Improving mass accuracy in matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of pathogenic proteins using 6xHIS-tagged internal calibration.

RATIONALE: Linear mode of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been routinely used for bacterial identification in the clinic, depending on the pattern analysis of spectral libraries rather than accurate mass measurement of ribosomal proteins (10-15 kDa). However, a demand for more accurate mass analysis of pathogens (e.g. KPC-2 carbapenemase) has been recently increasing for diagnostic purposes. METHODS: We introduced a 6xHIS-tagged KPC-2 (i.e. hKPC-2) and used it as an internal mass calibrator for the mass calibration of target proteins. After internal mass calibration (In-Cal), we evaluated the observed mass of KPC-2 against the theoretical mass of hKPC-2, which has 823 Da mass difference from the target protein. We further assessed the accuracy and precision of our calibration method regarding the identification of KPC-2 and other pathogens in clinical isolates (n = 42). RESULTS: Among several candidates for internal mass calibrators, the In-Cal using a 6xHIS-tagged protein on the target showed the highest mass accuracy and precision in the detection of target proteins (e.g. KPC-2). The application of hKPC-2 as an internal calibrator showed substantial improvement of mass accuracy, mass precision and also quantification of KPC in linearity and repeatability for KPC detection in the clinical isolates. CONCLUSIONS: Our In-Cal method using 6xHIS-tagged protein in MALDI-TOFMS allows successful mass calibration (<3.5 Da) of pathogenic proteins (>20 kDa) and provides high mass accuracy as much as that of medium- and high-resolution mass spectrometry.

A Graphene-Coated Silicon Wafer Plate Improves the Sensitivity and Reproducibility of MALDI-TOF MS Analysis of Proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used to analyze small and large molecules. However, proteins are difficult to analyze with MALDI-TOF MS in clinical applications because of their low ionization efficiency and heterogeneous crystallization with the matrix on the sample spots. Here, we investigate the potential of a customized graphene-coated silicon wafer (G/SiO2) plate for MALDI-TOF MS analysis of a clinically important protein, KPC-2, in comparison with a conventional stainless steel (SUS) plate. Our results demonstrate that the G/SiO2 plate outperforms the SUS plate in terms of sensitivity, reproducibility, and mass accuracy/precision across a wide range of molecular weights, even with highly complex samples. Furthermore, a five-day robustness test confirms the practical applicability of the G/SiO2 plate for the reliable identification of target protein(s) in MALDI-TOF MS analysis. Overall, our findings suggest that the use of the G/SiO2 plate holds great potential for improving the sensitivity and reproducibility of MALDI-TOF MS analysis for the identification of proteins, making it a promising tool for clinical applications.

Fast and straightforward simultaneous quantification of multiple apolipoproteins in human serum on a high-throughput LC-MS/MS platform.

PURPOSE: Apolipoprotein monitoring is useful for diagnosing cardiovascular diseases, as they are risk factors of arteriosclerosis and other neutral fat-related diseases. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is advantageous for simultaneous apolipoprotein quantification, differentiation, and standardization including their isoforms. However, fast and straightforward sample preparation that retains quantification accuracy remains challenging in clinical MS. EXPERIMENTAL DESIGN: We developed a simultaneous assay for serum apolipoprotein A-I (ApoA-I), apolipoprotein B100 family, and apolipoprotein C-III (ApoC-III) using a high-throughput LC-MS/MS platform coupled with a BRAVO system. The assay was simplified by using sodium deoxycholate and trypsin/lys-C without reduction and alkylation steps. RESULTS: Simple sample preparation reduced turnaround time by 1.5 h and neat goat serum was chosen as an optimal calibration matrix for accurate protein quantification. Assay precision, linearity, correlation, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and carryover were validated according to CLSI guidelines over 41 days using more than 100 human serum samples. Good correlation compared with turbidimetric immunoassay (TIA) was observed by Deming regression for all analytes. CONCLUSIONS AND CLINICAL RELEVANCE: A high-throughput LC-MS/MS and BRAVO assay for simultaneous apolipoprotein analysis was validated using a simple preparation method with a human serum calibrator in goat serum matrix. The assay is readily expandable to include other target serum proteins and/or their isoforms.

