The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

No single-omic approach completely elucidates the multitude of alterations taking place in Alzheimer's disease (AD). Here, we coupled transcriptomic and phosphoproteomic approaches to determine the temporal sequence of changes in mRNA, protein, and phosphopeptide expression levels from human temporal cortical samples, with varying degree of AD-related pathology. This approach highlighted fluctuation in synaptic and mitochondrial function as the earliest pathological events in brain samples with AD-related pathology. Subsequently, increased expression of inflammation and extracellular matrix-associated gene products was observed. Interaction network assembly for the associated gene products, emphasized the complex interplay between these processes and the role of addressing post-translational modifications in the identification of key regulators. Additionally, we evaluate the use of decision trees and random forests in identifying potential biomarkers differentiating individuals with different degree of AD-related pathology. This multiomic and temporal sequence-based approach provides a better understanding of the sequence of events leading to AD.

No single-omic approach completely elucidates the multitude of alterations taking place in Alzheimer's disease (AD). Here, we coupled transcriptomic and phosphoproteomic approaches to determine the temporal sequence of changes in mRNA, protein, and phosphopeptide expression levels from human temporal cortical samples, with varying degree of AD-related pathology. This approach highlighted fluctuation in synaptic and mitochondrial function as the earliest pathological events in brain samples with AD-related pathology. Subsequently, increased expression of inflammation and extracellular matrix-associated gene products was observed. Interaction network assembly for the associated gene products, emphasized the complex interplay between these processes and the role of addressing post-translational modifications in the identification of key regulators. Additionally, we evaluate the use of decision trees and random forests in identifying potential biomarkers differentiating individuals with different degree of AD-related pathology. This multiomic and temporal sequence-based approach provides a better understanding of the sequence of events leading to AD.

A subset of C9orf72 repeat expansion-carrying frontotemporal dementia patients display an Alzheimer-like decrease in cerebrospinal fluid amyloid-β (Aβ) biomarker levels. We report that downregulation of C9orf72 in non-neuronal human cells overexpressing amyloid-β protein precursor (AβPP) resulted i n increased levels of secreted AβPP fragments and Aβ, while levels of AβPP or its C-terminal fragments (CTFs) remained unchanged. In neuronal cells, AβPP and C83 CTF levels were decreased upon C9orf72 knockdown, but those of secreted AβPP fragments or Aβ remained unchanged. C9orf72 protein levels significantly increased in human brain with advancing neurofibrillary pathology and positively correlated with brain Aβ42 levels. Our data suggest that altered C9orf72 levels may lead to cell-type specific alterations in AβPP processing, but warrant further studies to clarify the underlying mechanisms.

Background It is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery. Methods We have applied TMTcalibrator™, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates. Results The biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients. Conclusions These findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model.

RATIONALE Ideal biomarkers are present in readily accessible samples such as plasma or CSF and are directly derived from the diseased tissue. As such they are likely to be of relatively low abundance as they are diluted into a large fluid volume. Traditional unbiased proteomic approaches for biomarker discovery have struggled to detect low abundance markers due to the high dynamic range of plasma and CSF proteins, the predominance of plasma albumin and other hyper-abundant proteins and the use of data dependent acquisition mass spectrometry. The use of data independent acquisition partially address sensitivity but are limited to analysis of single samples and require prior production of spectral libraries from an appropriate sample. METHODS To overcome these limitations and improve biomarker discovery in peripheral fluids, we have developed TMTcalibrator; a novel mass spectrometry workflow that allows the use of isobarically labeled diseased tissue digests in parallel with an appropriate set of labeled body fluids to increase the chance of identifying low abundance, tissue-derived biomarkers. Here the use of isobaric Tandem Mass Tags allows suitable tissue or cell line samples to be labelled and mixed into equivalently labelled samples of peripheral biofluids at a range of concentrations that provide a multi-point calibration curve to improve statistical analysis, and whose total amount is sufficient to ensure the vast majority of data dependent MS/MS acquisitions are made on tissue or cell-derived peptides. RESULTS For each peptide that is also present in the chosen body fluid, even when at a low abundance, the fragmentation of the unique TMT tag allows the relative abundance to be determined relative to the calibration curve created by the reference tissue calibrator. A further key aspect of this method is the use of Synchronous Precursor Selection (SPS) a form of MS3 analysis that eliminates quantitative interference from co-isolated ion species that is only available on the Orbitrap Fusion Tribrid. The use of SPS allows direct analysis of nondepleted, unfractionated CSF samples with the complete profiling of 6 individual patient samples requiring only 15 hours of mass spectrometer time, equivalent to 1.5 h per sample. CONCLUSIONS Here we apply the TMTcalibrator approach to identify markers of microglia activation in the cerebrospinal fluid of Alzheimer’s disease (AD) patients.

