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1.
Mol Cell Proteomics ; : 100176, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34774759

RESUMO

Urologic chronic pelvic pain syndrome (UCPPS) is a condition of unknown etiology characterized by pelvic pain, and urinary frequency and/or urgency. As the proximal fluid of this syndrome, urine is an ideal candidate sample matrix for an unbiased study of UCPPS. In this study, a large, discovery-phase, TMT-based quantitative urinary proteomics analysis of 244 subjects was performed. The subjects included patients with UCPPS (n=82), healthy controls (HC) (n=94) and disparate chronic pain diseases, termed positive controls (PC) (n=68). Utilizing training and testing cohorts, we identified and validated a small and distinct set of proteins that distinguished UCPPS from HC (n=9) and UCPPS from PC (n=3). Validated UCPPS: HC proteins were predominantly ECM/ECM modifying or immunomodulatory/host defense in nature. Significantly varying proteins in the UCPPS: HC comparison were overrepresented by members of several dysregulated biological processes including decreased immune cell migration, decreased development of epithelial tissue and increased bleeding. Comparison with the PC cohort enabled evaluation of UCPPS-specific upstream regulators, contrasting UCPPS with other conditions that cause chronic pain. Specific to UCPPS were alterations in the predicted signaling of several upstream regulators, including alpha-catenin, IL6, EGF, and TGFB1, among others. These findings advance our knowledge of the etiology of UCPPS and inform potential future clinical translation into a diagnostic panel for UCPPS.

2.
Neurology ; 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34675105

RESUMO

BACKGROUND AND OBJECTIVES: Sport-related concussions affect millions of individuals across the United States each year and current techniques to diagnose and monitor them rely largely on subjective measures. Our goal was to discover and validate objective, quantifiable non-invasive biomarkers with the potential to be used in sport-related concussion diagnosis. METHODS: Urine samples from a convenience series of healthy control collegiate athletes who had not sustained a concussion and athletes who sustained a concussion as diagnosed by a sports medicine physician within seven days were collected prospectively and studied. Participants also completed an instrumented single-task gait analysis as a functional measure. Participants were recruited from a single collegiate athletic program, were ≥18 years old, and were excluded if they had a concomitant injury, active psychiatric conditions or pre-existing neurological disorders. Using Tandem Mass Tags (TMT) mass spectroscopy and enzyme-linked immunosorbent assays (ELISA), urinary biomarkers of concussion were identified and validated. RESULTS: Forty-eight control and 47 concussion age- and sex-matched athletes were included in the study (51.6%F, 48.4%M, average age 19.6y). Participants represented both contact and non-contact sports. All but one of the post-concussion participants reported experiencing symptoms at the time of data collection. Insulin-like growth factor 1 (IGF-1) and IGF binding protein 5 (IGFBP5) were downregulated in the urine of athletes with concussions compared to healthy controls. Multivariable risk algorithms developed to predict the probability of sport-related concussion showed that IGF-1 multiplexed with single-task gait velocity predicts concussion risk across a range of post-injury timepoints (AUC=0.786; 95% CI:0.690-0.884). When IGF-1 and IGFBP5 are multiplexed with single-task gait velocity, they accurately distinguish between healthy controls and concussion at acute timepoints (AUC=0.835, 95% CI:0.701-0·968, p<0.001). DISCUSSION: These noninvasive biomarkers, discovered in an objective and validated manner, may be useful in diagnosing and monitoring sport-related concussions in both acute phases of injury in addition to several days post-injury. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that urinary IGF-1 and IGFBP5 multiplexed with single-task gait velocity may be useful in diagnosing sport-related concussion. TRIAL REGISTRATION INFORMATION: Clinicaltrials.gov identifier NCT02354469, submitted February 2015, first patient enrolled August 2015 (https://clinicaltrials.gov/ct2/show/NCT02354469).

3.
J Proteome Res ; 20(5): 2662-2672, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33650863

RESUMO

The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and N-glycans are critical for many biological functions of UMOD. Comprehensive glycan profiling of UMOD provides valuable information to understand the exact mechanisms of glycan-regulated functions. To perform comprehensive glycosylation analysis of UMOD from urine samples with limited volumes, we developed a streamlined workflow that included UMOD isolation from 5 mL of urine from 6 healthy adult donors (3 males and 3 females) and a glycosylation analysis using a highly sensitive and reproducible nanoLC-MS/MS based glycomics approach. In total, 212 N-glycan compositions were identified from the purified UMOD, and 17% were high-mannose glycans, 2% were afucosylated/asialylated, 3% were neutral fucosylated, 28% were sialylated (with no fucose), 46% were fucosylated and sialylated, and 4% were sulfated. We found that isolation of UMOD resulted in a significant decrease in the relative quantity of high-mannose and sulfated glycans with a significant increase of neutral fucosylated glycans in the UMOD-depleted urine relative to the undepleted urine, but depletion had little impact on the sialylated glycans. To our knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical workflow would be very beneficial for studies with limited sample size, such as pediatric studies, and can be applied to larger patient cohorts not only for UMOD interrogation but also for global glycan analysis.


