Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products.

Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.

Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations.

Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.

Identification of Recombinant Chimpanzee Adenovirus C68 Degradation Products Detected by AEX-HPLC.

Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer.

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.

Recombinant adeno-associated virus (rAAV) has emerged as an important gene therapy vector with many clinical trials currently in progress. Analytical characterization and quantitation of particle content remain challenges in both the development and production of rAAV vectors. In this study, charge detection mass spectrometry (CDMS) and gel electrophoresis are used to characterize the DNA content of recombinant AAV8 (rAAV8) vectors with a wide range of target genome sizes. We show that the differences between the masses of empty particles and particles with the genome of interest (GOI) are correlated with the expected genome mass. A small systematic deviation (around 2%) is attributed to the packaging of counterions along with the DNA. In addition to the GOI, a broad distribution of heterogeneous DNA is packaged. The distribution peaks are close to the packaging capacity of the rAAV8 vectors. There is also evidence for the co-packaging of small DNA fragments along with the GOI. Finally, we present evidence that incubation at an elevated temperature can reduce the heterogeneity of the packaged DNA. Taken together, these results show that CDMS is a viable tool for characterization of the packaged genome.

Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors.

Adeno-associated virus (AAV) vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. An inherent characteristic of the manufacturing process is production of empty capsids that lack the transgene and are therefore unable to provide the intended therapeutic benefit. The effect of empty capsids on clinical outcomes is not well understood, but there are immunogenicity and efficacy concerns, and these empty capsids are considered a product-related impurity. Therefore, empty capsids should be controlled during the manufacturing process and monitored through analytical testing, but there are limited techniques available that are capable of quantifying capsid content and even fewer that are amenable to validation and implementation as registered release tests in a regulated environment. In addition, there is currently not a widely accepted gold standard technique for quantifying capsid content, and the understanding of how the results compare between different orthogonal technologies is limited. The current study utilizes a comprehensive assessment to evaluate diverse analytical techniques for their ability to quantitate capsid content.

Enhancing the multi-attribute method through an automated and high-throughput sample preparation.

The multi-attribute method (MAM), a recent advance in the application of liquid chromatography-mass spectrometry within the pharmaceutical industry, enables the simultaneous monitoring of multiple product quality attributes in a single analytical method. While MAM is coupled with automated data processing and reporting, the sample preparation, based on proteolytic peptide mapping, remains cumbersome and low throughput. The standard sample preparation for MAM relies on protein denaturation, reduction, and alkylation prior to proteolytic digestion, but often a desalting step is required to maintain enzymatic activity. While most of the sample preparation can be automated on a standard robotic liquid handling system, a streamlined approach for protein desalting and temperature modulation is required for a viable, fully automated digestion. In this work, for the first time, a complete tip-based MAM sample preparation is automated on a single robotic liquid handling system, leveraging a deck layout that integrates both heating and cooling functionalities. The fully automated method documented herein achieves a high-throughput sample preparation for MAM, while maintaining superior method performance.Abbreviations: MAM: multi-attribute method; PQAs: product quality attributes; CE: capillary electrophoresis; IEX: ion-exchange chromatography; HILIC-FLR: hydrophilic interaction liquid chromatography coupled to a fluorescence detector; RP-LC/UV: reversed-phase liquid chromatography coupled to a UV detector; MS: mass spectrometry; NPD: new peak detection; GdnHCl: guanidine hydrochloride; TIC: total ion current; pAb: polyclonal antibody; IgG: immunoglobulin G; DTT: dithiothreitol; IAA: iodoacetic acid; TFA: trifluoroacetic acid; A280: absorbance at 280 nm wavelength; 96MPH: 96-channel multi-probe head; CPAC: Cold Plate Air Cooled; HHS: Hamilton Heater Shaker; DWP: Deep-Well Plate; PCR: Polymerase Chain Reaction; NTR: Nested Tip Rack; Met: methionine; Trp: tryptophan; N-term pQ: N-terminal glutamine cyclization; Lys: lysine; PAM: peptidylglycine α-amidating monooxygenase; G0F: asialo-, agalacto-, bi-antennary, core substituted with fucose; G1F: asialo-, mono-galactosylated bi-antennary, core substituted with fucose; G2F: asialo-, bi-galactosylated bi-antennary, core substituted with fucose; G0: asialo-, agalacto-, bi-antennary; Man5: oligomannose 5; Man8: oligomannose 8; TriF: asialo-, tri-galactosylated tri-antennary, core substituted with fucose.

Imaging Mass Spectrometry and Lectin Analysis of N-Linked Glycans in Carbohydrate Antigen-Defined Pancreatic Cancer Tissues.