Background: HER2 is an important target for antitumor therapy in several cancers, and while currently available HER2-targeting drugs provide a great treatment improvement they rarely achieve full disease control. We have developed a new HER2-targeting molecule with a unique pro-apoptotic mode of action that may provide additional benefit to patients. The DARPin® molecule MP0274 shows potency in several HER2-expressing PDX models and has been shown to directly induce apoptosis in cells that are addicted to HER2. Here we show through phospho-proteome analyses that MP0274 not only potently inhibits HER2/HER3 downstream signaling, but also shows a very distinct changes in the phospho-proteome pattern compared to approved HER2-targeting drugs. This provides mechanistic support to the unique mode of action of MP0274 that results in direct tumor cell killing without the need of effector-functions like ADCC. Methods: The effect of MP0274 on HER2 signaling was compared to trastuzumab, pertuzumab and a combination of both in the HER2-addicted cancer cell lines. Briefly, cells were incubated with drugs for 18 hours and then lysed. Lysates were analyzed for changes in the total proteome and phospho-proteome by Proteome Sciences’ proprietary SysQuant® Global Phosphoproteomics workflow. Results: Heat-maps of genes of interest indicate that MP0274 has a differential mode of action compared to trastuzumab, pertuzumab or a combination of both. On the total peptide and phospho-peptide level, the samples cluster specifically, based on the cancer cells used as well as drug treatment. Twenty nine unique global phosphorylation sites specific to HER2, including C-terminal tyrosines which are reported to recruit adaptor proteins starting signaling processes after auto-phosphorylation, were identified for MP0274. Several proteins were identified which were differentially expressed and phosphorylated after MP0274 treatment and which are involved in three key downstream signaling pathways activated by HER2/HER3 heterodimers: RAF/MAP kinase cascade, PI3K-induced AKT signaling, and signaling by PLCG1. Conclusions: MP0274 shows a unique and distinct inhibition of the HER2 signaling cascade, different from trastuzumab, pertuzumab and a combination of both. It induces a more profound inhibition of downstream signaling which provides mechanistic support to the finding that MP0274 direct cell killing by induction of apoptosis in HER2-addicted tumor cells. * DARPins are small repeat proteins, designed to bind targets with high affinity and specificity, and which can be combined in a modular fashion to produce multi-functional agents. Citation Format: Vikram Mitra, Ulrike Fiedler, Dan Snell, Keith M. Dawson, Stephan Jung, Ian Pike, Elmar vom Baur. Phospho-proteome analyses confirm the unique mode of action of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin® drug candidate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2017-4966

Label-free LC-MS(/MS) provides accurate quantitative profiling of proteins and metabolites in complex biological samples such as cell lines, tissues and body fluids. A label-free experiment consists of several LC-MS(/MS) chromatograms that might be acquired over several days, across multiple laboratories using different instruments. Single-stage part (MS1 map) of the LC-MS(/MS) contains quantitative information on all compounds that can be detected by LC-MS(/MS) and is the data of choice used by quantitative LC-MS(/MS) data pre-processing workflows. Differences in experimental conditions and fluctuation of analytical parameters influence the overall quality of the MS1 maps and are factors hampering comparative statistical analyses and data interpretation. The quality of the obtained MS1 maps can be assessed based on changes in the two separation dimensions (retention time, mass-to-charge ratio) and the readout (ion intensity) of MS1 maps. In this tutorial we discuss two types of changes, monotonic and non-monotonic shifts, which may occur in the two separation dimensions and the readout of MS1 map. Monotonic shifts of MS1 maps can be corrected, while non-monotonic ones can only be assessed but not corrected, since correction would require precise modelling of the underlying physicochemical effects, which would require additional parameters and analysis. We discuss reasons for monotonic and non-monotonic shifts in the two separation dimensions and readout of MS1 maps, as well as algorithms that can be used to correct monotonic or to assess the extent non-monotonic shifts. Relation of non-monotonic shift with peak elution order inversion and orthogonality as defined in analytical chemistry is discussed. We aim this tutorial for data generator and evaluators scientists who aim to known the condition and approaches to produce and pre-processed comparable MS1 maps.

