Dimethyl sulfoxide as a gas phase charge-reducing agent for the determination of PEGylated proteins' intact mass.

Determination of PEGylated proteins' intact mass by mass spectrometry is challenging due to the molecules' large size, excessive charges, and instrument limitations. Previous efforts have been reported. However, signal variability, ion coalescence, and a generally low degree of robustness have been observed. In this work, we have explored the capabilities of post-column infusion of dimethyl sulfoxide (DMSO) following reversed-phase liquid chromatography-mass spectrometry (RP-LCMS) to determine PEG-filgrastim' intact mass, and to characterize its PEG moiety. The method was optimized around reproducibility (six preparations, and three injection replicates) with an in-house prepared PEG-filgrastim standard. The method showed a mass accuracy of ≤1.2 Da. The average molecular weight (MWEO=483) was 40 147.9 Da. The number average molecular weight (Mn) and the weight average molecular weight (Mw) were observed to be 40 101.1 and 40 113.9 Da, respectively, both with an RSD of 0.03%. The molecular weight distribution of ethylene oxide (EO), the polydispersity index (PDI), was 1.0003 for all preparations with a minimum and maximum number of EO units of 448 ± 2 and 516 ± 2, respectively. The method was finally applied to commercially available Neulasta® lots where the Mn and Mw were 39 995.8 and 40 008.8 Da, respectively, both with an RSD of 0.1%. The minimum and maximum EO units across the lots were observed to be 444.5 ± 1.5 and 514 ± 3, respectively. The PDI for all Neulasta® lots was 1.0003. This study provides an insightful characterization of Neulasta® and describes a robust LC-MS methodology for the characterization of the PEGylated proteins.

Comparative analysis of Herceptin N-Linked glycosylation by HILIC-FLD and LC-MS/MS methods.

Monoclonal antibodies like Herceptin play a pivotal role in modern therapeutics, with their glycosylation patterns significantly influencing their bioactivity. To characterize the N-glycan profile and their relative abundance in Herceptin, we employed two analytical methods: hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) for released glycans and liquid chromatography tandem mass spectrometry (LC-MS/MS) for glycopeptides. Our analysis included 21 European Union (EU)-Herceptin lots and 14 United States (US)-Herceptin lots. HILIC-FLD detected 25 glycan species, including positional isomers, revealing comparable chromatographic profiles for both EU and US lots. On the other hand, LC-MS/MS identified 26 glycoforms within the glycopeptide EEQYNSTYR. Both methods showed that a subset of glycans dominated the total abundance. Notably, EU-Herceptin lots with an expiration date of October 2022 exhibited increased levels of afucosylated and high mannose N-glycans. Our statistical comparisons showed that the difference in quantitative results between HILIC-FLD and LC-MS/MS is significant, indicating that the absolute quantitative values depend on the choice of the analytical method. However, despite these differences, both methods demonstrated a strong correlation in relative glycan proportions. This study contributes to the comprehensive analysis of Herceptin's glycosylation, offering insights into the influence of analytical methods on glycan quantification and providing valuable information for the biopharmaceutical industry.

Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis.

In the development of biosimilar products to Neulasta, it is essential to determine the intact molecular mass and confirm precise PEGylation sites. In this study, we applied a combination of techniques, including post-column addition of triethylamine in reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) to determine the intact molecular mass, and in-source fragmentation (ISF) and higher-energy collision dissociation-tandem mass spectrometry (HCD-MS/MS) to identify the PEGylation site. Our results show that both the pegfilgrastim biosimilar candidate and Neulasta lots are mono-PEGylated at the N-terminal end. Furthermore, we show that the combined ISF and HCD-MS/MS method can be used for identifying the PEGylation sites in the diPEGylated variant of pegfilgrastim. The diPEGylated variant has modification sites at the N-terminal end and a lysine at position 35 in the protein sequence.

Nuclear translocation of an aminoacyl-tRNA synthetase may mediate a chronic "integrated stress response".

