Proteomic analysis in morquio a cells treated with immobilized enzymatic replacement therapy on nanostructured lipid systems

International Journal of Molecular Sciences Article Proteomic Analysis in Morquio A Cells Treated with Immobilized Enzymatic Replacement Therapy on Nanostructured Lipid Systems J. Víctor Álvarez 1,2,3,†, Susana B. Bravo 4,†, María García-Vence 4, María J. De Castro 2, Asteria Luzardo 5,6 , Cristóbal Colón2, Shunji Tomatsu 3, Francisco J. Otero-Espinar 1,6,* and María L. Couce 2,* 1 Department of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Campus Vida, University of Santiago de Compostela, 15872 Santiago de Compostela, Spain; josevictor[email protected]g 2Department of Forensic Sciences, Pathology, Gynecology and Obstetrics, Pediatrics, Neonatology Service, Department of Paediatrics, Hospital Clínico Universitario de Santiago de Compostela, Health Research Institute of Santiago de Compostela (IDIS), CIBERER, MetabERN, 15706 Santiago de Compostela, Spain; [email protected] (M.J.D.C.); [email protected] (C.C.) 3Skeletal Dysplasia Lab Nemours Biomedical Research Nemours/Alfred I. duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE 19803, USA; [email protected]g 4Proteomic Platform, Health Research Institute of Santiago de Compostela (IDIS), Hospital Clínico Universitario de Santiago de Compostela, 15706 Santiago de Compostea, Spain; [email protected] (S.B.B.); [email protected] (M.G.-V.) 5Department of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Sciences, Campus de Lugo, University of Santiago de Compostela, 27002 Lugo, Spain; [email protected] 6Paraquasil Platform, Health Research Institute of Santiago de Compostela (IDIS), Hospital Clínico Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain *Correspondence: [email protected] (F.J.O.-E.); [email protected] or [email protected] (M.L.C.); Tel.: +34-981563100 (ext. 24142; ext. 14878) (F.J.O.-E.); +34-981951100 (M.L.C.) †These authors contributed equally to this work. Received: 22 August 2019; Accepted: 13 September 2019; Published: 18 September 2019   Abstract: Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERT. Keywords: enzyme replacement therapy; lysosomal disorders; nanoparticles; proteomics Int. J. Mol. Sci. 2019,20, 4610; doi:10.3390/ijms20184610 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019,20, 4610 2 of 22 1. Introduction Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA, OMIM #253000), is an autosomal recessive disease, caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS), gene which result in deficient activity of N-acetylgalactosamine-6-sulfatase (GALNS, E.C.3.1.6.4), an enzyme that degrades glycosaminoglycan (GAG) keratan sulate (KS) and chondroitin-6-sulfate (C6S) [ 1 , 2 ]. The widespread accumulation of GAG, primarily in chondrocytes, and its extracellular matrix, leads to progressive cellular damage and organ dysfunction in the bone and cartilage. The classical phenotype is characterized by systemic skeletal dysplasia, including a short stature and neck, cervical instability and spinal cord compression, tracheal obstruction, prominent chest, kyphoscoliosis, laxity of joints, hip dysplasia, knock knee, etc. [ 3 ]. In severe forms, respiratory failure is the primary cause of death during the second and third decade of life if untreated [4,5]. There are two treatments for MPS IVA patients in clinical practice: enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation. ERT with elosulfase alpha, a recombinant human GALNS, is an established strategy for treating MPS IVA [ 1 , 6 , 7 ]. However, clinical trials have shown scarce improvement in growth and skeletal dysplasia [ 8 – 11 ]. In patients with MPS IVA, restrictive and obstructive lungs are due to an anatomical imbalance in growth (trachea and vessels grow while the spine and thoracic bones stop growing), leading to this life-threatening issue. ERT cannot affect bone deformity in the spine, ribs and sternum, and crowded thoracic cavity. Therefore, ERT may provide a limited impact on pulmonary function due to the remaining issue of skeletal dysplasia (restrictive lung). A recent long-term study of MPS IVA with ERT has shown that there was a global reduction in static spirometry values in all subjects with ERT, as well as cardiorespiratory function as assessed by 6MWT [ 12 , 13 ]. Similar to other ERTs for lysosomal storage diseases, elosulfase alpha has several pitfalls: (i) rapid clearance from circulation and a short half-life (2 min in mice; 35 min in humans) [ 10 , 12 , 14 ], (ii) limited penetration in the avascular cartilage, and (iii) immunological response against the infused enzyme [12,15,16]. Thus, therapies for resolving bone lesions remain an unmet need, and an alternative therapeutic approach is required to overcome such limitations. In this regard, one possible solution is a nanoparticular system that can protect the enzymes against their degradation or inactivation, minimize the associated immunological reactions, and increase cellular internalization to achieve maximum efficacy of the treatment [ 17 ]. Biodegradable nanoparticles of a different nature, such as polylactic-co-glycolic acid, have been used in ERT. Some studies have also shown the usefulness of solid lipid nanoparticles [ 8 , 12 , 15 , 16 ] to achieve the internalization of molecules, such as catalase, leuprolide, or insulin [ 18 – 21 ], to the cellular interior through their direct endocytosis towards lysosomes. One of the main limitations of these systems is their low capacity to immobilize and transport a protein into the lipid core. Some experiments have been carried out to absorb the protein on the surface of the lipid colloidal carriers, but in the case of enzymes, this approach can have drawbacks related to instability and the production of antibodies. Consequently, formulation design is a critical requirement to stabilize a high payload of the enzyme. We have recently developed a new drug delivery system to immobilize enzymes used for ERT and to target the lysosomes [nanostructure lipid carrier (NLC), patent registered number PCT/EP2019/068629]. Proteomics allows not only the identification but also the quantification of the overall proteins present in a cell, tissue, or organism in continuous change. One of the first