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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.

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 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., (Copyright © 2016 John Wiley & Sons, Ltd.)

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.

Chronic kidney disease (CKD) is a major public health concern with an increasing proportion of sufferers progressing to renal replacement therapy (RRT). Early identification of those at risk of disease progression could be key in improving outcomes. We hypothesise that urinary liver-type fatty acid binding protein (uL-FABP) may be a suitable biomarker for CKD progression and can add value to currently established biomarkers such as the urinary protein-to-creatinine ratio (uPCR). A total of 583 participants with CKD 1–5 (not receiving renal replacement therapy) entered a 2 yr prospective longitudinal study. UPCR and uL-FABP were measured at baseline and CKD progression was defined as either (i) a decline in eGFR of >5 mL/min/1.73 m2 or an increase in serum creatinine by 10% at 1 yr; (ii) a decline in eGFR of >6 mL/min/1.73 m2 or an increase in serum creatinine by 20% at 2 yrs; or (iii) the initiation of RRT. A combined outcome of initiating RRT or death was also included. Approximately 40% of participants showed CKD progression. uL-FABP predicted CKD progression at both years 1 and 2 (OR 1.01, p < 0.01). Sensitivity and specificity were comparable to those of uPCR (AUC 0.623 v 0.706) and heat map analysis suggested that uL-FABP in the absence of significant proteinuria can predict an increase in serum creatinine of 10% at 1 yr and 20% at 2 yrs. The risk of the combined outcome of initiating RRT or death was 23% higher in those with high uL-FABP (p < 0.01) independent of uPCR. uL-FABP appears to be a highly sensitive and specific biomarker of CKD progression. The use of this biomarker could enhance the risk stratification of CKD and its progression and should be assessed further. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (2IV7-3). 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).2 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. [ABSTRACT FROM AUTHOR]

Objective: LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application. Methods: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset. Results: Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power. Conclusion: Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case. [ABSTRACT FROM AUTHOR]