Various stress conditions are signaled through phosphorylation of translation initiation factor eukaryotic initiation factor 2α (eIF2α) to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. Here, we report that tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family that responds to diverse stress conditions through cytosol-nucleus translocation to activate stress-response genes, also inhibits global translation. However, it occurs at a later stage than eIF2α/ATF4 and mammalian target of rapamycin (mTOR) responses. Excluding TyrRS from the nucleus over-activates translation and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. We propose that TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and strategically located nuclear localization signal and integrate them by nucleus translocation to effect protective responses against chronic stress.

Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function.

Aurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and multifaceted regulation present challenges in its therapeutic targeting. Key to overcoming these challenges is to identify and characterize the full range of AURKA interactors, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non-mitotic AURKA interactions. Here, we generate the proximity interactome of AURKA in asynchronous cells, which consists of 440 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA has extensive proximate and physical interactions to centriolar satellites, key regulators of the primary cilium. Loss-of-function experiments identify satellites as negative regulators of AURKA activity, abundance, and localization in quiescent cells. Notably, loss of satellites activates AURKA at the basal body, decreases centrosomal IFT88 levels, and causes ciliogenesis defects. Collectively, our results provide a resource for dissecting spatiotemporal regulation of AURKA and uncover its proteostatic regulation by satellites as a new mechanism for its ciliary functions.

Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD).

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue.

Granulocyte-Colony-Stimulating Factor Alters the Proteomic Landscape of the Ventral Tegmental Area.

Cocaine addiction is characterized by aberrant plasticity of the mesolimbic dopamine circuit, leading to dysregulation of motivation to seek and take drug. Despite the significant toll that cocaine use disorder exacts on society, there are currently no available pharmacotherapies. We have recently identified granulocyte-colony stimulating factor (G-CSF) as a soluble cytokine that alters the behavioral response to cocaine and which increases dopamine release from the ventral tegmental area (VTA). Despite these known effects on behavior and neurophysiology, the molecular mechanisms by which G-CSF affects brain function are unclear. In this study mice were treated with repeated injections of G-CSF, cocaine or a combination and changes in protein expression in the VTA were examined using an unbiased proteomics approach. Repeated G-CSF treatment resulted in alterations in multiple signaling pathways related to synaptic plasticity and neuronal morphology. While the treatment groups had marked overlap in their effect, injections of cocaine and the combination of cocaine and G-CSF lead to distinct patterns of significantly regulated proteins. These experiments provide valuable information as to the molecular pathways that G-CSF activates in an important limbic brain region and will help to guide further characterization of G-CSF function and evaluation as a possible translational target.

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery.

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

Antigen-mediated regulation in monoclonal gammopathies and myeloma.

A role for antigen-driven stimulation has been proposed in the pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) based largely on the binding properties of monoclonal Ig. However, insights into antigen binding to clonal B cell receptors and in vivo responsiveness of the malignant clone to antigen-mediated stimulation are needed to understand the role of antigenic stimulation in tumor growth. Lysolipid-reactive clonal Ig were detected in Gaucher disease (GD) and some sporadic gammopathies. Here, we show that recombinant Ig (rIg) cloned from sort-purified single tumor cells from lipid-reactive sporadic and GD-associated gammopathy specifically bound lysolipids. Liposome sedimentation and binding assays confirmed specific interaction of lipid-reactive monoclonal Ig with lysolipids. The clonal nature of lysolipid-binding Ig was validated by protein sequencing. Gene expression profiling and cytogenetic analyses from 2 patient cohorts showed enrichment of nonhyperdiploid tumors in lipid-reactive patients. In vivo antigen-mediated stimulation led to an increase in clonal Ig and plasma cells (PCs) in GD gammopathy and also reactivated previously suppressed antigenically related nonclonal PCs. These data support a model wherein antigenic stimulation mediates an initial polyclonal phase, followed by evolution of monoclonal tumors enriched in nonhyperdiploid genomes, responsive to underlying antigen. Targeting underlying antigens may therefore prevent clinical MM.