The early detection of pancreatic ductal adenocarcinoma (PDAC) is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers carbohydrate antigen 19-9 (CA19-9) and sialylated tumor-related antigen (sTRA) are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and noncancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry (IMS) approach was used to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of patients with PDAC represented by tissue microarrays and whole-tissue sections. Orthogonally, these same tissues were characterized by multiround immunofluorescence that defined expression of CA19-9 and sTRA as well as other lectins toward carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated biantennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9-expressing tissues tended to be biantennary, triantennary, and tetra-antennary structures with both core and terminal fucose residues and bisecting GlcNAc. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored triantennary and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-immunohistochemistry and lectin-IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Defining the human kidney N-glycome in normal and cancer tissues using MALDI imaging mass spectrometry.

Clear-cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late-stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well-described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N-glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin-fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N-linked glycan-based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N-glycans with bisecting N-acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor-specific N-glycans were detected with tri- and tetra-antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N-glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N-glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers.

In Situ Imaging of N-Glycans by MALDI Imaging Mass Spectrometry of Fresh or Formalin-Fixed Paraffin-Embedded Tissue.

Glycosylation of cell surface, secreted, and circulating proteins is one of the most common types of post-translational modification. These modifications occur most commonly as one of three major classes: N-linked glycosylation on asparagine residues, O-linked glycosylation on serine or threonine residues, or as glycosaminoglycan oligosaccharide polymers on serine. Specifically, for N-linked glycans, an endoglycosidase enzyme, peptide N-glycosidase F (PNGase F), cleaves the attached oligosaccharides between the asparagine and first sugar. A method to analyze released N-glycans and map them to specific locations within a tissue is presented here. The PNGase F is applied by solvent sprayer as a molecular layer on frozen or formalin-fixed tissues and all released N-glycans in a given region of tissue are detected using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (MALDI-IMS). Using the described MALDI-IMS protocol, at least 40 or more individual N-glycans can be mapped to tissue histopathology and extracted for further structural analysis approaches. © 2018 by John Wiley & Sons, Inc.

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing.

Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 mm × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 ng. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 µL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3-linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.

FLI1 Levels Impact CXCR3 Expression and Renal Infiltration of T Cells and Renal Glycosphingolipid Metabolism in the MRL/lpr Lupus Mouse Strain.

The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.

Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry.

A new mass spectrometry imaging approach to simultaneously map the two-dimensional distribution of N-glycans in tissues has been recently developed. The method uses Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) to spatially profile the location and distribution of multiple N-linked glycan species released by peptide N-glycosidase F in frozen or formalin-fixed tissues. Multiple formalin-fixed human hepatocellular carcinoma tissues were evaluated with this method, resulting in a panel of over 30 N-glycans detected. An ethylation reaction of extracted N-glycans released from adjacent slides was done to stabilize sialic acid containing glycans, and these structures were compared to N-glycans detected directly from tissue profiling. In addition, the distribution of singly fucosylated N-glycans detected in tumor tissue microarray cores were compared to the histochemistry staining pattern of a core fucose binding lectin. As this MALDI-IMS workflow has the potential to be applied to any formalin-fixed tissue block or tissue microarray, the advantages and limitations of the technique in context with other glycomic methods are also summarized.

Altered glycosylation in prostate cancer.

Prostate cancer is annually the most common newly diagnosed cancer in men. The prostate functions as a major secretory gland for the production of glycoproteins critical to sperm activation and reproduction. Prostate-specific antigen (PSA), produced by the prostate, is one of the most commonly assayed glycoproteins in blood, serving as a biomarker for early detection and progression of prostate cancer. The single site of N-glycosylation on PSA has been the target of multiple glycan characterization studies. In this review, the extensive number of studies that have characterized the changes in O-linked and N-linked glycosylations associated with prostate cancer development and progression will be summarized. This includes analysis of the glycosylation of PSA, and other prostate glycoproteins, in tissues, clinical biofluids, and cell line models. Other studies are summarized in the context of understanding the complexities of these glycan changes in order to address the many confounding questions associated with prostate cancer, as well as efforts to improve prostate cancer biomarker assays using targeted glycomic-based strategies.

Renal glycosphingolipid metabolism is dysfunctional in lupus nephritis.