RATIONALE:Ideal biomarkers are present in readily accessible samples including plasma and cerebrospinal fluid (CSF), and are directly derived from diseased tissue, therefore likely to be of relatively low abundance. Traditional unbiased proteomic approaches for biomarker discovery have struggled to detect low-abundance markers due to the high dynamic range of proteins, the predominance of hyper-abundant proteins, and the use of data-dependent acquisition mass spectrometry (MS). To overcome these limitations and improve biomarker discovery in peripheral fluids, we have developed TMTcalibrator™; a novel MS workflow combining isobarically labelled diseased tissue digests in parallel with an appropriate set of labelled body fluids to increase the chance of identifying low-abundance, tissue-derived biomarkers.METHODS:A disease relevant cell line was labelled with TMT® in a range of concentrations generating a multi-point calibration curve. Peripheral biofluid samples were labelled with the remaining tags and quantitative analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer with a Top10 CID-HCD MS3 synchronous precursor selection (SPS) method. SPS allowed direct analysis of non-depleted, unfractionated CSF samples with complete profiling of six individual samples requiring only 15 hours of MS time, equivalent to 1.5 h per sample.RESULTS:Using the TMTcalibrator™ workflow allowed the identification of several markers of microglia activation that are differentially quantified in the CSF of patients with Alzheimer's disease (AD). We report peptides from 41 proteins that have not previously been detected in the CSF, that appear to be regulated by at least 60% in AD.CONCLUSIONS:This study has demonstrated the benefits of the new TMTcalibrator™ workflow and the results suggest this is a suitable and efficient method of detecting low-abundance peptides within biological fluids. The use of TMTcalibrator™ in further biomarker discovery studies should be considered to overcome some of the limitations commonly associated with more conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.

Aberrant tau phosphorylation is a hallmark in Alzheimer's disease (AD), believed to promote formation of paired helical filaments, the main constituent of neurofibrillary tangles in the brain. While cerebrospinal fluid (CSF) levels of total tau and tau phosphorylated at threonine residue 181 (pThr181) are established core biomarkers for AD, the value of alternative phosphorylation sites, which may have more direct relevance to pathology, for early diagnosis is not yet known, largely due to their low levels in CSF and lack of standardized detection methods. To overcome sensitivity limitations for analysis of phosphorylated tau in CSF, we have applied an innovative mass spectrometry (MS) workflow, TMTcalibratortrademark, to enrich and enhance the detection of phosphoproteome components of AD brain tissue in CSF, and enable the quantitation of these analytes. We aimed to identify which tau species present in the AD brain are also detectable in CSF and which, if any, are differentially regulated with disease. Over 75% coverage of full-length (2N4R) tau was detected in the CSF with 47 phosphopeptides covering 31 different phosphorylation sites. Of these, 11 phosphopeptides were upregulated by at least 40%, along with an overall increase in tau levels in the CSF of AD patients relative to controls. Use of the TMTcalibratortrademark workflow dramatically improved our ability to detect tau-derived peptides that are directly related to human AD pathology. Further validation of regulated tau peptides as early biomarkers of AD is warranted and is currently being undertaken.