proteomic approaches includes a one or two-dimensional polyacrylamide gel system using sodium dodecyl sulfate (1D SDS-PAGE and 2D SDS-PAGE) and provides a powerful tool to monitor protein purification and rapidly profile the global protein expression and posttranscriptional modifications [ 22 ]. Although 1D and 2D-PAGE are common proteomic approaches for high throughput screening of putative biomarkers in several disorders, particularly in animal studies [ 23 , 24 ], they have not been used in lysosomal diseases. On the other hand, mass spectrometry (MS) is a beneficial proteomic technology that allows proteomic pattern visualization and the identification of proteins [ 25 , 26 ] and their post-transcriptional modifications, such as phosphorylation, glycosylation, similar to 2D-Page approach [ 27 ]. In the last decade,MShasmadesignificantprogressbyincreasingthenumberofidentifiedproteins[ 26 ]. Therefore, Int. J. Mol. Sci. 2019,20, 4610 3 of 22 liquid chromatography (LC) coupled with MS is widely used for searching disease biomarkers [ 26 ] in samples such as biological fluids, tissues, cells, etc. [28,29]. Most quantitative methods involve labeling proteins with heavy and light stable-isotope pairs (SILAC, I-TRAQ). Other quantitation techniques eliminate labeling (label-free) and rely on advanced software analysis as a new technology named SWATH-MS (sequential window acquisition of all theoretical mass spectra) [ 30 , 31 ]. These methods measure the relative concentrations of peptide analytes within two or more samples. In contrast, absolute quantitation techniques use internal standard peptides that have been synthetically prepared for selected or multiple reaction monitoring (selected reaction monitoring or multiple reaction monitoring, respectively) analysis [25]. In general, disease states involve alterations in protein expression levels. Therefore, proteomics provides important biological information that contributes to the advance in our understanding of pathophysiological mechanisms and aid in the identification of novel biomarkers for different diseases [ 30 , 32 , 33 ]. Specific biomarkers identified from proteomics may be useful in diagnosis, prognosis, and as evaluators of treatment outcomes [34–42]. In this study, we have analyzed the effects of protein expression levels in the MPS IVA fibroblasts by providing the immobilized and free enzyme (GALNS) and by using qualitative and quantitative proteomic techniques. 2. Results 2.1. Qualitative Analysis Liquid Chromatography—Mass Spectrometry (DDA-LC-MS/MS) By using DDA-LC-MS/MS, we identified 287 to 1623 proteins in untreated cells and 586 to 1817 proteins in fibroblasts treated with NLC +ERT or ERT alone (Table 1). Table 1. The number of proteins identified in the qualitative (DDA-LC-MSMS) assay. Number of Proteins. 1st Analysis Number of Proteins. 2nd Analysis Number of Proteins. 3rd Analysis Samples No Treat NLC + ERT ERT No Treat NLC + ERT ERT No Treat NLC + ERT ERT HC 683 1395 1612 1561 1668 919 737 1183 S.1 MoC 730 960 923 1304 1623 1817 S.2 MoC 1220 1255 832 1022 793 1078 S.3 MoC 793 1658 1251 1594 1419 1104 S.4 MoC 611 1445 287 870 1403 586 Note: All the assays were made in triplicate. HC: healthy cells; treat: treatment; NLC: nanostructure lipid carrier; LC: lipid chromatography; ERT: Enzyme replacement therapy; MoC: Morquio A cells; S.1: Sample 1. To obtain a better representation of all the proteins identified in each treatment, we selected only those that were present in at least 2 of the replicates per treatment (Figure 1and Supplementary Figure S1). To analyze the origin of the identified proteins under no treatment, NLC +ERT, and ERT alone, we used FunRich analysis. The majority of proteins in all the samples were found to be sourced from four different cellular components: mitochondrion, lysosomal membrane, lysosome, and endosome (Figure 2A). In healthy cells (HC) with ERT (HC +ERT), we found an increase in a small percentage of proteins compared with untreated healthy cells (UHC), particularly for the mitochondrion and endosome. In the comparison between untreated Morquio A cells (UMoC) vs. Morquio A cells with ERT (MoC +ERT), no changes (or a minimal change) were found (Figure 2B and Supplementary Figure S2). When we compared HC vs. HC with NLC +ERT (HC NLC +ERT) (Figure 2C and Supplementary Figure S2), we found only a small decrease in the percentage of mitochondrion and endosome. When we compared UMoC vs. the MoC NLC +ERT (Figure 2D and Supplementary Int. J. Mol. Sci. 2019,20, 4610 4 of 22 Figure S2), small differences in the percentage of proteins (a slight increase in mitochondrion and endosome and a slight decrease in lysosomal membrane and lysosome) were found. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 22 Most quantitative methods involve labeling proteins with heavy and light stable-isotope pairs (SILAC, I-TRAQ). Other quantitation techniques eliminate labeling (label-free) and rely on advanced software analysis as a new technology named SWATH-MS (sequential window acquisition of all theoretical mass spectra) [30,31]. These methods measure the relative concentrations of peptide analytes within two or more samples. In contrast, absolute quantitation techniques use internal standard peptides that have been synthetically prepared for selected or multiple reaction monitoring (selected reaction monitoring or multiple reaction monitoring, respectively) analysis [25]. In general, disease states involve alterations in protein expression levels. Therefore, proteomics provides important biological information that contributes to the advance in our understanding of pathophysiological mechanisms and aid in the identification of novel biomarkers for different diseases [30,32,33]. Specific biomarkers identified from proteomics may be useful in diagnosis, prognosis, and as evaluators of treatment outcomes [34–42]. In this study, we have analyzed the effects of protein expression levels in the MPS IVA fibroblasts by providing the immobilized and free enzyme (GALNS) and by using qualitative and quantitative proteomic techniques. 2. Results 2.1. Qualitative Analysis Liquid Chromatography – Mass Spectrometry (DDA-LC-MS/MS) By using DDA-LC-MS/MS, we identified 287 to 1623 proteins in untreated cells and 586 to 1817 proteins in fibroblasts treated with NLC+ERT or ERT alone (Table 1). Table 1. The number of proteins identified in the qualitative (DDA-LC-MSMS) assay. Number of proteins. 1st Analysis Number of Proteins. 2nd Analysis Number of Proteins. 3rd Analysis Samples No treat NLC+ERT ERT No treat NLC+ERT ERT No treat NLC+ERT ERT HC 683 1395 1612 1561 1668 919 737 1183 S.1 MoC 730 960 923 1304 1623 1817 S.2 MoC 1220 1255 832 1022 793 1078 S.3 MoC 793 1658 1251 1594 1419 1104 S.4 MoC 611 1445 287 870 1403 586 Note: All the assays were made in triplicate. HC: healthy cells; treat: treatment; NLC: nanostructure lipid carrier; LC: lipid chromatography; ERT: Enzyme replacement therapy; MoC: Morquio A cells; S.1: Sample 1. To obtain a better representation of all the proteins identified in each treatment, we selected only those that were present in at least 2 of the replicates per treatment (Figure 1 and Supplementary Figure S1). Figure 1. Venn diagram from the triplicates studies of proteins in Morquio A cells (Sample 1) after 24 h of NLC +ERT treatment. The common proteins identified in 2 or 3 of the replicates are shown. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 22 Figure 1. Venn diagram from the triplicates studies of proteins in Morquio A cells (Sample 1) after 24 h of NLC+ERT treatment. The common proteins identified in 2 or 3 of the replicates are shown. To analyze the origin of the identified proteins under no treatment, NLC+ERT, and ERT alone, we used FunRich analysis. The majority of proteins in all the samples were found to be sourced from four different cellular components: mitochondrion, lysosomal membrane, lysosome, and endosome (Figure 2A). In healthy cells (HC) with ERT (HC+ERT), we found an increase in a small percentage of proteins compared with untreated healthy cells (UHC), particularly for the mitochondrion and endosome. In the comparison between untreated Morquio A cells (UMoC) vs. Morquio A cells with ERT (MoC+ERT), no changes (or a minimal change) were found (Figure 2B and Supplementary Figure 2). When we compared HC vs. HC with NLC+ERT (HC NLC+ERT) (Figure 2C and Supplementary Figure 2), we found only a small decrease in the percentage of mitochondrion and endosome. When we compared UMoC vs. the MoC NLC+ERT (Figure 2D and Supplementary Figure S2), small differences in the percentage of proteins (a slight increase in mitochondrion and endosome and a slight decrease in lysosomal membrane and lysosome) were found. Figure 2. FunRich functional analysis results of expressed proteins from cellular components. (A) HC and HC + ERT. (B) UMoC (Sample 2) and MoC + ERT (Sample 2). (C) HC and HC with NLC+ ERT. (D) UMoC (Sample 1) and MoC with NLC+ERT (Sample 1). Regarding the biological process of the mitochondrion, we found changes in the proteins to be related to the interaction between the mitochondria and the lysosome (Figure 3). These changes were more prominent in the cells treated with NLC+ERT (Figure 3A) or ERT (Figure 3B). The analysis indicates that the expression level of proteins related to cell redox homeostasis, the response to oxidative stress, the response to calcium ion, transferrin transport, amino acid transport, and mitochondrion to lysosome transport were decreased in both treatments, while the expression levels of proteins related to fatty acid β-oxidation were increased. Figure 2. FunRich functional analysis results of expressed proteins from cellular components. ( A ) HC and HC +ERT. ( B ) UMoC (Sample 2) and MoC +ERT (Sample 2). ( C ) HC and HC with NLC +ERT. (D) UMoC (Sample 1) and MoC with NLC +ERT (Sample 1). Regarding the biological process of the mitochondrion, we found changes in the proteins to be related to the interaction between the mitochondria and the lysosome (Figure 3). These changes were more prominent in the cells treated with NLC +ERT (Figure 3A) or ERT (Figure 3B). The analysis indicates that the expression level of proteins related to cell redox homeostasis, the response to oxidative stress, the response to calcium ion, transferrin transport, amino acid transport, and mitochondrion to lysosome transport were decreased in both treatments, while the expression levels of proteins related to fatty acid β-oxidation were increased. Int. J. Mol. Sci. 2019,20, 4610 5 of 22 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 22 Figure 3. FunRich functional analysis results of the expressed proteins from the metabolic process of mitochondrion. (A) Comparison between untreated and treated for 24 h with NLC + ERT in HC and MoC. (B) Comparison between untreated and treated for 24 h with ERT alone in HC and MoC. When we analyzed the biological process of the endosome (Figure 4), the changes in the proteins involved in the endosome-to-lysosome transport and iron ion homeostasis were observed. We found an increase in endosome-to-lysosome transport in both HC and MoC with NLC+ERT (Figure 4A) but only in HC with ERT alone (Figure 4B). In the case of the iron ion homeostasis transport, we found a significant increase in MoC with both NLC+ERT and ERT alone (Figure 4A,B) and only a small increase in HC with ERT (Figure 4B). Figure 4. FunRich functional analysis results of expressed proteins from the metabolic process of the endosome. (A) comparison between untreated and treated for 24 h with NLC + ERT in HC and MoC; (B) comparison between untreated and treated for 24 h with ERT in HC and MoC. In the lysosomal membrane (Figure 5), we observed a decrease of the expression level in the proteins related to targeting the lysosome and to the lysosomal membrane organization in NLC+ERT (Figure 5A). However, in ERT treatments, we only found a decrease in the lysosomal membrane (Figure 5B). Moreover, it should be noted that in HC, both HC alone or with treatment (NLC+ERT or ERT), we found that the targeted lysosome and lysosomal membrane organization protein levels decrease (Figure 5A,B). Figure 3. FunRich functional analysis results of the expressed proteins from the metabolic process of mitochondrion. ( A ) Comparison between untreated and treated for 24 h with NLC +ERT in HC and MoC. (B) Comparison between untreated and treated for 24 h with ERT alone in HC and MoC. When we analyzed the biological process of the endosome (Figure 4), the changes in the proteins involved in the endosome-to-lysosome transport and iron ion homeostasis were observed. We found an increase in endosome-to-lysosome transport in both HC and MoC with NLC +ERT (Figure 4A) but only in HC with ERT alone (Figure 4B). In the case of the iron ion homeostasis transport, we found a significant increase in MoC with both NLC +ERT and ERT alone (Figure 4A,B) and only a small increase in HC with ERT (Figure 4B). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 22 Figure 3. FunRich functional analysis results of the expressed proteins from the metabolic process of mitochondrion. (A) Comparison between untreated and treated for 24 h with NLC + ERT in HC and MoC. (B) Comparison between untreated and treated for 24 h with ERT alone in HC and MoC. When we analyzed the biological process of the endosome (Figure 4), the changes in the proteins involved in the endosome-to-lysosome transport and iron ion homeostasis were observed. We found an increase in endosome-to-lysosome transport in both HC and MoC with NLC+ERT (Figure 4A) but only in HC with ERT alone (Figure 4B). In the case of the iron ion homeostasis transport, we found a significant increase in MoC with both NLC+ERT and ERT alone (Figure 4A,B) and only a small increase in HC with ERT (Figure 4B). Figure 4. FunRich functional analysis results of expressed proteins from the metabolic process of the endosome. (A) comparison between untreated and treated for 24 h with NLC + ERT in HC and MoC; (B) comparison between untreated and treated for 24 h with ERT in HC and MoC. In the lysosomal membrane (Figure 5), we observed a decrease of the expression level in the proteins related to targeting the lysosome and to the lysosomal membrane organization in NLC+ERT (Figure 5A). However, in ERT treatments, we only found a decrease in the lysosomal membrane (Figure 5B). Moreover, it should be noted that in HC, both HC alone or with treatment (NLC+ERT or ERT), we found that the targeted lysosome and lysosomal membrane organization protein levels decrease (Figure 5A,B). Figure 4. FunRich functional analysis results of expressed proteins from the metabolic process of the endosome. ( A ) comparison between untreated and treated for 24 h with NLC +ERT in HC and MoC; (B) comparison between untreated and treated for 24 h with ERT in HC and MoC. In the lysosomal membrane (Figure 5), we observed a decrease of the expression level in the proteins related to targeting the lysosome and to the lysosomal membrane organization in NLC +ERT (Figure 5A). However, in ERT treatments, we only found a decrease in the lysosomal membrane (Figure 5B). Moreover, it should be noted that in HC, both HC alone or with treatment (NLC +ERT or ERT), we found that the targeted lysosome and lysosomal membrane organization protein levels decrease (Figure 5A,B). Related to the biological processing of proteins in the lysosome (Figure 6), we analyzed both the biosynthesis and the catabolism of C6S and KS. We observed that in cells treated with NLC +ERT (Figure 6A) or ERT (Figure 6B), the proteins related to these processes were decreased. Int. J. Mol. Sci. 2019,20, 4610 6 of 22 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 22 Figure 5. FunRich functional analysis results of expressed proteins from the metabolic process of the lysosomal membrane. (A) comparison between no treatment and 24 h treatment with NLC + ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. Related to the biological processing of proteins in the lysosome (Figure 6), we analyzed both the biosynthesis and the catabolism of C6S and KS. We observed that in cells treated with NLC+ERT (Figure 6A) or ERT (Figure 6B), the proteins related to these processes were decreased. Figure 6. FunRich functional analysis results of the expressed proteins from the metabolic process of the lysosome. (A) comparison between no treatment and 24 h treatment with NLC + ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. We also analyzed the biological processes of proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (Figure 7), specifically looking for changes in the metabolic pathway of cholesterol and SNARE complex disassembly. The percentage of proteins involved in these processes was increased with both treatments. Figure 5. FunRich functional analysis results of expressed proteins from the metabolic process of the lysosomal membrane. ( A ) comparison between no treatment and 24 h treatment with NLC +ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 22 Figure 5. FunRich functional analysis results of expressed proteins from the metabolic process of the lysosomal membrane. (A) comparison between no treatment and 24 h treatment with NLC + ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. Related to the biological processing of proteins in the lysosome (Figure 6), we analyzed both the biosynthesis and the catabolism of C6S and KS. We observed that in cells treated with NLC+ERT (Figure 6A) or ERT (Figure 6B), the proteins related to these processes were decreased. Figure 6. FunRich functional analysis results of the expressed proteins from the metabolic process of the lysosome. (A) comparison between no treatment and 24 h treatment with NLC + ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. We also analyzed the biological processes of proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (Figure 7), specifically looking for changes in the metabolic pathway of cholesterol and SNARE complex disassembly. The percentage of proteins involved in these processes was increased with both treatments. Figure 6. FunRich functional analysis results of the expressed proteins from the metabolic process of the lysosome. ( A ) comparison between no treatment and 24 h treatment with NLC +ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. We also analyzed the biological processes of proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (Figure 7), specifically looking for changes in the metabolic pathway of cholesterol and SNARE complex disassembly. The percentage of proteins involved in these processes was increased with both treatments. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 22 Figure 5. FunRich functional analysis results of expressed proteins from the metabolic process of the lysosomal membrane. (A) comparison between no treatment and 24 h treatment with NLC + ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. Related to the biological processing of proteins in the lysosome (Figure 6), we analyzed both the biosynthesis and the catabolism of C6S and KS. We observed that in cells treated with NLC + ERT (Figure 6A) or ERT (Figure 6B), the proteins related to these processes were decreased. Figure 6. FunRich functional analysis results of the expressed proteins from the metabolic process of the lysosome. (A) comparison between no treatment and 24 h treatment with NLC + ERT in HC and MoC (B) comparison between no treatment and 24 h treatment with ERT in HC and MoC. We also analyzed the biological processes of proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (Figure 7), specifically looking for changes in the metabolic pathway of cholesterol and SNARE complex disassembly. The percentage of proteins involved in these processes was increased with both treatments. Figure 7. FunRich functional analysis results of expressed proteins from the metabolic process of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. ( A ) comparison between non-treatment and 24 h treatment with NLC+ERT in HC and MoC ( B ) comparison between non-treatment and 24 h treatment with ERT in HC and MoC. Int. J. Mol. Sci. 2019,20, 4610 7 of 22 With regards to the GAG degradation route (Figure 8), our results showed that, in UMoC only three of the four proteins implicated in the pathway were present. However, when we added NCL+ERT or ERT (data not shown), all the proteins appeared, thus completing the pathway and confirming that ERT provides an impact on the GAG degradation pathway. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 22 Figure 7. FunRich functional analysis results of expressed proteins from the metabolic process of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. (A) comparison between non-treatment and 24 h treatment with NLC+ ERT in HC and MoC (B) comparison between non-treatment and 24 h treatment with ERT in HC and MoC. With regards to the GAG degradation route (Figure 8), our results showed that, in UMoC only three of the four proteins implicated in the pathway were present. However, when we added NCL+ERT or ERT (data not shown), all the proteins appeared, thus completing the pathway and confirming that ERT provides an impact on the GAG degradation pathway. Figure 8. DAVID functional analysis of the glycosaminoglycan (GAG) degradation pathway in mucopolysaccharidosis type IVA (MPS IVA) fibroblasts without treatment (A) or NLC+ERT (B). Red stars correspond to proteins identified by the DDA-LC-MS/MS analysis. Green color of the boxes: sure confidence, black solid arrows: high confidence; black dotted arrows: moderate confidence. Accordingly, we also tested the vesicles’ internalization, which was previously described as mannose-6-phosphate receptor-dependent [43,44]. Our results indicate that NLC encapsulation of the drug induces internalization of vesicles in a different way through endocytosis, as we found proteins involved in the initial and final phases of endosome formation ( Figure 2; Figure 9). Figure 8. DAVID functional analysis of the glycosaminoglycan (GAG) degradation pathway in mucopolysaccharidosis type IVA (MPS IVA) fibroblasts without treatment ( A ) or NLC +ERT ( B ). Red stars correspond to proteins identified by the DDA-LC-MS/MS analysis. Green color of the boxes: sure confidence, black solid arrows: high confidence; black dotted arrows: moderate confidence. Accordingly, we also tested the vesicles’ internalization, which was previously described as mannose-6-phosphate receptor-dependent [ 43 , 44 ]. Our results indicate that NLC encapsulation of the drug induces internalization of vesicles in a different way through endocytosis, as we found proteins involved in the initial and final phases of endosome formation (Figures 2and 9). We obtained thequantitativemeasurementof proteinsbyanother method (describedinSection 2.2). We analyzed the same specimens with a new database (shown in Supplementary Table S1). In Figure 10, we show the percentage of upregulated proteins, comparing HC, UMoC, and MoC with NLC +ERT; MoC with ERT alone; HC with NLC +ERT; and HC with ERT alone in different organelles. The protein pattern expressions in the endolysosomal membrane are similar to those in the untreated affected cells and the HC. However, proteins related to the mitochondrion, lysosomal membrane, lysosome, and endosome were upregulated in the HC. Likewise, 17% of proteins in MoC treated with NLC +ERT were elevated in the mitochondrion when compared with the untreated cells. No significant difference was found in the remaining organelles. Int. J. Mol. Sci. 2019,20, 4610 8 of 22 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 22 Figure 9. DAVID functional analysis of vesicle internalization. Red stars correspond to proteins identified by the DDA-LC-MS/MS analysis. Green color of the boxes: sure confidence, black solid arrows: high confidence; black dotted arrows: moderate confidence. We obtained the quantitative measurement of proteins by another method (described in Section 3.2). We analyzed the same specimens with a new database (shown in Supplementary Table 1). In Figure 10, we show the percentage of upregulated proteins, comparing HC, UMoC, and MoC with NLC+ERT; MoC with ERT alone; HC with NLC+ERT; and HC with ERT alone in different organelles. The protein pattern expressions in the endolysosomal membrane are similar to those in the untreated affected cells and the HC. However, proteins related to the mitochondrion, lysosomal membrane, lysosome, and endosome were upregulated in the HC. Likewise, 17% of proteins in MoC treated with NLC+ERT were elevated in the mitochondrion when compared with the untreated cells. No significant difference was found in the remaining organelles. Figure 10. FunRich Functional analysis results of upregulated proteins showing the results related to protein internalization. 2.2. Protein Quantification by SWATH-MS Figure 9. DAVID functional analysis of vesicle internalization. Red stars correspond to proteins identified by the DDA-LC-MS/MS analysis. Green color of the boxes: sure confidence, black solid arrows: high confidence; black dotted arrows: moderate confidence. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 22 Figure 9. DAVID functional analysis of vesicle internalization. Red stars correspond to proteins identified by the DDA-LC-MS/MS analysis. Green color of the boxes: sure confidence, black solid arrows: high confidence; black dotted arrows: moderate confidence. We obtained the quantitative measurement of proteins by another method (described in Section 3.2). We analyzed the same specimens with a new database (shown in Supplementary Table 1). In Figure 10, we show the percentage of upregulated proteins, comparing HC, UMoC, and MoC with NLC+ERT; MoC with ERT alone; HC with NLC+ERT; and HC with ERT alone in different organelles. The protein pattern expressions in the endolysosomal membrane are similar to those in the untreated affected cells and the HC. However, proteins related to the mitochondrion, lysosomal membrane, lysosome, and endosome were upregulated in the HC. Likewise, 17% of proteins in MoC treated with NLC+ERT were elevated in the mitochondrion when compared with the untreated cells. No significant difference was found in the remaining organelles. Figure 10. FunRich Functional analysis results of upregulated proteins showing the results related to protein internalization. 2.2. Protein Quantification by SWATH-MS Figure 10. FunRich Functional analysis results of upregulated proteins showing the results related to protein internalization. 2.2. Protein Quantification by SWATH-MS We employed this approach based on the use of a library in which quantification is performed to study deregulated proteins. To generate this library, the samples were pooled, and 4 µ L of each sample was loaded into an DDA-LC-MS/MS system. A total of 1019 different proteins were identified (Supplementary Data 1). These proteins were selected because they had a global false discovery rate <1% and an unused score (protein score) of >1.2. We found 11 upregulated proteins in UMoC, compared in ERT treated cells and, conversely, 43 upregulated proteins in MoC with ERT, compared in UMoC. These proteins had a p<0.05 and a fold change (FC) >1.2 (Table 2). Int. J. Mol. Sci. 2019,20, 4610 9 of 22 Table 2. Quantitative study of proteins in UMoC vs. MoC with ERT (A) and vice versa (B). A. Upregulated Proteins in UMoC, Compared in MoC with ERT Protein Group p-Value FC P29692 Elongation factor 1-delta 0.0341 2.1053 P54727 UV excision repair protein RAD23 homolog B 0.0340 1.9325 P16401 Histone H1.5 0.0357 1.8159 P26373 60S ribosomal protein L13 0.0449 1.8140 O15143 Actin-related protein 2/3 complex subunit 1B 0.0329 1.7815 P31942 Heterogeneous nuclear ribonucleoprotein H3 0.0234 1.6284 P07437 Tubulin beta chain 0.0361 1.6138 Q07020 Q9Y4L1 60S ribosomal protein L18 Hypoxia up-regulated protein 1 0.0477 0.0104 1.4902 1.4313 P13674 Prolyl 4-hydroxylase subunit alpha-1 0.0299 1.3145 P68104 Elongation factor 1-alpha 1 0.0342 1.2451 B. Upregulated Proteins in MoC with ERT, Compared in UMoC Protein Group p-Value FC P07237 Protein disulfide-isomerase 0.0408 1.2058 P78417 Glutathione S-transferase omega-1 0.0425 1.2224 O00299 Chloride intracellular channel protein 1 0.0195 1.2303 P23526 Adenosylhomocysteinase 0.0443 1.2358 Q96S97 Myeloid-associated differentiation marker 0.0496 1.2446 P62258 14-3-3 protein epsilon 0.0079 1.2498 P16152 Carbonyl reductase [NADPH] 1 0.0199 1.2521 P32119 Peroxiredoxin-2 0.0385 1.2540 P00387 NADH-cytochrome b5 reductase 3 0.0087 1.2629 P17655 Calpain-2 catalytic subunit 0.0287 1.2708 P61981 14-3-3 Protein gamma 0.0482 1.2743 P60900 Proteasome subunit alpha type-6 0.0270 1.2756 O43707 Alpha-actinin-4 0.0309 1.2781 P27824 Calnexin 0.0254 1.2798 P11413 Glucose-6-phosphate 1-dehydrogenase 0.0215 1.2906 P08133 Annexin A6 0.0055 1.2937 P12814 Alpha-actinin-1 0.0146 1.3017 P22314 Ubiquitin-like modifier-activating enzyme 1 0.0015 1.3163 P54578 Ubiquitin carboxyl-terminal hydrolase 14 0.0386 1.3214 P49721 Proteasome subunit beta type-2 0.0414 1.3568 Q96AG4 Leucine-rich repeat-containing protein 59 0.0085 1.3642 P67812 Signal peptidase complex catalytic subunit SEC11A 0.0153 1.3671 Q15404 Ras suppressor protein 1 0.0172 1.3808 P07996 Thrombospondin-1 0.0379 1.3992 O00629 Importin subunit alpha-3 0.0386 1.4218 Q9BS26 Endoplasmic reticulum resident protein 44 0.0170 1.4754 Q99798 Aconitate hydratase, mitocondrial 0.0357 1.4799 P60953 Cell division control protein 42 homolog 0.0215 1.4806 Q9Y3I0 tRNA-splicing ligase RtcB homolog 0.0506 1.4807 P2484 Myosin regulatory light polypeptide 9 0.0344 1.5135 Q15758 Neutral amino acid transporter B(0) 0.0076 1.5970 Q13724 Mannosyl-oligosaccharide glucosidase 0.0351 1.6011 P08727 Keratin, type I cytoskeletal 19 0.0315 1.6256 P24941 Cyclin-dependent kinase 2 0.0114 1.6367 P61619 Protein transport protein Sec61 subunit alpha isoform 1 0.0398 1.7619 P08195 4F2 cell-surface antigen heavy chain 0.0151 1.7870 Q16881 Thioredoxin reductase 1, cytoplasmic 0.0472 1.8402 P55060 Exportin-2 0.0229 1.9550 P04179 Superoxide dismutase [Mn], mitocondrial 0.0066 2.3668 Q9NVD7 Alpha-parvin 0.0339 2.3802 Q9HDC9 Adipocyte plasma membrane-associated protein 0.0129 2.4386 Int. J. Mol. Sci. 2019,20, 4610 16 of 22 Table 6. Values of enzyme activity. Samples Before Treatment (nM/h/mg) After Treatment (nM/h/mg) S.1 MoC 0.0 1.6 S.2 MoC 0.0 4.8 S.3 MoC 0.0 6.5 S.4 MoC 0.0 8.9 4.7. Proteomic Analysis 4.7.1. In Gel Protein Digestion To ensure global and quantitative (by SWATH-MS) protein identification, an equal amount of proteins from the treated patients and controls were loaded on a 10% SDS-PAGE gel. The run was stopped as soon as the front had penetrated 3 mm into the resolving gel [ 59 , 60 ]. The protein band was detected by Sypro-Ruby fluorescent staining (Lonza, Basel, Switzerland), excised, and processed for in-gel and manual tryptic digestion, as described [ 61 ]. Gel pieces were reduced with 10 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) and alkylated with 55 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM ammonium bicarbonate. Then, the gel pieces were rinsed with 50 mM ammonium bicarbonate in 50% methanol (HPLC grade, Scharlau, Barcelona, Spain), dehydrated via the addition of acetonitrile (HPLC grade, Scharlau, Barcelona, Spain), and dried in a SpeedVac. Modified porcine trypsin (Promega, Madison, WI, USA) was added to the dry gel pieces at a final concentration of 20 ng/ µ L in 20 mM ammonium bicarbonate, incubating them at 37 ◦ C for 16 h. The peptides were extracted thrice by 20 min incubation in 40 µ L of 60% acetonitrile in 0.5% formic acid. The resulting peptide extracts were pooled, concentrated in a SpeedVac (TermoFisher Scientific: Haysham, Lancashire), and stored at −20 ◦C. 4.7.2. Mass Spectrometric Analysis (DDA acquisition) Digested peptides were separated using Reverse Phase Chromatography. The gradient was created using a micro liquid chromatography system (Eksigent Technologies nanoLC 400, ABSciex, (Warrington, Cheshire, UK) coupled to high-speed Triple TOF 6600 mass spectrometer (ABSciex, Foster City, CA, USA), with a micro flow source, as described previously [ 26 , 27 ]. The chosen analytical column was a silica-based reversed-phase column Eksigent C18CL 150 × 0.30 mm, 3 µ m particle size, and 120 Å pore size (Eksigent, ABSciex, Woodlands Central Indus. Estate, Singapore). The trap column was a YMC-TRIART C18 (YMC Technologies, Teknokroma, Barcelona, Spain) with a 3 µ m particle size and 120 Å pore size, switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 10 µ L/min. The micro-pump generated a flow-rate of 5 µ L/min and operated under gradient elution conditions, using 0.1% formic acid in the water as mobile phase A, and 0.1% formic acid in acetonitrile as mobile phase B. The peptides were separated using a 90 min gradient ranging from 2% to 90% of mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid). The injection volume was 4 µ L (more or less 4 µ g of protein). Data acquisition was performed in a TripleTOF 6600 System (ABSciex, Foster City, CA, USA) using a data dependent workflow. Source and interface conditions were the following: ion spray voltage floating (ISVF) 5500 V, curtain gas (CUR) 25, collision energy (CE) 10, and ion source gas 1 (GS1) 25. The instruments were operated with the Analyst TF 1.7.1 software (ABSciex, Woodlands Central Indus. Estate, Singapore). The switching criteria were set to ions greater than the mass to charge ratio (m/z) 350 and smaller than m/z1400, with a charge state of 2–5, a mass tolerance of 250 ppm, and an abundance threshold of more than 200 counts (cps). Former target ions were excluded for 15 s. Int. J. Mol. Sci. 2019,20, 4610 17 of 22 The instruments were automatically calibrated every 4 h using external calibrant tryptic peptides from PepCalMix (Sciex, Warrington, Cheshire, UK). 4.7.3. Data Analysis After the MS/MS analysis, the data files were processed using the ProteinPilotTM 5.0.1 software from ABSciex (Woodlands Central Indus Estate, Singapore), which uses the algorithm ParagonTM for database searching and ProgroupTM for data grouping. Data were searched using a Human-specific Uniprot database (www.uniprot.org). The false discovery rate was determined using a non-linear fitting method displaying only those results that reported a 1% Global false discovery rate or better [ 28 ]. Functionalanalysiswasperformedbydifferentopenaccesssoftware. Weused FunRich (functional enrichment analysis tool) for functional enrichment and interaction network analysis (Available online: http://funrich.org/index.html). For statistics, we used FunRich (hypergeometric test), Bonferroni [ 29 , 30 ], network construction, and clustering. We also used DAVID (Available online: https://david.ncifcrf.gov/) for GAG degradation pathways and functional analysis of the vesicle internalization. 4.8. Protein Quantification by SWATH-MS 4.8.1. Creation of the Spectral Library To build the MS/MS spectral libraries, the peptide solutions were analyzed by a shotgun data-dependent acquisition (DDA) approach using micro-LC-MS/MS (ABSciex, Redwood City, CA, USA), as described previously by us and other authors [27,46,47,62]. To obtain a good representation of the peptides and proteins present in all samples, pooled vials of the samples from each group were prepared using equal mixtures of the original samples. A total of 4 µ L from each pool was separated into a micro-LC system Ekspert nLC425 (Eksigen, Dublin, CA, USA) using an Eksigent C18 150 ×0.30 mm , with 3 mm particle size and 120 Å pore size (Eksigent, ABSciex), at a flow rate of 5 mL/min. Water and ACN, both containing 0.1% formic acid, were used as solvents A and B, respectively. The gradient run consisted of 5% to 95% B for 30 min, 5 min at 90% B, and, finally, 5 min at 5% B for column equilibration, for a total run time of 40 min. As the peptides eluted, they were directly injected into a hybrid quadrupole-TOF mass spectrometer Triple TOF 6600 (ABSciex, Redwood City, CA, USA) operated with a data-dependent acquisition system in positive ion mode. A Micro source (ABSciex) was used for the interface between microLC and MS, with an application of 2600 V voltage. The acquisition mode consisted of a 250 ms survey MS scan from 400 to 1250 m/z, followed by an MS/MS scan from 100 to 1500 m/z(25 ms acquisition time) of the top 65 precursor ions from the survey scan, for a total cycle time of 2.8 s. The fragmented precursors were added to a dynamic exclusion list for 15 s. Any singly charged ions were excluded from the MS/MS analysis. The peptide and protein identifications were performed using Protein Pilot software (version 5.0.1, ABSciex), and the data were searched using a Human-specific Uniprot database, specifying iodoacetamide as the Cys alkylation. The false discovery rate (FDR) was set to 1 for both peptides and proteins. The MS/MS spectra of the identified peptides were then used to generate the spectral library for the SWATH-MS peak extraction using the add-in for the PeakView Software (version 2.2, ABSciex) MS/MSALL with the SWATH-MS Acquisition MicroApp (version 2.0, ABSciex). Peptides with a confidence score above 99% (as obtained from Protein Pilot database search) were included in the spectral library). 