Simultaneous Quantification of Apolipoprotein C-III O-Glycoforms by Protein-MRM.

Apolipoprotein C-III (APOC-III) regulates triglyceride levels, associated with a risk of cardiovascular disease. One gene generates several proteoforms, each with a different molecular mass and a unique function. Unlike peptide multiple reaction monitoring (MRM), protein-MRM without digestion is required to analyze clinically relevant individual proteoforms. We developed a protein-MRM method without digestion to individually quantify APOC-III proteoforms in human serum. We optimized the protein-MRM method following 60% acetonitrile extraction with C18 filtration. Bovine serum and myoglobin served as supporting cushions and the internal standard during sample preparation, respectively. Furthermore, we evaluated the LOD, lower limit of quantification, linearity, accuracy, and precision. Good correlation compared with turbidimetric immunoassay (TIA) and peptide-MRM was observed using 30 clinical sera. Individual APOC-III O-glycoforms were identified by top-down proteomics and simultaneously quantified using the protein-MRM method. The sum abundance of APOC-III proteoforms was significantly correlated with TIA and peptide-MRM. Our protein-MRM method provides an affordable and rapid quantification of potential disease-specific proteoforms. Precise quantification of each proteoform allows investigators to identify novel biological roles potentially related to cardiovascular disease or novel biomarkers. We expect our protein-oriented method to be more clinically useful than antibody-based immunoassays and peptide-oriented MRM analysis, especially for quantification of a biomarker proteoform with certain post-translational modifications.

Clinical Performance of the Osmotic Shock-MALDI MS Method to Detect Klebsiella pneumoniae Carbapenemase in Clinical Isolates.

The World Health Organization recently highlighted the serious worldwide problem of the emergence of antibiotic-resistant or antibiotic multidrug-resistant bacteria. Carbapenem-resistant Enterobacterales, including carbapenemase-producing Enterobacterales (CPE), are major antibiotic-resistant bacteria that can be identified by various methods, including antibiotic susceptibility testing, PCR, and immunologic assays. However, there is a need for a faster, more accurate, low-cost, and easy method to detect CPE strains. We previously developed an osmotic shock matrix-assisted laser desorption/ionization mass spectrometry (OS-MALDI MS) method for directly detecting intact Klebsiella pneumoniae carbapenemase (KPC) using osmotic shock cell lysis. In this study, we evaluated the OS-MALDI MS method and compared it with two other methods (octyl-glucoside-aided direct KPC detection method [OG-MALDI MS] and Bruker's MBT subtyping module indirect method [MBT-SM MALDI MS]). We first completed an analytical performance evaluation of the OS-MALDI MS method according to Clinical and Laboratory Standards Institute guidelines. Clinical testing was performed with 437 clinical isolates, including 292 KPC-producing bacteria and 145 non-KPC-producing bacteria. The OS-MALDI MS method exhibited 95.9% sensitivity, 100.0% specificity, and 100.0% precision for detecting KPC. Accuracy of the OS-MALDI MS, OG-MALDI MS, and MBT-SM MALDI MS methods was 97.3%, 55.9%, and 50.2%, respectively. In conclusion, the OS-MALDI MS method clearly outperformed the other methods, exhibiting the highest accuracy and sensitivity of the three methods. We propose the OS-MALDI MS method as a practical, useful method for clinic environments, which may help guide appropriate antibiotic treatment and contribute to the prevention of the spread of CPE.

Nutrient-specific proteomic analysis of the mucin degrading bacterium Akkermansia muciniphila.