Nearly one half of patients with lupus develop glomerulonephritis (GN), which often leads to renal failure. Although nephritis is diagnosed by the presence of proteinuria, the pathology of nephritis can fall into one of five classes defined by different forms of tissue injury, and the mechanisms involved in pathogenesis are not completely understood. Glycosphingolipids are abundant in the kidney, have roles in many cellular functions, and were shown to be involved in other renal diseases. Here, we show dysfunctional glycosphingolipid metabolism in patients with lupus nephritis and MRL/lpr lupus mice. Specifically, we found that glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels are significantly higher in the kidneys of nephritic MRL/lpr lupus mice than the kidneys of non-nephritic lupus mice or healthy controls. This elevation may be, in part, caused by altered transcriptional regulation and/or activity of LacCer synthase (GalT5) and neuraminidase 1, enzymes that mediate glycosphingolipid metabolism. We show increased neuraminidase 1 activity early during the progression of nephritis (before significant elevation of GlcCer and LacCer in the kidney). Elevated levels of urinary LacCer were detected before proteinuria in lupus mice. Notably, LacCer levels were higher in the urine and kidneys of patients with lupus and nephritis than patients with lupus without nephritis or healthy controls. Together, these results show early and significant dysfunction of the glycosphingolipid metabolic pathway in the kidneys of lupus mice and patients with lupus nephritis and suggest that molecules in this pathway may serve as early markers in lupus nephritis.

MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

MALDI imaging mass spectrometry profiling of proteins and lipids in clear cell renal cell carcinoma.

Reducing the incidence and mortality rates for clear cell renal cell carcinoma (ccRCC) remains a significant clinical challenge with poor 5-year survival rates. A unique tissue cohort was assembled of matched ccRCC and distal nontumor tissues (n = 20) associated with moderate risk of disease progression, half of these from individuals who progressed to metastatic disease and the other half who remained disease free. These tissues were used for MALDI imaging MS profiling of proteins in the 2-20 kDa range, resulting in a panel of 108 proteins that had potential disease-specific expression patterns. Protein lysates from the same tissues were analyzed by MS/MS, resulting in identification of 56 proteins of less than 20 kDa molecular weight. The same tissues were also used for global lipid profiling analysis by MALDI-FT-ICR MS. From the cumulative protein and lipid expression profile data, a refined panel of 26 proteins and 39 lipid species was identified that could either distinguish tumor from nontumor tissues, or tissues from recurrent disease progressors from nonrecurrent disease individuals. This approach has the potential to not only improve prognostic assessment and enhance postoperative surveillance, but also to inform on the underlying biology of ccRCC progression.

Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.

A new matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for 2 h, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure, and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug, and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N-glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.

Increased bisecting N-acetylglucosamine and decreased branched chain glycans of N-linked glycoproteins in expressed prostatic secretions associated with prostate cancer progression.

PURPOSE: Using prostatic fluids rich in glycoproteins like prostate-specific antigen and prostatic acid phosphatase (PAP), the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging MS-based glycopeptide analysis platforms. EXPERIMENTAL DESIGN: A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. RESULTS: Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. CONCLUSION AND CLINICAL RELEVANCE: Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis.

Bronchoscopy simulator effectively prepares junior residents to competently perform basic clinical bronchoscopy.

BACKGROUND: Teaching procedural skills in a clinical setting is becoming increasingly difficult. Simulators can provide safe and inexpensive skills training. This randomized study was conducted to evaluate the effectiveness of a bronchoscopy simulator in teaching clinical bronchoscopy. METHODS: Three groups of surgical residents were evaluated while performing an intraoperative flexible bronchoscopy. First year (PGY1) residents were randomly assigned to perform bronchoscopy either with (n = 5) or without (n = 5) preprocedural bronchoscopic simulator training (PreOp flexible bronchoscopic simulator, Immersion Medical, Gaithersburg, MD). Residents PGY2 to 3 (n = 3) with prior bronchoscopic experience (> or = 10 bronchoscopies) underwent evaluation without simulator training. Subjects were required to complete a systematic airway examination through a laryngeal mask airway with patients under general anesthesia. Evaluation criteria included procedure time, number of verbal and physical interventions by evaluator, and a rating of exam thoroughness, proficiency, and confidence. RESULTS: The PGY1 subjects who trained on the simulator required significantly fewer verbal (6.2 +/- 1.6 vs 3.2 +/- 0.8) and physical (1.6 +/- 0.2 vs 0.2 +/- 0.4) cues and performed more systematic examinations (2.6 +/- 0.5 vs 4.4 +/- 0.9 on scale 1 to 5) than those who did not use the trainer. The skill level of PGY1 subjects who worked with the simulator was similar to that of PGY2 to 3 residents experienced in bronchoscopy. Procedural times were not different between groups as the evaluator maintained the pace of the examination using verbal and physical assistance. CONCLUSIONS: One hour of training with the bronchoscopic simulator effectively taught residents basic bronchoscopy and familiarity with airway anatomy. Residents using the trainer performed first-time bronchoscopy nearly as competently as residents experienced with bronchoscopy.