4.8.2. Relative Quantification by SWATH-MS Acquisition SWATH–MS acquisition was performed on a TripleTOF ® 6600 LC-MS/MS system (ABSciex). Each sample (4 µ L) was analyzed using the LC-MS/MS equipment (Sciex, Warrington, Cheshire, UK) and the LC gradient described above for building the spectral library, but instead used the SWATH-MS acquisition method. The method consisted of repeating a cycle that consisted of the acquisition of 65 TOF MS/MS scans (400 to 1500 m/z, high sensitivity mode, 50 ms acquisition time) of overlapping Int. J. Mol. Sci. 2019,20, 4610 18 of 22 sequential precursor isolation windows of variable widths (1 m/zoverlap), covering the 400 to 1250 m/zmass range with a previous TOF MS scan (400 to 1500 m/z, 50 ms acquisition time) for each cycle. The total cycle time was 6.3 s. For each sample set, the width of the 65 variable windows was optimized according to the ion density found in the DDA runs using a SWATH-MS variable window calculator worksheet from Sciex. 4.8.3. Data Analysis The targeted data extraction of the fragment ion chromatogram traces from the SWATH-MS runs was performed by Peak View (version 2.2, ABSciex) using the SWATH-MS Acquisition Micro App (version 2.0, Sciex, Warrington, Cheshire, UK). This application processed the data using the spectral library created from the shotgun data. Up to ten peptides per protein and seven fragments per peptide were selected, based on signal intensity. Any shared and modified peptides were excluded from the processing. Five-minute windows and 30 ppm widths were used to extract the ion chromatograms. SWATH-MS quantization was attempted for all proteins in the ion library that were identified by Protein Pilot with an FDR below 1%. The retention times from the peptides that were selected for each protein were realigned in each run, according to the iRT peptides that spiked in each sample, and eluted along the whole-time axis. The extracted ion chromatograms were then generated for each selected fragment ion. The peak areas for the peptides were obtained by summing the peak areas from the corresponding fragment ions. Peak View computed an FDR and a score for each assigned peptide according to the chromatographic and spectra components. Only peptides with an FDR below 5% were used for protein quantization. Protein quantization was calculated by adding the peak areas of the corresponding peptides. The integrated peak areas were directly exported to the Marker View software (ABSciex) for relative quantitative analysis. The export generated three files containing quantitative information about individual ions, the summed intensity of the different ions for a particular peptide, and the summed intensity of different peptides for a particular protein. Marker View was used for analysis of the SWATH-MS data reported in other proteomics studies [ 34 , 62 – 64 ] because of its data-independent method of quantization. Marker View uses processing algorithms that accurately find chromatographic and spectral peaks direct from the raw SWATH-MS data. Data alignment by Marker View compensated for minor variations in both the mass and retention time values, ensuring that identical compounds in different samples were accurately compared to one another. To control for possible uneven sample loss across the different samples during the sample preparation process, we performed a global normalization based on the total sum of all the peak areas extracted from all the peptides and transitions across the replicates of each sample [ 65 ]. An unsupervised multivariate statistical analysis using principal component analysis (PCA) was performed to compare the data across the samples. The average MS peak area of each protein was derived from the replicates of the SWATH-MS of each sample followed by a Student’s t-test analysis using the Marker View software for comparison among the samples based on the averaged area sums of all the transitions derived for each protein. The t-test indicated how well each variable distinguished the two groups, reported as a p-value. For each library, each set of differentially expressed proteins (p-value <0.05) with 1.2 up-regulated or down-regulated proteins was selected. 5. Conclusions We have demonstrated that our new delivery system, through a nanostructure lipid carrier, can induce changes in proteins that are involved in the disease. This system used a different internalization pathway (not saturable) that seems to induce better access to the lysosome. Using a low enzyme dosage, we obtained excellent C6S and KS degradation, greater than with the free enzyme. Therefore, this system can improve the lysosome-endosome-mitochondria machine in the MoC. Supplementary Materials: Supplementary materialscan be found at http://www.mdpi.com/1422-0067/20/18/4610/s1. Int. J. Mol. Sci. 2019,20, 4610 19 of 22 Author Contributions: Contributions were provided by the authors in the statements: conceptualization, J.V. Á ., S.B.B., F.J.O.-E., and M.L.C.; methodology, S.B.B., J.V. Á ., M.G.-V.; validation, J.V. Á ., and S.B.B.; formal analysis, J.V. Á ., S.B.B. and C.C.; writing—original draft preparation, J.V. Á ., S.B.B.; writing—review and editing, M.L.C., M.J.D.C, F.J.O.-E., A.L., and S.T.; visualization, S.B.B.; supervision, M.L.C., F.J.O.-E., A.L., and S.T. Conflicts of Interest: J. V í ctor Á lvarez, Asteria Luzardo, Crist ó bal Col ó n, Francisco J. Otero-Espinar, and Mar í a L. Couce declare competing financial interests: They have a patent application, PCT/EP2019/068629. This patent application covers the underlying concept of immobilized ERT on nanostructured lipid systems to treat MPS IVA, as described in the manuscript. Abbreviations C6S Chondroitin 6 sulphate ERT Enzyme replacement therapy FC Fold change GAG Glycosaminoglycan GALNS N-acetylgalactosamine-6-sulfatase KS Keratan sulphateJ LC Liquid chromatography MPS IVA Mucopolysaccharidosis type IVA MRM Multiple reaction monitoring MS Mass spectrometry NLC Nanostructure lipid carrier S Sample SDC Sodium dodecyl sulfate SRM Selected reaction monitoring SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor SWATH-MS Sequential window acquisition of all theoretical mass spectra UMoC Untreated Morquio A cells References 1. Hendriksz, C.J.; Berger, K.I.; Giugliani, R.; Harmatz, P.; Kampmann, C.; Mackenzie, W.G.; Raiman, J.; Villarreal, M.S.; Savarirayan, R. International Guidelines for the Management and Treatment of Morquio A Syndrome. Am. J. Med. Genet. A 2015,167, 11–25. [CrossRef] [PubMed] 2. Khan, S.; Alm é ciga-D í az, C.J.; Sawamoto, K.; Mackenzie, W.G.; Theroux, M.C.; Pizarro, C.; Mason, R.W.; Orii, T.; Tomatsu, S. 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