Quantification of Urinary Protein Biomarkers of Autosomal Dominant Polycystic Kidney Disease by Parallel Reaction Monitoring.

PURPOSE: Autosomal dominant polycystic kidney disease (ADPKD) is a life-long disease in which the genes responsible are known, but the pathogenesis of cyst formation and cyst growth are not understood. Cyst growth ultimately leads to end-stage renal failure in most patients. Analysis of the urinary proteome offers the potential to identify proteins that indicate the presence of cysts (and thus provides diagnosis) as well as the rates of cyst growth (providing prognostic information). EXPERIMENTAL DESIGN: A scheduled parallel reaction monitoring (sPRM) assay is performed on urine samples from 14 patients and 18 normal controls. For relative quantification, stable isotope-labeled synthetic peptides are spiked in the urinary protein digests prior to data collection. The data are subsequently normalized to creatinine and protein concentration in the respective urine samples to control for variations in water intake between individuals. RESULTS: Out of the 143 urinary proteins targeted for sPRM assay, 69 proteins are observed to be significantly dysregulated in ADPKD. The dysregulated proteins are used to cluster ADPKD patients into those who are more or less similar to normal controls. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that sPRM is a promising approach to rapidly screen large numbers of proteins in urine in order to provide earlier diagnosis and potentially better understand the pathogenesis of ADPKD development and progression.

MELK Promotes Melanoma Growth by Stimulating the NF-κB Pathway.

Melanoma accounts for more than 80% of skin cancer-related deaths, and current therapies provide only short-term benefit to patients. Here, we show in melanoma cells that maternal embryonic leucine zipper kinase (MELK) is transcriptionally upregulated by the MAPK pathway via transcription factor E2F1. MELK knockdown or pharmacological inhibition blocked melanoma growth and enhanced the effectiveness of BRAFV600E inhibitor against melanoma cells. To identify mediators of MELK function, we performed stable isotope labeling with amino acids in cell culture (SILAC) and identified 469 proteins that had downregulated phosphorylation after MELK inhibition. Of these proteins, 139 were previously reported as substrates of BRAF or MEK, demonstrating that MELK is an important downstream mediator of the MAPK pathway. Furthermore, we show that MELK promotes melanoma growth by activating NF-κB pathway activity via Sequestosome 1 (SQSTM1/p62). Altogether, these results underpin an important role for MELK in melanoma growth downstream of the MAPK pathway.

Quantitative Analysis of the Proteome Response to the Histone Deacetylase Inhibitor (HDACi) Vorinostat in Niemann-Pick Type C1 disease.

Niemann-Pick type C (NPC) disease is an inherited, progressive neurodegenerative disorder principally caused by mutations in the NPC1 gene. NPC disease is characterized by the accumulation of unesterified cholesterol in the late endosomes (LE) and lysosomes (Ly) (LE/Ly). Vorinostat, a histone deacetylase inhibitor (HDACi), restores cholesterol homeostasis in fibroblasts derived from NPC patients; however, the exact mechanism by which Vorinostat restores cholesterol level is not known yet. In this study, we performed comparative proteomic profiling of the response of NPC1I1061T fibroblasts to Vorinostat. After stringent statistical criteria to filter identified proteins, we observed 202 proteins that are differentially expressed in Vorinostat-treated fibroblasts. These proteins are members of diverse cellular pathways including the endomembrane dependent protein folding-stability-degradation-trafficking axis, energy metabolism, and lipid metabolism. Our study shows that treatment of NPC1I1061T fibroblasts with Vorinostat not only enhances pathways promoting the folding, stabilization and trafficking of NPC1 (I1061T) mutant to the LE/Ly, but alters the expression of lysosomal proteins, specifically the lysosomal acid lipase (LIPA) involved in the LIPA->NPC2->NPC1 based flow of cholesterol from the LE/Ly lumen to the LE/Ly membrane. We posit that the Vorinostat may modulate numerous pathways that operate in an integrated fashion through epigenetic and post-translational modifications reflecting acetylation/deacetylation balance to help manage the defective NPC1 fold, the function of the LE/Ly system and/or additional cholesterol metabolism/distribution pathways, that could globally contribute to improved mitigation of NPC1 disease in the clinic based on as yet uncharacterized principles of cellular metabolism dictating cholesterol homeostasis.

