whereas in the minimally invasive group, no statistically significant risk factors for incisional hernia were found. CONCLUSIONS: Open surgery for renal cell carcinoma is associated with a significantly higher risk for developing incisional hernia. If open surgery is the only option, care should be taken when choosing the approach and closing the wound. More studies are needed to find strategies to reduce the risk of abdominal wall complications following open kidney surgery.

Androgen Deprivation Therapy and the Risk for Inguinal Hernia: An Observational Nested Case Control Study.

It has been suggested that hypogonadism increases the risk for inguinal hernia (IH). The aim of this study was to investigate any association between androgen deprivation therapy (ADT) for prostate cancer and increased risk for IH. The study population in this population-based nested case-control study was based on data from the Prostate Cancer Database Sweden. The cohort included all men with prostate cancer who had not received curative treatment. Men who had been diagnosed or had undergone IH repair (n = 1,324) were cases and controls, where not diagnosed, nor operated on for IH, matched only on birth year (n = 13,240). Conditional multivariate logistic regression models were used to assess any temporal association between ADT and IH, adjusting for marital status, education level, prostate cancer risk category, Charlson Comorbidity Index, ADT, time since prostate cancer diagnosis, and primary prostate cancer treatment. Odds ratio (OR) for diagnosis/repair of IH 0 to 1 year from start of ADT was 0.5 (95% confidence interval [CI] = [0.38, 0.68]); between 1 and 3 years after, the OR was 0.35 (95% CI = [0.26, 0.47]); between 3 and 5 years after, the OR was 0.39 (95% CI = [0.26, 0.56]); between 5 and 7 years after, the OR was 0.6 (95% CI = [0.41, 0.97]); and >9 years after, the OR was 3.68 (95% CI = [2.45, 5.53]). The marked increase in OR for IH after 9 years of ADT supports the hypothesis that low testosterone levels increase the risk for IH. The low risk for IH during the first 8 years on ADT is likely caused by selection of men with advanced cancer unlikely to be diagnosed or treated for IH.