Akkermansia muciniphila is a prominent mucin-degrading bacterium that acts as a keystone species in regulating the human gut microbiota. Despite recently increasing research into this bacterium and its relevance to human health, a high-resolution database of its functional proteins remains scarce. Here, we provide a proteomic overview of A. muciniphila grown in different nutrient conditions ranging from defined to complex. Of 2318 protein-coding genes in the genome, we identified 841 (40%) that were expressed at the protein level. Overall, proteins involved in energy production and carbohydrate metabolism indicate that A. muciniphila relies mainly on the Embden-Meyerhof-Parnas pathway, and produces short-chain fatty acids through anaerobic fermentation in a nutrient-specific manner. Moreover, this bacterium possesses a broad repertoire of glycosyl hydrolases, together with putative peptidases and sulfatases, to cleave O-glycosylated mucin. Of them, putative mucin-degrading enzymes (Amuc_1220, Amuc_1120, Amuc_0052, Amuc_0480, and Amuc_0060) are highly abundant in the mucin-supplemented media. Furthermore, A. muciniphila uses mucin-derived monosaccharides as sources of energy and cell wall biogenesis. Our dataset provides nutrient-dependent global proteomes of A. muciniphila ATCC BAA-835 to offer insights into its metabolic functions that shape the composition of the human gut microbiota via mucin degradation.

Optimization of a lysis method to isolate periplasmic proteins from Gram-negative bacteria for clinical mass spectrometry.

PURPOSE: Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram-negative bacteria and apply to clinical mass spectrometry. EXPERIMENTAL DESIGN: The Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC-MS/MS and MALDI-TOF MS. The effectiveness of the OS lysis method for KPC-2-producing Enterobacteriaceae clinical isolates were then confirmed by MALDI-TOF MS. RESULTS: The optimized OS lysis using KPC-2 producing E. coli standard cells showed a high yield of KPC-2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC-2 protein significantly increased in MALDI-TOF MS analysis. Nineteen clinical isolates were validated by MALDI-TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n-octyl-ß-D-glucopyranoside) (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: This study provides a straightforward, rapid, affordable, and detergent-free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS-based clinical diagnostics.

Record of North American boreal forest fires in northwest Greenland snow.

We present boreal forest fire proxies in a northwest Greenland snowpit spanning a period of six years, from spring 2003 to summer 2009. Levoglucosan (C6H10O5) is a specific organic molecular marker of biomass burning caused by boreal forest fires. In this study, levoglucosan was determined via liquid chromatography/negative ion electrospray ionization-tandem mass spectrometry, wherein isotope-dilution and multiple reaction monitoring methods are employed. Ammonium (NH4+) and oxalate (C2O42-), traditional biomass burning proxies, were determined using two-channel ion chromatography. In the northwest Greenland snowpit, peaks in levoglucosan, ammonium, and oxalate were observed in snow layers corresponding to the summer-fall seasons of 2004 and 2005. Considered together, these spikes are a marker for large boreal forest fires. The levoglucosan deposited in the Greenland snow was strongly dependent on long-range atmospheric transportation. A 10-day backward air mass trajectory analysis supports that the major contributors were air masses from North America. In addition, satellite-derived carbon monoxide (CO) and ammonia (NH3) concentrations suggest that chemicals from North American boreal forest fires during the summer-fall of 2004 and 2005 were transported to Greenland. However, large boreal fires in Siberia in 2003 and 2008 were not recorded in the snowpit. The sub-annual resolution measurements of levoglucosan and ammonium can distinguish between the contributions of past boreal forest fires and soil emissions from anthropogenic activity to Greenland snow and ice.

Measuring fucosylated alpha-fetoprotein in hepatocellular carcinoma: A comparison of µTAS and parallel reaction monitoring.

PURPOSE: Fucosylation of alpha-fetoprotein (AFP) is closely correlated with the diagnosis of patients with hepatocellular carcinoma (HCC). In current, a micro-total analysis system (µTAS) using immunoassay has been developed for determining fucosylated AFP EXPERIMENTAL DESIGN: We compared two analytical methods, µTAS and liquid chromatography-parallel reaction monitoring mass spectrometry (LC-PRM MS), for the measurement of fucosylated AFP in serum to evaluate the usefulness of the results. For this purpose, serum samples were used (cirrhosis, n = 105; HCC, n = 105), and we have discussed the analytical performance of these two methods RESULTS: We observed a correlation (R2  = 0.84) between LC-PRM MS and µTAS using samples where fucosylated levels were measured by both methods. The fucosylated level of AFP by LC-PRM MS better differentiated between cirrhosis and HCC patients than those by µTAS (AUC = 0.910 vs. 0.861), particularly in subgroups with a level of total AFP < 20 ng/mL (0.973 vs. 0.874) and in early stage (I and II) patients (0.922 vs. 0.835) CONCLUSIONS AND CLINICAL RELEVANCE: From this comparative study we can suggest that the LC-PRM MS is applicable in the measurement of fucosylated AFP from human serum and is more useful for early diagnosis of HCC. CLINICAL RELEVANCE: Fucosylation of AFP is used for the detection of HCC. A micro-total analysis system (µTAS) has been only developed for measuring fucosylation of AFP in clinical research. This study reports the fucosylation of AFP in human serum samples from cirrhosis and HCC patients using the µTAS and a LC-PRM MS to evaluate fucosylation of AFP from each method. As a result, LC-PRM MS is complementary to the conventional µTAS method. Furthermore, LC-PRM MS provides a higher diagnostic accuracy than the µTAS in patients with low AFP levels and an early stage.

SWATH-MS analysis of cerebrospinal fluid to generate a robust battery of biomarkers for Alzheimer's disease.

Cerebrospinal fluid (CSF) Aß42 and tau protein levels are established diagnostic biomarkers of Alzheimer's disease (AD). However, their inadequacy to represent clinical efficacy in drug trials indicates the need for new biomarkers. Sequential window acquisition of all theoretical fragment ion spectra (SWATH)-based mass spectrometry (MS) is an advanced proteomic tool for large-scale, high-quality quantification. In this study, SWATH-MS showed that VGF, chromogranin-A, secretogranin-1, and opioid-binding protein/cell adhesion molecule were significantly decreased in 42 AD patients compared to 39 controls, whereas 14-3-3ζ was increased (FDR < 0.05). In addition, 16 other proteins showed substantial changes (FDR < 0.2). The expressions of the top 21 analytes were closely interconnected, but were poorly correlated with CSF Aß42, tTau, and pTau181 levels. Logistic regression analysis and data mining were used to establish the best algorithm for AD, which created novel biomarker panels with high diagnostic value (AUC = 0.889 and 0.924) and a strong correlation with clinical severity (all p < 0.001). Targeted proteomics was used to validate their usefulness in a different cohort (n = 36) that included patients with other brain disorders (all p < 0.05). This study provides a list of proteins (and combinations thereof) that could serve as new AD biomarkers.

Direct detection of intact Klebsiella pneumoniae carbapenemases produced by Enterobacterales using MALDI-TOF MS.