BACKGROUND: There are currently no accurate diagnostic or prognostic tools for ALS, resulting in delayed diagnosis and poor disease stratification. Although the aetiology of ALS is still unknown, a hallmark of this neurological condition is the presence of protein aggregates within neurons (1). In addition, ALS and other neurodegenerative diseases are associated with a compromised blood brain barrier (BBB), resulting in leakage of brain-derived proteins into plasma (2). Thus, plasma could represent a readily accessible source of diagnostic and prognostic biomarkers. We and others have shown that Neurofilaments (NFs) in cerebrospinal fluid (CSF) and plasma are a promising ALS biomarker, but suffer from high variability within and between patients. Interestingly, our data suggest that this could in part be due to epitope masking caused by protein aggregation. We propose that these circulating NF aggregates will have multiple protein components, reflecting both mechanistic and random associations. Thus, the identification of these aggregate components may provide a source of sensitive and specific biomarkers for the early diagnosis and prognostic monitoring of ALS patients. OBJECTIVES: To develop and validate a method for the isolation of circulating blood protein aggregates and to characterise the composition of these aggregates by Mass Spectrometry (MS) based Proteomics. METHODS: In order to establish the best isolation method, a Pooled Plasma sample (PP) has been created using plasma from six Healthy Controls between 51.2-62.9 years of age and with known blood level of NF heavy chain (between 7.0 and 42.9ng/ml). Two different conditions for aggregate isolation have been evaluated, Sedimentation velocity and Seprion TM ligand interaction. RESULTS: Western blot (WB) showed a detectable amount of NFs in the aggregate fractions. Tandem MS data showed the presence of 651 and 1068 proteins (Protein Grouping: True; Peptide Confidence: High; Minimal number of peptides: 1) for the sedimentation and ligand methods, respectively. 380 common proteins were found between the two methods. DISCUSSION: We showed that sedimentation is a good methodology for isolation of aggregates circulating in blood. The detection of high molecular weight bands by WB suggests the presence of aggregates containing NFs in our pellets. Also, from proteomics data it is clear that these two methods isolate cytoskeletal and other intracellular proteins, potential allowing the detection of CNS derived material. Further analysis is ongoing to evaluate the best methods for isolation and analysis. AKNOLEDGMENTS: This project is funded by an MRC-Industry CASE Studentship. Plasma samples obtained from study 09/H0703/27. REFERENCES: Blokhuis AM, Groen EJ, Koppers M, van den Berg LH, Pasterkamp RJ. Protein aggregation in amyotrophic lateral sclerosis. Acta neuropathologica. 2013;125(6):777-94. Garbuzova-Davis S, Hernandez-Ontiveros DG, Rodrigues MC, Haller E, Frisina-Deyo A, Mirtyl S, et al. Impaired blood-brain/spinal cord barrier in ALS patients. Brain research. 2012;1469:114-28.

Complex shotgun proteomics peptide profiles obtained in quantitative differential protein expression studies, such as in biomarker discovery, may be affected by multiple experimental factors. These preanalytical factors may affect the measured protein abundances which in turn influence the outcome of the associated statistical analysis and validation. It is therefore important to determine which factors influence the abundance of peptides in a complex proteomics experiment and to identify those peptides that are most influenced by these factors. In the current study we analyzed depleted human serum samples to evaluate experimental factors that may influence the resulting peptide profile such as the residence time in the autosampler at 4 °C, stopping or not stopping the trypsin digestion with acid, the type of blood collection tube, different hemolysis levels, differences in clotting times, the number of freeze-thaw cycles, and different trypsin/protein ratios. To this end we used a two-level fractional factorial design of resolution IV (2(IV)(7-3)). The design required analysis of 16 samples in which the main effects were not confounded by two-factor interactions. Data preprocessing using the Threshold Avoiding Proteomics Pipeline (Suits, F.; Hoekman, B.; Rosenling, T.; Bischoff, R.; Horvatovich, P. Anal. Chem. 2011, 83, 7786-7794, ref 1) produced a data-matrix containing quantitative information on 2,559 peaks. The intensity of the peaks was log-transformed, and peaks having intensities of a low t-test significance (p-value > 0.05) and a low absolute fold ratio (<2) between the two levels of each factor were removed. The remaining peaks were subjected to analysis of variance (ANOVA)-simultaneous component analysis (ASCA). Permutation tests were used to identify which of the preanalytical factors influenced the abundance of the measured peptides most significantly. The most important preanalytical factors affecting peptide intensity were (1) the hemolysis level, (2) stopping trypsin digestion with acid, and (3) the trypsin/protein ratio. This provides guidelines for the experimentalist to keep the ratio of trypsin/protein constant and to control the trypsin reaction by stopping it with acid at an accurately set pH. The hemolysis level cannot be controlled tightly as it depends on the status of a patient's blood (e.g., red blood cells are more fragile in patients undergoing chemotherapy) and the care with which blood was sampled (e.g., by avoiding shear stress). However, its level can be determined with a simple UV spectrophotometric measurement and samples with extreme levels or the peaks affected by hemolysis can be discarded from further analysis. The loadings of the ASCA model led to peptide peaks that were most affected by a given factor, for example, to hemoglobin-derived peptides in the case of the hemolysis level. Peak intensity differences for these peptides were assessed by means of extracted ion chromatograms confirming the results of the ASCA model.