Data-Independent Acquisition and Parallel Reaction Monitoring Mass Spectrometry Identification of Serum Biomarkers for Ovarian Cancer.

A data-independent acquisition (DIA)/parallel reaction monitoring (PRM) workflow was implemented to identify improved ovarian cancer biomarkers. Data-independent acquisition on ovarian cancer versus control sera and literature searches identified 50 biomarkers and indicated that apolipoprotein A-IV (ApoA-IV) is the most significantly differentially regulated protein. Parallel reaction monitoring with Targeted Ovarian Cancer Proteome Assay validated differential ApoA-IV expression and quantified 9 other biomarkers. Random Forest (RF) analyses achieved 92.3% classification accuracy and confirmed ApoA-IV as the leading biomarker. Indeed, all samples were classified correctly with an [ApoA-IV] breakpoint. The next best biomarkers were C-reactive protein, transferrin, and transthyretin. The Targeted Ovarian Cancer Proteome Assay suggests that ApoA-IV is a more reliable biomarker than had been determined by immunological assays and it is a better biomarker than ApoA-I, which is in the OVA1 test for ovarian cancer. This research provides a PRM/RF approach together with 4 promising biomarkers to speed the development of a clinical assay for ovarian cancer.

Proteomics data on MAP Kinase Kinase 3 knock out bone marrow derived macrophages exposed to cigarette smoke extract.

This data article reports changes in the phosphoproteome and total proteome of cigarette smoke extract (CSE) exposed WT and MAP Kinase Kinase 3 knock out (MKK3-/-) bone marrow derived macrophages (BMDM). The dataset generated is helpful for understanding the mechanism of CSE induced inflammation and the role of MAP kinase signaling pathway. The cellular proteins were labeled with isobaric tags for relative and absolute quantitation (iTRAQ®) reagents and analyzed by LC-MS/MS. The standard workflow module for iTRAQ® quantification within the Proteome Discoverer was utilized for the data analysis. Ingenuity Pathway Analysis (IPA) software and Reactome was used to identify enriched canonical pathways and molecular networks (Mannam et al., 2016) [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs.

The centriolar satellite protein CCDC66 interacts with CEP290 and functions in cilium formation and trafficking.

Centriolar satellites are membrane-less structures that localize and move around the centrosome and cilium complex in a microtubule-dependent manner. They play important roles in centrosome- and cilium-related processes, including protein trafficking to the centrosome and cilium complex, and ciliogenesis, and they are implicated in ciliopathies. Despite the important regulatory roles of centriolar satellites in the assembly and function of the centrosome and cilium complex, the molecular mechanisms of their functions remain poorly understood. To dissect the mechanism for their regulatory roles during ciliogenesis, we performed an analysis to determine the proteins that localize in close proximity to the satellite protein CEP72, among which was the retinal degeneration gene product CCDC66. We identified CCDC66 as a microtubule-associated protein that dynamically localizes to the centrosome, centriolar satellites and the primary cilium throughout the cell cycle. Like the BBSome component BBS4, CCDC66 distributes between satellites and the primary cilium during ciliogenesis. CCDC66 has extensive proximity interactions with centrosome and centriolar satellite proteins, and co-immunoprecipitation experiments revealed interactions between CCDC66, CEP290 and PCM1. Ciliogenesis, ciliary recruitment of BBS4 and centriolar satellite organization are impaired in cells depleted for CCDC66. Taken together, our findings identify CCDC66 as a targeting factor for centrosome and cilium proteins.

MKK3 influences mitophagy and is involved in cigarette smoke-induced inflammation.