Assuntos
Glicômica , Espectrometria de Massas em Tandem , Adulto , Criança , Feminino , Glicosilação , Humanos , Masculino , Polissacarídeos , Uromodulina
4.
Mol Cell Proteomics ; 19(11): 1767-1776, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32737218

RESUMO

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1-10 years, n = 21) and adult (21-50 years, n = 22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared with the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose and tetrasialylated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.


Assuntos
Glicômica/métodos , Polissacarídeos/análise , Polissacarídeos/urina , Espectrometria de Massas em Tandem/métodos , Adulto , Criança , Pré-Escolar , Cromatografia Líquida , Estudos de Coortes , Feminino , Fucose/urina , Glicosilação , Humanos , Lactente , Masculino , Manose/metabolismo , Pessoa de Meia-Idade
5.
Mol Cell Proteomics ; 19(3): 456-466, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896675

RESUMO

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1-3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) ≥2 in at least 7 patients in either VUR or control cohort. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly up-regulated and 36 downregulated (q <0.075, FC ≥1.2). Urinary proteins involved in inflammation, acute phase response (APR), modulation of extracellular matrix (ECM), and carbohydrate metabolism were altered among the study cohort.


Assuntos
Proteoma , Infecções Urinárias/urina , Refluxo Vesicoureteral/urina , Feminino , Humanos , Masculino , Peptídeos/urina , Projetos Piloto , Recidiva , Infecções Urinárias/metabolismo , Urina/química , Refluxo Vesicoureteral/metabolismo
6.
Mol Cell Proteomics ; 19(1): 11-30, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31591262

RESUMO

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.


Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos , Biofarmácia/métodos , Anticorpos Monoclonais/metabolismo , Glicômica/métodos , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Laboratórios , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos
7.
BJU Int ; 120(1): 130-142, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28263447

RESUMO

OBJECTIVE: To examine a series of candidate markers for urological chronic pelvic pain syndrome (UCPPS), selected based on their proposed involvement in underlying biological processes so as to provide new insights into pathophysiology and suggest targets for expanded clinical and mechanistic studies. METHODS: Baseline urine samples from Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network study participants with UCPPS (n = 259), positive controls (PCs; chronic pain without pelvic pain, n = 107) and healthy controls (HCs, n = 125) were analysed for the presence of proteins that are suggested in the literature to be associated with UCPPS. Matrix metalloproteinase (MMP)-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex (also known as Lipocalin 2), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF-R1) and NGAL were assayed and quantitated using mono-specific enzyme-linked immunosorbent assays for each protein. Log-transformed concentration (pg/mL or ng/mL) and concentration normalized to total protein (pg/µg) values were compared among the UCPPS, PC and HC groups within sex using the Student's t-test, with P values adjusted for multiple comparisons. Multivariable logistic regression and receiver-operating characteristic curves assessed the utility of the biomarkers in distinguishing participants with UCPPS and control participants. Associations of protein with symptom severity were assessed by linear regression. RESULTS: Significantly higher normalized concentrations (pg/µg) of VEGF, VEGF-R1 and MMP-9 in men and VEGF concentration (pg/mL) in women were associated with UCPPS vs HC. These proteins provided only marginal discrimination between UCPPS participants and HCs. In men with UCCPS, pain severity was significantly positively associated with concentrations of MMP-9 and MMP-9/NGAL complex, and urinary severity was significantly positively associated with MMP-9, MMP-9/NGAL complex and VEGF-R1. In women with UCPPS, pain and urinary symptom severity were associated with increased normalized concentrations of MMP-9/NGAL complex, while pain severity alone was associated with increased normalized concentrations of VEGF, and urinary severity alone was associated with increased normalized concentrations of MMP-2. Pain severity in women with UCPPS was significantly positively associated with concentrations of all biomarkers except NGAL, and urinary severity with all concentrations except VEGF-R1. CONCLUSION: Altered levels of MMP-9, MMP-9/NGAL complex and VEGF-R1 in men, and all biomarkers in women, were associated with clinical symptoms of UCPPS. None of the evaluated candidate markers usefully discriminated UCPPS patients from controls. Elevated VEGF, MMP-9 and VEGF-R1 levels in men and VEGF levels in women may provide potential new insights into the pathophysiology of UCPPS.


Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Dor Pélvica/fisiopatologia , Dor Pélvica/psicologia , Sistema Urinário/patologia , Doenças Urológicas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Biomarcadores/metabolismo , Pesquisa Biomédica , Dor Crônica , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Comunicação Interdisciplinar , Masculino , Projetos de Pesquisa , Síndrome , Estados Unidos , Doenças Urológicas/fisiopatologia
8.
Mol Cell Proteomics ; 15(8): 2607-15, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27215552

RESUMO

Prenatal hydronephrosis is a common condition that may spontaneously resolve after birth. However, this condition can result in renal damage and requires surgical correction in a number of cases. Preventing renal damage is paramount, but existing diagnostic technology is invasive, exposes infants to radiation, is costly, and is often indeterminate. A better understanding of the pathophysiology of renal obstruction as reflected in the urinary proteome may provide new insights into the disease that could potentially alter the clinical management of hydronephrosis. We performed a quantitative proteomics study of urine that was surgically obtained from eight clinically significant, unilaterally obstructed infants versus eight healthy controls, with the goal of identifying quantitatively varying proteins and the biological networks associated with them. Notably, urine was obtained from both the obstructed kidney and the bladder. Over 1100 proteins were identified, and a total of 76 quantitatively varying proteins were identified. Proteins involved in oxidative stress, inflammation, and renal disease pathways showed the most significant abundance differences. This study gives a deeper understanding of the critical proteomic changes associated with renal obstruction and represents the deepest proteomic profile of renal obstruction to date.


Assuntos
Biomarcadores/urina , Rim/metabolismo , Proteômica/métodos , Obstrução Ureteral/metabolismo , Bexiga Urinária/metabolismo , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Mapas de Interação de Proteínas
9.
J Proteome Res ; 13(10): 4377-87, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25160569

RESUMO

Synovial fluid in an articulating joint contains proteins derived from the blood plasma and proteins that are produced by cells within the joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid and the cellular origins of many synovial fluid components are not fully understood. Here, we present a normative proteomics study using porcine synovial fluid. Using our optimized method, we identified 267 proteins with high confidence in healthy synovial fluid. We also evaluated mRNA expression data from tissues that can contribute to the synovial fluid proteome, including synovium, cartilage, blood, and liver, to better estimate the relative contributions from these sources to specific synovial fluid components. We identified 113 proteins in healthy synovial fluid that appear to be primarily derived from plasma transudates, 37 proteins primarily derived from synovium, and 11 proteins primarily derived from cartilage. Finally, we compared the identified synovial fluid proteome to the proteome of human plasma, and we found that the two body fluids share many similarities, underlining the detected plasma derived nature of many synovial fluid components. Knowing the synovial fluid proteome of a healthy joint will help to identify mechanisms that cause joint disease and pathways involved in disease progression.


Assuntos
Articulação do Joelho/metabolismo , Proteoma , Líquido Sinovial/metabolismo , Animais , Cromatografia Líquida , Processamento de Proteína Pós-Traducional , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Suínos , Porco Miniatura , Espectrometria de Massas em Tandem
10.
Anal Chem ; 86(13): 6277-84, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24766348

RESUMO

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.


Assuntos
Fetuínas/química , Imunoglobulina G/química , Ácido N-Acetilneuramínico/análise , Polissacarídeos/análise , Polissacarídeos/urina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Sequência de Carboidratos , Bovinos , Humanos , Dados de Sequência Molecular , Polissacarídeos/química
11.
Biochim Biophys Acta ; 1844(5): 1044-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23707565

RESUMO

In this study, we performed an in-depth characterization of the male pediatric infant urinary proteome by parallel proteomic analysis of normal healthy adult (n=6) and infant (n=6) males and comparison to available published data. A total of 1584 protein groups were identified. Of these, 708 proteins were identified in samples from both cohorts. Although present in both cohorts, 136 of these common proteins were significantly enriched in urine from adults and 94 proteins were significantly enriched in urine from infants. Using Gene Ontology, we found that the infant-enriched or specific subproteome (743 proteins) had an overrepresentation of proteins that are involved in translation and transcription, cellular growth and metabolic processes. In contrast, the adult enriched or specific subproteome (364 proteins) showed an overexpression of proteins involved in immune response and cell adhesion. This study demonstrates that the non-diseased male urinary proteome is quantitatively affected by age, has age-specific subproteomes, and identifies a common subproteome with no age-dependent abundance variations. These findings highlight the importance of age-matching in urinary proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.