Aberrant tau phosphorylation is a pathological hallmark in Alzheimer's disease (AD) promoting formation of paired helical filaments, the main constituent of neurofibrillary tangles. Whilst CSF levels of total tau and pThr181 tau are considered biomarkers for AD the value of alternative phosphorylation sites, that may have more direct relevance to pathology, for early diagnosis is not yet known, largely due to their low levels in CSF and lack of standardised detection methods. To overcome sensitivity limitations for analysis of phosphorylated tau in CSF we have applied a combination of our SysQuant® and TMTcalibrator+™ mass spectrometry (MS) workflows, to enrich and quantify the phosphoproteome of AD post mortem brain tissue, and identify which tau species present in the brain are also in CSF and which, if any, are differentially regulated with disease. Using isobaric TMT®, peptide preparations from AD brain tissue were labelled to form a standard reference peptide mix. Similarly, CSF samples (600μl) from three patients with clinically and biomarker defined AD, and three non-AD controls were digested and labelled with the remaining tags within the TMT 10-plex set. All labelled digests were pooled together to generate the TMTcalibrator+ analytical sample. The labelled brain tissue samples were mixed with the CSF at a concentration sufficient to ensure the vast majority of MS/MS acquisitions are made on tissue-derived peptides. The sample was fractionated and phosphopeptides were enriched by TiO2 and IMAC. Additionally, an un-enriched portion of the sample was also retained and analysed. The fractionated, enriched and un-enriched samples were analysed by LC-MS/MS (LTQ-Orbitrap Fusion - Thermo Scientific). Over 85% coverage of full length (2N4R) tau was detected in the CSF with 47 phosphopeptides covering 31 different phosphorylation sites. Of these, 11 phosphopepitdes were up-regulated by at least 40%, along with an overall increase in tau levels in the CSF of AD patients relative to controls. Use of the SysQuant/TMTcalibrator+ hybrid workflow dramatically improved our ability to detect tau-derived peptides that are directly related to human AD pathology. Further validation of regulated tau peptides as early biomarkers of AD will be undertaken.

Neurodegenerative diseases (NDG's) typically feature aggregates of one or more abnormally modified proteins and, by mechanisms not yet fully understood, extensive neuronal cell death. Phosphorylation is a prevalent modification in NDG's, with phosphorylated forms of tau (AD), TDP-43 (ALS, FTLD) and alpha-synuclein (PD) all associated with disease. In addition, the role of phosphorylation in regulating cell signalling pathways is increasingly recognised as modulating key aspects of neurodegeneration such as re-activation of cell cycle in neuronal cells and activation of microglia and astrocytes. Accordingly, there is a strong rationale to develop and apply global phosphorylation profiling in neurodegenerative disease research. We have applied SysQuant®, a global [phospho]proteomic workflow using isobaric TMT® and differential chromatography, to measure non-phosphorylated and phosphorylated peptides in brain cortex of nine patients with different levels of tau pathology (Braak stage 0 – VI) to determine changes in expression correlating with increasing disease severity. 1 mg of brain digest from each patient and a 10th pooled sample were labelled with TMT 10plex and mixed to form a single analytical sample. Fractionation into 6 fractions by SCX was followed by direct analysis of a small aliquot of each fraction on an Orbitrap Fusion Tribrid MS with Easymate 1000 UHPLC (Thermo Scientific). The remaining material was enriched using TiO2 and IMAC before analysis of phosphopeptides on the same system. In a single experiment we quantified 7,342 proteins and 11,972 phosphorylation sites. Along with anticipated effects on the phosphorylation of aggregating proteins such as tau, and alpha-synuclein, we identified significant regulation of several other proteins associated with AD in a phospho-tau dose-dependent manner. Furthermore, many regulated proteins were associated with mediating neurotoxic response to amyloid and tau. Analysis of the SysQuant® data at the pathway level showed regulation of key processes such as calcium signalling, glutamatergic signalling and oxidative phosphorylation, along with neurodegenerative KEGG pathways. This represents the most comprehensive pathway analysis of the effects of increasing tau toxicity in AD. By using a TMT® 10plex labelling strategy we were able to generate a detailed, quantitative phosphoproteomic map for nine brain samples in approximately 8 weeks.