OBJECTIVES: A MALDI-TOF MS-based identification method for KPC-producing Enterobacterales was developed. METHODS: The molecular mass of the intact KPC-2 polypeptide was estimated for blaKPC-2 transformants using MALDI Microflex and the exact mass was confirmed by LC and a high-resolution MS/MS system. A total of 1181 clinical Enterobacterales strains, including 369 KPC producers and 812 KPC non-producers, were used to set up the methodology and the results were compared with those from PCR analyses. For external validation, a total of 458 Enterobacterales clinical isolates from a general hospital between December 2018 and April 2019 were used. RESULTS: The exact molecular mass of the intact KPC-2 protein was 28 718.13 Da and KPC peaks were observed at m/z 28 708.87-28 728.34 using MALDI Microflex. Most of the KPC-2 (99.1%, 335/338) and KPC-3 (100%, 6/6) producers presented a clear peak via this method, while 12.0% (3/25) of the KPC-4 producers had a peak of weak intensity associated with low levels of gene expression. It took less than 20 min for the entire assay to be performed with colonies on an agar plate. External validation showed that the analytical sensitivity and specificity of the method compared with PCR were 100% (59/59) and 99.50% (397/399), respectively. CONCLUSIONS: The MALDI-TOF MS-based method for directly detecting the intact KPC protein is applicable to routine tests in clinical microbiology laboratories, supported by its speed, low cost and excellent sensitivity and specificity.

Subunits of the vacuolar H+-ATPase complex, Vma4 and Vma10, are essential for virulence and represent potential drug targets in Candida albicans.

Hyphal morphogenesis of Candida albicans is important for its pathogenesis. Here, we showed that the filamentous growth of C. albicans requires vacuolar H+-ATPase function. Results showed that levels of Vma4 and Vma10 increased in cells undergoing hyphal growth compared to those undergoing yeast growth. Deleting VMA4 or VMA10 abolished vacuolar functions and hyphal morphogenesis. These deletion mutants were also characterized as avirulent in a mouse model of systemic infection. Furthermore, VMA4 and VMA10 deletion strains showed hypersensitivity to fluconazole, terbinafine, and amphotericin B. Based on these findings, Vma4 and Vma10 are not only involved in vacuole biogenesis and hyphal formation, but also are good targets for antifungal drug development in C. albicans.

Identification of Matrix Metalloproteinase-1-Suppressive Peptides in Feather Keratin Hydrolysate.

Inhibition of matrix metalloproteinases (MMPs), which degrade collagen and elastin in the dermis of normal skin, is a key strategy for anti-skin aging. In this study, we identified five low-molecular-weight (LMW, <1 kDa) MMP-1-suppressive peptides in feather keratin hydrolysate (FKH) obtained by anaerobic digestion with an extremophilic bacterium. FKH was first subjected to ultrafiltration, followed by size-exclusion chromatography and liquid chromatography/electrospray ionization tandem mass spectrometry analysis. Chemically synthesized peptides identical to the sequences identified suppressed MMP expression in human dermal fibroblasts (HDFs). To investigate the impact of the MMP-1-suppressive peptides on the signaling pathway, we performed antibody array phosphorylation profiling of HDFs. The results suggested that the peptide GGFDL regulates ultraviolet-B-induced MMP-1 expression by inhibiting mitogen-activated protein kinases and nuclear factor κB signaling pathways as well as histone modification. Thus, LMW feather keratin peptides could serve as novel bioactive compounds to protect the skin against intrinsic and extrinsic factors.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration.

BACKGROUND: To identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls. METHODS: After uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups). RESULTS: Increased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen. CONCLUSIONS: Differential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Ssb2 is a novel factor in regulating synthesis and degradation of Gcn4 in Saccharomyces cerevisiae.

Yeast cells respond to environmental stress by inducing the master regulator Gcn4 to control genes involved in biosynthesis of amino acids and purine pathways. Gcn4 is a member of the basic leucine Zipper family and binds directly as a homodimer to a conserved regulatory region of target genes. Ssb2 was discovered to rescue the mutant Gcn4 which has a point mutation that decreases DNA-binding affinity. Ssb2 is part of the Hsp70 protein family responsible for protein quality control and it is thought that Ssb2 assists the passage of nascent polypeptide chains from the ribosomes. To characterize the mechanism behind the rescue of the mutant gcn4 phenotype, transcriptional activity and protein levels of Gcn4 were analyzed. We found that Ssb2 improved the expression of Gcn4 target genes by increasing the DNA-binding affinity of gcn4 mutants to target gene promoters under conditions of amino acid starvation. Gcn4 levels increased at both translational and post-translational levels without regulating GCN4 steady-state mRNA levels. We also found that the nuclear export signal of Ssb2 is required for interaction with Gcn4 and rescue of the gcn4 mutant phenotype. These findings suggest that Ssb2 is a critical factor that modulates Gcn4 functions in the nucleus and cytosol.