Assuntos
Biomarcadores/urina , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Urina/química , Adulto , Cromatografia Líquida , Estudos de Coortes , Humanos , Lactente , Masculino , Frações Subcelulares , Espectrometria de Massas em Tandem , Adulto Jovem
12.
Mol Cell Proteomics ; 12(10): 2981-91, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23820512

RESUMO

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.


Assuntos
Glicômica/métodos , Glicoproteínas/metabolismo , Proteômica/métodos , Animais , Bovinos , Cromatografia Líquida , Fetuínas/metabolismo , Glicosilação , Humanos , Imunoglobulina G/metabolismo , Polissacarídeos/metabolismo , Ribonucleases/metabolismo , Espectrometria de Massas em Tandem
13.
Mol Cell Proteomics ; 12(4): 1017-25, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23438733

RESUMO

Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses.


Assuntos
Glicoproteínas/química , Processamento de Proteína Pós-Traducional , Algoritmos , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Simulação por Computador , Glicoproteínas/metabolismo , Glicosilação , Humanos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteólise , Proteômica , Espectrometria de Massas em Tandem/métodos
14.
Mol Cell Proteomics ; 12(6): 1735-40, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23443135

RESUMO

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.


Assuntos
Cromatografia Líquida/métodos , Glicoproteínas/isolamento & purificação , Anotação de Sequência Molecular/métodos , Polissacarídeos/isolamento & purificação , Software , Espectrometria de Massas em Tandem/métodos , Glicoproteínas/urina , Humanos , Lactente , Anotação de Sequência Molecular/normas , Polissacarídeos/urina , Análise de Sequência de Proteína
15.
Anal Biochem ; 427(1): 33-5, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22516523

RESUMO

Common de-N-glycosylation protocols usually require a lengthy incubation time. Although pressure cycling technology or scientific microwave reactors can accelerate this enzyme reaction, they may not be easily accessible. In this brief report, we employed an alternative strategy using a standard domestic microwave oven to perform the de-N-glycosylation. Model glycoproteins (bovine RNase B, bovine fetuin, and human IgG) and a complex mixture from human plasma were fully deglycosylated in 20 min, without any apparent adverse affects on the glycans or protein backbones. This new method provides a simple and inexpensive solution to achieve rapid de-N-glycosylation.


Assuntos
Glicosilação/efeitos da radiação , Micro-Ondas , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Animais , Catálise , Bovinos , Fetuínas/análise , Glicoproteínas/química , Humanos , Imunoglobulina G/análise , Polissacarídeos/química , Ribonuclease Pancreático/análise
16.
Mol Cell Proteomics ; 11(6): M111.015248, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22261723

RESUMO

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.


Assuntos
Interações Hospedeiro-Patógeno , Lactação , Lactoferrina/metabolismo , Leite Humano/metabolismo , Processamento de Proteína Pós-Traducional , Aderência Bacteriana , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Escherichia coli O157/fisiologia , Feminino , Análise de Fourier , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicosilação , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Polissacarídeos/química , Salmonella/fisiologia , Análise de Sequência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Anal Chem ; 83(14): 5541-7, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21661761

RESUMO

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.


Assuntos
Glicopeptídeos/análise , Glicoproteínas/química , Espectrometria de Massas em Tandem/métodos , Animais , Galinhas , Cromatografia Líquida/métodos , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Polissacarídeos/análise , Proteólise , Espectrometria de Massas em Tandem/economia
18.
Anal Biochem ; 408(1): 136-46, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20804719

RESUMO

Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, ß-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, ß-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lactação , Proteínas do Leite/química , Leite Humano/química , Fosfopeptídeos/análise , Espectrometria de Massas em Tandem/métodos , Automação , Feminino , Humanos , Lactalbumina/química , Fosforilação , Titânio/química
19.
J Agric Food Chem ; 58(21): 11234-42, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-20925428

RESUMO

Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed "lactosomes" that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼ 25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.


Assuntos
Glicolipídeos/química , Glicoproteínas/química , Leite Humano/química , Adulto , Feminino , Humanos , Gotículas Lipídicas , Lipídeos/química , Tamanho da Partícula , Proteínas/química
20.
J Agric Food Chem ; 58(10): 6440-8, 2010 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-20415418

RESUMO

While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.


Assuntos
Glicoproteínas/análise , Lactação/metabolismo , Proteínas do Leite/análise , Leite Humano/química , Eletroforese em Gel de Poliacrilamida , Feminino , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lactoferrina/análise , Lactoferrina/metabolismo , Proteínas do Leite/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo
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