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  • Pediatric cancers are fundamentally different from


    Pediatric cancers are fundamentally different from adult tumors and usually display a lower mutational burden and a faster growth rate. About 30% of all childhood malignancies are solid tumors and, in a majority of cases, respond well to chemotherapy. However, in particular in neuroblastoma the success of chemotherapy is hampered by the occurrence of MDR which represents a major clinical problem leading to poor outcome associated with chemoresistant disease [33]. In Digoxigenin-11-dUTP to adult tumors, MDR has not yet been studied intensively in pediatric malignancies [34]. Here, we provide evidence showing that p-glycoprotein is highly relevant in neuroblastoma and RMS, two of the most common childhood cancers. Targeted therapies are being discussed as treatment options for chemoresistant pediatric cancers [33,35], and hence our findings suggesting that Smac mimetics are substrates for p-glycoprotein are important to guide the design of clinical studies.
    Conflicts of interest
    Funding This work has been partially supported by grants from the BMBF and the Deutsche Kinderkrebsstiftung (to S. F.).
    Introduction Glycoproteins represent a major class of the human plasma proteome with more than 50% of proteins being glycosylated [1] and are involved in many important biological processes such as cell signalling pathways, cell division and immunological reactions [2]. Specific glycoproteins have been identified as potential disease biomarkers and therapeutic targets in a range of human diseases (e.g Her 2/neu in breast cancer [3], prostate-specific antigen in prostate cancer [4], and vascular cell adhesion molecule 1 in rheumatoid arthritis [5]). These glycoproteins have the potential to provide a better understanding of the biochemical mechanisms underlying pathologies [[5], [6], [7], [8]]. Therefore, measuring the changes in glycoprotein profiles between diseased and non-diseased states can make a fundamental contribution to diagnostic and prognostic clinical science. Most glycoprotein analysis has focused on N-glycoproteins which are predominant in the extracellular matrix (e.g. secreted forms) and in biofluids such as blood plasma, cerebrospinal fluid, urine and saliva [9]. Two strategies namely, lectin affinity chromatography and hydrazide chemistry, have demonstrated effective extraction of N-glycoproteins and N-glycopeptides from complex biological samples [10,11]. In hydrazide chemistry, N-glycoproteins are extracted by a chemical reaction between the hydrazide group of the immobilised bead and the dialdehyde group derived from the glycan moiety of the glycoprotein [9]. The second strategy, lectin affinity, has become a common and powerful tool for the selective capture of a wide range of glycoproteins [6]. Different lectins with variable, but partially overlapping, binding profiles can recognise and bind to specific carbohydrate residues on glycoproteins [12]. Commonly, two or more lectins with complementary specificities for different glycosylation structures are used in multi-lectin affinity enrichment to provide a more comprehensive coverage for the glycoproteome of interest [13]. Concanavalin A (ConA) is a lectin which preferentially recognises mannosyl oligosaccharides, and to lesser extent glucosyl oligosaccharides, attached to proteins through asparagine residues. This type of carbohydrate is commonly present in a wide variety of serum glycoproteins and, therefore, ConA is the most commonly used lectin for N-glycoprotein isolation [14]. ConA is a readily available, low cost, high affinity ligand with a relatively broad specificity which allows for the selective isolation of the majority of glycoproteins in a plasma sample [15]. Given their high sensitivity and selectivity, mass spectrometry-based methods are widely used to identify and quantify glycoproteins, recognise glycosylation sites, and characterise the attached oligosaccharides [16]. Targeted glycoprotein quantification by LC-MS/MS provides an improved sensitivity and specificity when compared to shotgun glycoproteomics workflows [7,17]. A targeted approach can be used for relative or absolute protein quantification applying either stable isotope standards (SIS), reference-labelled peptides or label-free workflows [18]. SIS is considered the gold standard method for absolute Digoxigenin-11-dUTP quantification and, therefore, most of the reported targeted glycoprotein profiling studies used this workflow for plasma glycoprotein measurements. However, SIS method is limited by the high cost of the labelled peptide standards which creates an obstacle for the routine use of this methodology, especially for large numbers of clinical samples and/or when a large number of glycoproteins require simultaneous monitoring [19]. As a result, a relatively small numbers of plasma glycoproteins (less than 15 glycoproteins) have been profiled simultaneously by previous glycoprotein methods [20,21]. The reference-labelled peptide strategy involves a single isotopically labelled peptide used as a reference for the normalisation of all peptides monitored whilst in the label-free strategy, the raw peak areas of the monitored peptides are compared between different conditions [18]. Although label-free methodology is generally associated with higher variability than SIS [18], careful method development and validation can provide acceptable analytical precision. Additionally, a label free approach has the advantage of not being limited to a specific set of glycoproteins. This prompted us to develop a simple and more cost-effective targeted LC-MS/MS methodology using a label-free workflow to provide a cost- and time-effective solution to the measurement of a large number of glycoproteins simultaneously. Here we present a reproducible label-free targeted LC-MS/MS method adapted to a standard microflow LC-MS/MS system with the ability to achieve high throughput profiling of more than fifty plasma glycoproteins most of which are biomarkers of disease and very few of them have been jointly investigated in the same sample set previously. We also show the application of the developed method to modified blood plasma and serum intended to represent perturbed change.