AimsThis combined proteomic and histopathological study was aimed to compare tissue characteristics of immunoglobulin (Ig)G4‐related sclerosing cholangitis (ISC) and primary sclerosing cholangitis (PSC) in a global, non‐biased manner.Methods and resultsTissue proteomes and phosphorylomes of frozen large bile duct samples were analysed by a conventional liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) protocol and additional phosphopeptide enrichment methods. The proteomic examination identified 23 373 peptides and 4870 proteins, including 4801 phosphopeptides and 1121 phosphoproteins. The expression profiles of phosphopeptides discriminated ISC from PSC more clearly than those of non‐phosphopeptides. In the pathway analysis, ISC was found to have 11 more activated signal cascades, including three immunological pathways, all B cell‐ or immunoglobulin‐related. On immunostaining, two immunological markers (FYN‐binding protein and allograft inflammatory factor‐1) up‐regulated in ISC were expressed mainly in M2 macrophages, consistent with increased phagocytotic activity induced by the immunoglobulin (Ig)G‐Fcγ receptor interaction. In contrast, PSC had two more activated signal pathways related to extracellular matrix (ECM) remodelling. Filamin‐A involved in ECM remodelling was expressed aberrantly in injured bile ducts and associated cholangiocarcinomas in PSC, suggesting its possible roles in periductal fibrosis and carcinogenesis in PSC.ConclusionsThis study suggested crucial roles of B cells and macrophages in ISC, and more dynamic ECM remodelling in PSC.

Abstract Targeted kinase inhibition is a promising treatment modality for melanoma, which has significantly increased available clinical strategies and improved survival outcomes for patients, whose tumors harbor the BRAF V600E mutation. However, responses are transient and multiple mechanisms for drug resistance can contribute to therapeutic escape. Therefore, a novel multiplexed proteomics approach provides an optimal choice for comprehensively dissecting multiple mechanisms of resistance to BRAFi and combination therapy within one experiment. A large-scale quantitative expression proteomics and phosphoproteomics analysis (SysQuant) was carried out on BRAF V600E mutant melanoma cell lines treated with two different clinically relevant kinase inhibitor combinations: 1) BRAFi/MEKi and 2) BRAFi/PI3Ki. Changes in protein expression and phosphorylation in response to each treatment are determined by comparison to vehicle controls. Cell line models all harbor BRAF V600E mutations, but differ in PTEN status (A375 is WT, while WM793 is PTEN null). Briefly, cells were treated with either drug combination (as above) and harvested at 1hr, 6hrs, 24hrs and 48hrs post-treatment. Control cells were treated with DMSO. Samples were lysed, reduced, alkylated and digested with trypsin. Tryptic peptides from each sample were chemically labeled or “barcoded” with TMT-10plex reagents (4 BRAFi/MEKi time points, 4 BRAFi/PI3Ki time points, 1 DMSO control and a pooled reference sample) and combined for LC-MS/MS. After peptide fractionation with strong cation exchange chromatography and phosphopeptide enrichment, LC-MS/MS peptide sequencing and relative quantification was performed using an Orbitrap Fusion mass spectrometer (Thermo). Raw MS data was searched by Proteome Discoverer and analyzed by in-house R-scripts and Perseus statistical software package. Pathway analysis was done in GeneGO (Metacore). The SysQuant workflow identified >9,000 protein groups and >17,000 unique phosphosites per cell line across different treatments. Principal component analysis of the phosphoproteomics data revealed signaling differences across different treatment conditions and drug combinations are mainly driven by BRAFi and the time post-treatment. K-means clustering was also used to examine trends in the data; as an example, this technique could be used to track signaling changes that correlate with reduction and recovery in ERK signaling. The SysQuant approach provides a systems view of global signaling changes occurring in response to drug treatment. The ability to multiplex samples with TMT allows quantitative deduction of protein expression and phosphorylation patterns that are common or unique in different cell lines, time post-treatment and the effect of combination treatments. This proteomics approach ties protein expression and phosphorylation status in response to combination therapy and generates several hypotheses for further testing with the goal of developing novel combination therapy strategies. Citation Format: Ritin Sharma, Inna Fedorenko, Sasa Koncarevic, Vikram Mitra, Stefan Selzer, Gitte Boehm, Ian Pike, Keiran Smalley, John M. Koomen. Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C12.

