Development and evaluation of a neonatal intensive care unit medication safety simulation for nursing students in South Korea: a quasi-experimental study.

PURPOSE: Nursing students are susceptible to medication safety incidents in the neonatal intensive care unit (NICU) related to a lack of communication experience. The purpose of the present study was to investigate the impact of a NICU medication safety simulation (NMSS) focusing on communication clarity, patient hand-off confidence, and patient safety competency in senior-year nursing students. METHODS: The study utilized a nonequivalent control group pretest-posttest design. In total, 60 nursing students were assigned to two groups. The experimental group participated in the NMSS, which included three medication error scenarios. Pairs of students completed the scenarios together in 10 to 20 minutes. Data were analyzed using the chi-squared test, independent t test, and ANCOVA. RESULTS: The experimental group showed significant improvements in communication clarity (p=.015), and patient safety competency (p<.001) compared to the control group. Using the pretest values as covariates, patient hand-off confidence scores significantly increased (p=.027). CONCLUSION: Implementing the NMSS focusing on communication in the pediatric nursing curriculum helped students to communicate clearly and concisely about medication errors, and its use is recommended to promote patient safety competency in the NICU.

Targeting extracellular matrix glycation to attenuate fibroblast activation.

The extracellular matrix (ECM) of the tumor microenvironment undergoes constant remodeling that alters its biochemical and mechano-physical properties. Non-enzymatic glycation can induce the formation of advanced glycation end-products (AGEs), which may cause abnormal ECM turnover with excessively cross-linked collagen fibers. However, the subsequent effects of AGE-mediated matrix remodeling on the characteristics of stromal cells in tumor microenvironments remain unclear. Here, we demonstrate that AGEs accumulated in the ECM alter the fibroblast phenotype within a three-dimensional collagen matrix. Both the AGE interaction with its receptor (RAGE) and integrin-mediated mechanotransduction signaling were up-regulated in glycated collagen matrix, leading to fibroblast activation to acquire a cancer-associated fibroblast (CAF)-like phenotype. These effects were blocked with neutralizing antibodies against RAGE or the inhibition of focal adhesion (FA) signaling. An AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711), also reduced the transformation of fibroblasts into the CAF-like phenotype because of its dual inhibitory role in the AGE-modified matrix. Apart from targeting the AGE-RAGE interaction directly, the decreased matrix stiffness attenuated fibroblast activation by inhibiting the downstream cellular response to matrix stiffness. Our results suggest that indirect/direct targeting of accumulated AGEs in the ECM has potential for targeting the tumor stroma to improve cancer therapy. STATEMENT OF SIGNIFICANCE: Advanced glycated end-products (AGEs)-modified extracellular matrix (ECM) is closely associated with pathological states and is recognized as a critical factor that precedes tumorigenesis. While increased matrix stiffness is known to induce fibroblast activation, less is known about how both biochemical and mechano-physical changes in AGE-mediated matrix-remodeling cooperate to produce a myofibroblastic cancer-associated fibroblast (CAF)-like phenotype. For the first time, we found that both the AGE interaction with its receptor (RAGE) and integrin-mediated mechanotransduction were up-regulated in glycated collagen matrix, leading to fibroblast activation. We further demonstrated that an AGE cross-link breaker, ALT-711, reduced the CAF-like transformation because of its dual inhibitory role in the AGE-modified matrix. Our findings offer promising extracellular-reversion strategies targeting the non-enzymatic ECM glycation, to regulate fibroblast activation.

Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction.

Disorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem's biological activity, the target protein was identified via combined drug affinity responsive target stability and LC-MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

DNA Polymerase Alpha Subunit B Is a Binding Protein for Erlotinib Resistance in Non-Small Cell Lung Cancer.

Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Effects of a Community-based Follow-up Program for Parents with Premature Infants on Parenting Stress, Parenting Efficacy, and Coping.

PURPOSE: This study was conducted to evaluate the efficacy of a community-based follow-up program on parenting stress, parenting efficacy, and coping among parents with premature infants. METHODS: A non-equivalent control group pre-post quasi-experimental design was used. This program consisted of structured home visits and self-help group meetings for 6 months. The experimental group (n=29) received visits by an experienced neonatal intensive care unit (NICU) nurse and the control group (n=27) was visited by a visiting nurse. Data were analyzed using the x 2 test, t-test, and analysis of covariance. RESULTS: Parents' coping behavior significantly differed in the experimental group compared to the control group (t=3.14, p=.003). In particular, coping subscale I, for maintaining the family situation (t=2.63, p=.011), and subscale III, for understanding the infant's medical situation (t=4.30, p<.001), showed significant differences in the experimental group. There were no significant between-group differences in parenting stress or parenting efficacy. CONCLUSION: The findings of this study suggest that home visits by an experienced NICU nurse provided through a community-based follow-up program were an effective intervention to improve coping behavior among parents with premature infants.

Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma.

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers. Graphical abstract.

Experiences of Mothers' Attachment in a Follow-Up Program Using Early Intervention for Low-Birth-Weight Infants.

PURPOSE: Mothers who give birth prematurely experience parenting stress after their babies are discharged and find it difficult to emotionally bond with them. Forming an emotional bond with a baby promotes the baby's growth and development, helps the mother cope with parenting stress after discharge, and is important for maintaining family functioning. This study aimed to identify the attachment experiences of mothers with low-birth-weight infants (LBWIs) in a follow-up program using early intervention. METHODS: A phenomenological perspective was used for this qualitative research. Data were collected from in-depth interviews with twelve mothers who participated in a follow-up program using early intervention for mothers with LBWIs from September 2017 to December 2017. Colaizzi's method was used to analyze the data. RESULTS: The experience of mothers' attachment was investigated on the basis of three categories: 'beginning of changes in parenting methods,' 'forming an intimate mother-child bond,' and 'concerns and expectation about the child's development.' CONCLUSION: The results indicate that the follow-up program using an early intervention designed to increase mothers' confidence in their parenting skills can promote mother' attachment and the quality of life of families with LBWIs.

Isomer separation of sialylated O- and N-linked glycopeptides using reversed-phase LC-MS/MS at high temperature.

Analyses of intact glycopeptides using mass spectrometry is challenging due to the numerous types of isomers of glycan moieties attached to the peptide backbone. Here, we demonstrate that high-temperature reversed-phase liquid chromatography (RPLC) can be used to separate isomeric O- and N-linked glycopeptides. In general, high column temperatures enhanced the resolution for separation of sialylated O- and N-linked glycopeptide isomers with decreased retention times. Using the high-temperature RPLC method, α2-6-linked sialylated N-glycopeptides were eluted first, followed by α2-3-linked isomers. However, highly sialylated N-glycopeptides containing hydrophobic amino acids exhibited increased retention times at high temperature. The separation of sialylated O- and N-glycopeptides with different glycan isoforms using a high-temperature RPLC method was demonstrated. This study indicates that reversed-phase chromatographic separation at high column temperatures is suitable for the separation of glycopeptide structural isomers.

Systematic Review of Behavioral Therapy to Improve Swallowing Functions of Patients With Parkinson's Disease.

Decreased swallowing function is a common and main cause of malnutrition and aspiration pneumonia in patients with Parkinson's disease. The aims of this systematic review were to summarize and qualitatively analyze the studies that have been published on behavioral therapies for improving swallowing functions in patients with Parkinson's disease. Studies published from January 2000 to December 2015 were identified via electronic database searches using Ovid-MEDLINE, Ovid-EMBASE, the Cochrane Library, and 8 Korean databases. Two reviewers independently evaluated the studies using inclusion criteria. Nine studies were included, of which 6 evaluated rehabilitation technique studies and 3 evaluated compensatory strategies. The 9 studies were evaluated qualitatively using a methodology checklist of the Scottish Intercollegiate Guideline Network, according to which all of the studies had acceptable quality. The available data on the effects of rehabilitation techniques and compensatory strategies remain insufficient. Further randomized controlled studies should be done to investigate the effect of behavioral therapy on improving swallowing functions in patients with Parkinson's disease.

[A Structural Model on the Nursing Competencies of Nursing Simulation Learners].

PURPOSE: The purpose of this study was to test a model of nursing competencies of nursing simulation learners. The conceptual model was based on the theory of Jeffries's simulaton theory. METHODS: Data collection was conducted in October 2017 for 310 students from two nursing universities in Kyungbuk area for 20 days. Data analysis methods were covariance structure analysis using SPSS 21.0 and AMOS 22.0 statistical programs. RESULTS: The hypothetical model was a good fit for the data. The model fit indices were comparative fit index=.97, normed fit index=.94, Tucker-Lewis Index=.97, root mean square error of approximation=.44, and standardized root mean square residual=.04. Teacher factors were directly related to simulation design characteristics, and it was confirmed that the curriculum, classroom operation and teaching method of the instructors were important factors. Learner factors were found to have a direct effect on nursing competence, self-confidence, and clinical performance that belong to nursing capacity. In particular, the results of this study indicate that the simulation design characteristics have a partial mediating effect on learner factors and clinical performance, and a complete mediating effect on learner factors and clinical judgment ability. CONCLUSION: In order to improve the learner's clinical performance and clinical judgment ability, it is necessary to conduct practical training through nursing simulation besides preparing the learner and the educator.

Clinical Nurses' Resilience Skills for Surviving in a Hospital Setting: A Q-methodology Study.

PURPOSE: Resilience relates to coping with stressful hospital environment. The purpose of this study was to identify the types of resilience skills of clinical nurses for surviving in a hospital setting. METHODS: The Q methodology was used as it helps analyze the participants' subjective perspective on each item. Participants were 32 registered nurses who sorted 38 selected Q statements that were then plotted on a normal distribution using a 9-point scale. The subjective perspectives on the resilience of clinical nurses were analyzed using the PC-QUANAL program. RESULTS: This study revealed four types of resilience in clinical nurses, accounting for 65.2% of the variance: Type I: Reality-harmonic type; Type II: Own will type; Type III: Professionalism-oriented type; and Type IV: Relation-oriented type. CONCLUSION: The present findings suggest the need to develop interventions for improving clinical nurses' resilience according to their types. Following further investigation of nurses' resilience, it may be necessary for organizations to develop several resilience strategies.

Lysosomal Targeting Enhancement by Conjugation of Glycopeptides Containing Mannose-6-phosphate Glycans Derived from Glyco-engineered Yeast.

Many therapeutic enzymes for lysosomal storage diseases require a high content of mannose-6-phosphate (M6P) glycan, which is important for cellular uptake and lysosomal targeting. We constructed glyco-engineered yeast harboring a high content of mannosylphosphorylated glycans, which can be converted to M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural M6P glycan source for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion steps. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting. The purified M6PgPs were successfully conjugated to a recombinant acid α-glucosidase (rGAA), used for the treatment of Pompe disease, by two-step reactions using two hetero-bifunctional crosslinkers. First, rGAA and M6PgPs were modified with crosslinkers containing azide and dibenzocyclooctyne, respectively. In the second reaction using copper-free click chemistry, the azide-functionalized rGAA was conjugated with dibenzocyclooctyne-functionalized M6PgPs without the loss of enzyme activity. The M6PgP-conjugated rGAA had a 16-fold higher content of M6P glycan than rGAA, which resulted in greatly increased cellular uptake and efficient digestion of glycogen accumulated in Pompe disease patient fibroblasts.

Direct Monitoring of Fucosylated Glycopeptides of Alpha-Fetoprotein in Human Serum for Early Hepatocellular Carcinoma by Liquid Chromatography-Tandem Mass Spectrometry with Immunoprecipitation.

PURPOSE: Alpha-fetoprotein (AFP) is a widely used serological marker that is associated with hepatocellular carcinoma (HCC). Although the level of AFP is increased in HCC, its sensitivity for diagnosis is poor because AFP levels are also increased in liver diseases. Changes in glycoform, especially fucosylation, have been reported to be associated with the development of HCC. EXPERIMENTAL DESIGN: The authors introduce the monitoring of fucosylated glycopeptides by liquid chromatography (LC)-mass spectrometry (MS) combined with immunoprecipitation, where glycan-cleaved fragments with an amino acid sequence are used as transitions. Furthermore, neuraminidase for desialylation is useful to improve the MS detection limit (limit of detection [LOD] <2 ng mL-1 ) in 0.1 µL of serum. RESULTS: The performance of the relative percentage of fucosylated AFP (AFP-fuc%) for differentiating between early HCC and cirrhosis is better than that of serum AFP levels as indicated by a greater area under the receiver operator characteristic curve (area under the curve = 0.962 vs. 0.628) and sensitivity (92.3% vs. 53.9%), respectively. Furthermore, the inter- and intraday repeatability of AFP-fuc% in serum is less than 2.1%. CONCLUSIONS AND CLINICAL RELEVANCE: These findings suggest that glycopeptide-based LC-MS/MS is a promising method and that AFP-fuc% is a clinically useful parameter for differentiating between early HCC and liver cirrhosis.

Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples.

Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell-cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without centrifugation of blood samples, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method, we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS samples, from immunoglobulin gamma 1 (IgG-1, 10 mg/mL) down to complement component C7 (50 µg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180 days stored at room temperature. The major abundant glycoproteins in the DBS samples were IgG-1 and IgG-2, which contain nine asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, alpha-1-antitypsin, alpha-2-macroglobulin, haptoglobin, hemopexin, Ig alpha 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS samples by LC-MS/MS with minimal sample preparation.

Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.

Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate.

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

Analysis of fucosylation in liver-secreted N-glycoproteins from human hepatocellular carcinoma plasma using liquid chromatography with tandem mass spectrometry.

Fucosylation of N-glycoproteins has been implicated in various diseases, such as hepatocellular carcinoma (HCC). However, few studies have performed site-specific analysis of fucosylation in liver-secreted proteins. In this study, we characterized the fucosylation patterns of liver-secreted proteins in HCC plasma using a workflow to identify site-specific N-glycoproteins, where characteristic B- and/or Y-ion series with and without fucose in collision-induced dissociation were used in tandem mass spectrometry. In total, 71 fucosylated N-glycopeptides from 13 major liver-secreted proteins in human plasma were globally identified by LC-MS/MS. Additionally, 37 fucosylated N-glycopeptides were newly identified from nine liver-secreted proteins, including alpha-1-antichymotrypsin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, ceruloplasmin, alpha-1-acid glycoprotein 1/2, alpha-2-macroglobulin, serotransferrin, and beta-2-glycoprotein 1. Of the fucosylated N-glycopeptides, bi- and tri-antennary glycoforms were the most common ones identified in liver-secreted proteins from HCC plasma. Therefore, we suggest that this analytical method is effective for characterizing fucosylation in liver-secreted proteins. Graphical abstract A global map of fucosylated and non-fucosylated glycopeptides from 13 liver-secreted glycoproteins in hepatocellular carcinoma plasma.

[Effects of a Hospital Based Follow-Up Program for Mothers with Very Low Birth Weight Infants].

PURPOSE: This paper reports the results of a hospital centered follow-up program on parenting stress, parenting efficacy and coping for mothers with very low birth weight (VLBW) infants. METHODS: The follow-up program consisted of home visiting by an expert group and self-help program for 1 year. A non-equivalent control group pre-post quasi-experimental design was used. Participants were 70 mothers with low birth weight infants and were assigned to one of two groups, an experimental groups (n=28), which received the family support program; and a control group (n=27), which received the usual discharge education. Data were analyzed using χ²-test, t-test, and ANCOVA with IBM SPSS statistics 20.0. RESULTS: Mothers' parenting stress (F=5.66, p=.004) was significantly decreased in the experimental group. There were also significant increases in parenting efficacy (F=13.05, p<.001) and coping (F=8.91, p=.002) in the experimental group. CONCLUSION: The study findings suggest that a follow-up program for mothers with VLBW infants is an effective intervention to decrease mothers' parenting stress and to enhance parenting efficacy and coping.

Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.

Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.

Chromosome-Based Proteomic Study for Identifying Novel Protein Variants from Human Hippocampal Tissue Using Customized neXtProt and GENCODE Databases.

The goal of the Chromosome-Centric Human Proteome Project (C-HPP) is to fully provide proteomic information from each human chromosome, including novel proteoforms, such as novel protein-coding variants expressed from noncoding genomic regions, alternative splicing variants (ASVs), and single amino acid variants (SAAVs). In the 144 LC/MS/MS raw files from human hippocampal tissues of control, epilepsy, and Alzheimer's disease, we identified the novel proteoforms with a workflow including integrated proteomic pipeline using three different search engines, MASCOT, SEQUEST, and MS-GF+. With a <1% false discovery rate (FDR) at the protein level, the 11 detected peptides mapped to four translated long noncoding RNA variants against the customized databases of GENCODE lncRNA, which also mapped to coding-proteins at different chromosomal sites. We also identified four novel ASVs against the customized databases of GENCODE transcript. The target peptides from the variants were validated by tandem MS fragmentation pattern from their corresponding synthetic peptides. Additionally, a total of 128 SAAVs paired with their wild-type peptides were identified with FDR <1% at the peptide level using a customized database from neXtProt including nonsynonymous single nucleotide polymorphism (nsSNP) information. Among these results, several novel variants related in neuro-degenerative disease were identified using the workflow that could be applicable to C-HPP studies. All raw files used in this study were deposited in ProteomeXchange (PXD000395).