S-1-Induced Lacrimal Drainage Obstruction and Its Association with Ingredients/Metabolites of S-1 in Tears and Plasma: A Prospective Multi-institutional Study.

PURPOSE: This prospective study was conducted to determine the incidence of lacrimal drainage obstruction (LDO) during S-1 chemotherapy and evaluate the association between the development of LDO and the concentrations of ingredients/metabolites of S-1 in tears and plasma. MATERIALS AND METHODS: A total of 145 patients with gastric cancer who received adjuvant S-1 therapy were enrolled. Ophthalmologic examinations were performed regularly during S-1 chemotherapy. Concentrations of tegafur, 5-chloro-2,4-dihydroxypyridine (CDHP), and 5-fluorouracil at steady-state trough level were measured in both tears and plasma. RESULTS: Fifty-three patients (37%) developed LDO. The median time to the onset of LDO was 10.9 weeks, and LDO developed most frequently in the nasolacrimal duct. Univariable analyses revealed that an older age (≥ 70 years), creatinine clearance rate (Ccr) < 80 mL/min, 5-fluorouracil concentration in plasma ≥ 22.3 ng/mL (median), CDHP concentration in plasma ≥ 42.0 ng/mL (median), and tegafur concentration in tears ≥ 479.2 ng/mL (median) were related to increased development of LDO. Multivariable analysis indicated that a high plasma 5-fluorouracil concentration was predictive of increased development of LDO (hazard ratio, 2.02; p=0.040), along with older age and decreased Ccr. Patients with LDO also developed S-1-related non-hematologic toxicity more frequently than those without LDO (p=0.016). CONCLUSION: LDO is a frequent adverse event during S-1 chemotherapy. An older age, decreased Ccr, and high plasma 5-fluorouracil concentration were found to be independent risk factors for LDO. The high incidence of LDO warrants regular ophthalmologic examination and early intervention in patients receiving S-1 therapy.

PiB-PET Imaging-Based Serum Proteome Profiles Predict Mild Cognitive Impairment and Alzheimer's Disease.

Development of a simple, non-invasive early diagnosis platform of Alzheimer's disease (AD) using blood is urgently required. Recently, PiB-PET imaging has been shown to be powerful to quantify amyloid-ß plaque loads leading to pathophysiological alterations in AD brains. Thus, there has been a need for serum biomarkers reflecting PiB-PET imaging data as an early diagnosis platform of AD. Here, using LC-MS/MS analysis coupled with isobaric tagging, we performed comprehensive proteome profiling of serum samples from cognitively normal controls, mild cognitive impairment (MCI), and AD patients, who were selected using PiB-PET imaging. Comparative analysis of the proteomes revealed 79 and 72 differentially expressed proteins in MCI and AD, respectively, compared to controls. Integrated analysis of these proteins with genomic and proteomic data of AD brain tissues, together with network analysis, identified three biomarker candidates representing the altered proteolysis-related process in MCI or AD: proprotein convertase subtilisin/kexin type 9 (PCSK9), coagulation factor XIII, A1 polypeptide (F13A1), and dermcidin (DCD). In independent serum samples of MCI and AD, we confirmed the elevation of the candidates using western blotting and ELISA. Our results suggest that these biomarker candidates can serve as a potential non-invasive early diagnosis platform reflecting PiB-PET imaging for MCI and AD.

NEDDylation promotes stress granule assembly.

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Reciprocal changes in phosphorylation and methylation of mammalian brain sodium channels in response to seizures.

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.

Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.