Neuroinflammation is an early event in AD pathology and active secretion of microglial proteins into the brain and subsequently into CSF may therefore provide a source of early diagnostic biomarkers. However, their low abundance represents a technical challenge for current proteomic methods. TMTcalibrator+ is a novel Mass Spectrometry workflow to overcome the sensitivity challenges involved in early biomarker discovery. We used TMTcalibrator+ to analyse protein digests of CSF from AD and non-AD patients into which a calibration curve of digested microglia cells had been added in a 2:1 excess, to determine which activated microglial proteins are found in CSF and may serve as early biomarkers of AD. Microglial cell-line (BV2) was activated in vitro to mimic microglia in disease. Using isobaric TMT®, peptide preparations from activated BV2 cells were labelled and mixed to form a four point calibration curve reference. Similarly, CSF samples from three patients with clinically and biomarker defined AD, and three non-AD controls were digested and labelled with the remaining tags within the TMT 10-plex set. All labelled digests were pooled together to generate the TMTcalibrator+ analytical sample, and analysed by LC-MS/MS (LTQ-Orbitrap Fusion - Thermo Scientific). By comparing the levels of each TMT reporter ion generated upon fragmentation, we can determine which activated microglial proteins are also found in CSF and which are differentially expressed in AD compared to individuals with normal cognitive function. We identified 2,849 unique peptides from 1,109 protein groups present in all CSF samples and microglial cell digests. Of these 84 unique peptide sequences from 77 protein groups were significantly up-regulated by at least 60% in AD CSF compared to controls, and 34 peptides from 26 proteins that were significantly down-regulated by at least 60%. These regulated proteins may represent a cellular response specific to AD and have potential as early disease biomarkers. Low abundance proteins derived from diseased tissue that are released into an available body fluid represent the best potential for early diagnostic biomarkers. TMTcalibrator+ provides unparalleled gains in sensitivity over traditional proteomic methods and makes these rare biomarkers readily available for further evaluation.

Specific glycosylated peptides of clusterin are found associated with hippocampal atrophy. The glycosylation of clusterin from human plasma was comprehensively analyzed and characterized using mass spectrometry (MS)-based glycoproteomics analysis. All six known N-glycosylation sites are covered, three in the alpha subunit (α64N, α81N and α123N) and three in the beta subunit (β64N, β127N, and β147N). More detailed structural characterization of clusterin glycopeptides was also performed, demonstrating the presence of glycosylated peptides and their corresponding glycans. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have determined the differences in the glycoforms associated at each of the different glycosylation sites in plasma clusterin obtained from subjects of low hippocampal atrophy (n = 13) and high hippocampal atrophy (n = 14). In our pilot study, the β64N site shows the most significant regulations between clinical groups. Eight β64N glycoforms are significantly reduced in patients with high atrophy compared with those with low atrophy, which demonstrates the utility of clusterin isoforms as diagnostic and prognostic Alzheimer's disease (AD) markers. These results provide a novel and robust workflow suitable for rapid verification of specific clusterin glycoforms with utility as AD biomarkers.