Cigarette smoking is the primary risk factor for COPD which is characterized by excessive inflammation and airflow obstruction of the lung. While inflammation is causally related to initiation and progression of COPD, the mitochondrial mechanisms that underlie the associated inflammatory responses are poorly understood. In this context, we have studied the role played by Mitogen activated protein (MAP) kinase kinase 3 (MKK3), a dual-specificity protein kinase, in cigarette smoke induced-inflammation and mitochondrial dysfunction. Serum pro-inflammatory cytokines were significantly elevated in WT but not in MKK3-/- mice exposed to Cigarette smoke (CS) for 2 months. To study the cellular mechanisms of inflammation, bone marrow derived macrophages (BMDMs), wild type (WT) and MKK3-/-, were exposed to cigarette smoke extract (CSE) and inflammatory cytokine production and mitochondrial function assessed. The levels of IL-1ß, IL-6, and TNFα were increased along with higher reactive oxygen species (ROS) and P-NFκB after CSE treatment in WT but not in MKK3-/- BMDMs. CSE treatment adversely affected basal mitochondrial respiration, ATP production, maximum respiratory capacity, and spare respiratory capacity in WT BMDMs only. Mitophagy, clearance of dysfunctional mitochondria, was up regulated in CS exposed WT mice lung tissue and CSE exposed WT BMDMs, respectively. The proteomic analysis of BMDMs by iTRAQ (isobaric tags for relative and absolute quantitation) showed up regulation of mitochondrial dysfunction associated proteins in WT and higher OXPHOS (Oxidative phosphorylation) and IL-10 signaling proteins in MKK3-/- BMDMs after CSE exposure, confirming the critical role of mitochondrial homeostasis. Interestingly, we found increased levels of p-MKK3 by immunohistochemistry in COPD patient lung tissues that could be responsible for insufficient mitophagy and disease progression. This study identifies MKK3 as a negative regulator of mitochondrial function and inflammatory responses to CS and suggests that MKK3 could be a therapeutic target.

Parallel Reaction Monitoring: A Targeted Experiment Performed Using High Resolution and High Mass Accuracy Mass Spectrometry.

The parallel reaction monitoring (PRM) assay has emerged as an alternative method of targeted quantification. The PRM assay is performed in a high resolution and high mass accuracy mode on a mass spectrometer. This review presents the features that make PRM a highly specific and selective method for targeted quantification using quadrupole-Orbitrap hybrid instruments. In addition, this review discusses the label-based and label-free methods of quantification that can be performed with the targeted approach.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses.

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.

Quantitative Proteomics of Human Fibroblasts with I1061T Mutation in Niemann-Pick C1 (NPC1) Protein Provides Insights into the Disease Pathogenesis.

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in the late endosomal/lysosomal compartments. Mutations in the NPC1 protein are implicated in 95% of patients with NPC disease. The most prevalent mutation is the missense mutation I1061T that occurs in ∼ 15-20% of the disease alleles. In our study, an isobaric labeling-based quantitative analysis of proteome of NPC1(I1061T) primary fibroblasts when compared with wild-type cells identified 281 differentially expressed proteins based on stringent data analysis criteria. Gene ontology enrichment analysis revealed that these proteins play important roles in diverse cellular processes such as protein maturation, energy metabolism, metabolism of reactive oxygen species, antioxidant activity, steroid metabolism, lipid localization, and apoptosis. The relative expression level of a subset of differentially expressed proteins (TOR4A, DHCR24, CLGN, SOD2, CHORDC1, HSPB7, and GAA) was independently and successfully substantiated by Western blotting. We observed that treating NPC1(I1061T) cells with four classes of seven different compounds that are potential NPC drugs increased the expression level of SOD2 and DHCR24. We have also shown an abnormal accumulation of glycogen in NPC1(I1061T) fibroblasts possibly triggered by defective processing of lysosomal alpha-glucosidase. Our study provides a starting point for future more focused investigations to better understand the mechanisms by which the reported dysregulated proteins triggers the pathological cascade in NPC, and furthermore, their effect upon therapeutic interventions.

Isobaric labeling-based relative quantification in shotgun proteomics.

Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined.