RESUMO
Insects are a sustainable protein source for food and feed. The yellow mealworm (Tenebrio molitor L.) is a promising candidate for industrial insect rearing and was the focus of this study. This research revealed the diversity of Tenebrio molitor larvae in the varying larval instars in terms of the nutritional content. We hypothesized that water and protein are highest in the earlier instar, while fat content is very low but increases with larval development. Consequently, an earlier instar would be a good choice for harvest, since proteins and amino acids content decrease with larval development. Near-infrared reflectance spectroscopy (NIRS) was represented in this research as a tool for predicting the amino and fatty acid composition of mealworm larvae. Samples were scanned with a near-infrared spectrometer using wavelengths from 1100 to 2100 nm. The calibration for the prediction was developed with modified partial least squares (PLS) as the regression method. The coefficient for determining calibration (R2C) and prediction (R2P) were >0.82 and >0.86, with RPD values of >2.20 for 10 amino acids, resulting in a high prediction accuracy. The PLS models for glutamic acid, leucine, lysine and valine have to be improved. The prediction of six fatty acids was also possible with the coefficient of the determination of calibration (R2C) and prediction (R2P) > 0.77 and >0.66 with RPD values > 1.73. Only the prediction accuracy of palmitic acid was very weak, which was probably due to the narrow variation range. NIRS could help insect producers to analyze the nutritional composition of Tenebrio molitor larvae fast and easily in order to improve the larval feeding and composition for industrial mass rearing.
RESUMO
Background: Fish and fish products are one of the most important food sources of high commercial interest. The global food trade and the associated risks are constantly presenting new challenges to consumer protection and public authorities, which, among other things, demand state-of-the-art analytical methods to ensure food authenticity. Objective: The establishment of MS-based strategies plays a decisive role alongside the (further) development of ELISA- or DNA-oriented methods. Methods: In the present work, therefore, the development and in-house validation of an LC-MS and LC-MS/MS-based assay for authenticity testing of certain fish species is described. Results: Based on the execution of a validated bottom-up LC-electrospray-MS and MS/MS assay and multivariate analysis, the commercially available species Lutjanus malabaricus (red snapper) and Sebastes spp. (redfish) are distinguished from each other, whereas an additional 68 samples [nine additional marine species such as pangasius (Pangasianodon hypophthalmus), salmon (Salmo salar), turbot (Scophthalmus maximus), plaice (Pleuronectes platessa), sole (Solea solea), lemon sole (Glyptocephalus cynoglossus), halibut (Reinhardtius hypoglossoides), red salmon (Oncorhynchus nerka), and great scallop (Pecten jacobaeus)] served as blinded negative controls to ensure the specificity of the assay. Conclusions and Highlights: A promising LC-MS and LC-MSMS based assay has been developed that could enable the detection of fish fraud at the protein level in the future.
Assuntos
Cromatografia Líquida/métodos , Proteínas de Peixes/análise , Peixes/classificação , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Moluscos/classificação , Redes Neurais de Computação , Análise de Componente PrincipalRESUMO
The detection of food fraud and undeclared food allergens is one of the major challenges for competent authorities. Because adulterations are continuously adapted to the methods used to uncover them, the accomplishment of this task has become increasingly difficult over time. In recent years, various new promising methods for the detection of multiple food adulterants and multiple food allergens have been developed. Some of them utilize LC-MS to identify specific marker peptides. However, these methods have yet to be validated and standardized. For this reason, the German officials have established a working group with the objective of validating methods through multilaboratory validation studies. The experts of the working group also aim for the first time to standardize validated methods and to develop general validation criteria. This manuscript will highlight the current work of the group. For this purpose, an overview is given on the principles and applications of the new mass spectrometric methods. Moreover, requirements and the present work of other institutions regarding method validation are described.
Assuntos
Alérgenos/análise , Contaminação de Alimentos/análise , Espectrometria de Massas/métodos , Proteínas/análise , Animais , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Alemanha , Órgãos Governamentais , Humanos , Pessoal de Laboratório , Espectrometria de Massas/normas , Carne/análise , Plantas/química , Frutos do Mar/análise , Estudos de Validação como AssuntoRESUMO
PURPOSE: An autologous tumor vaccine already used successfully in the immune therapy of renal cell carcinoma was investigated in detail. The evaluation of potential tumor markers should allow for the assessment of potency according to pharmaceutical regulations. METHODS: A panel of 36 tumor-associated antigens and cellular marker proteins was characterized in a total of 133 tumor cell lysates by methods such as ELISA, Western blots, and topological proteomics. The induction of tumor-associated antigen-specific antibodies was demonstrated by immunization in mice. RESULTS: Tumor heterogeneity was demonstrated: none of the tumor-associated antigens investigated were detectable in each tumor lysate. In parallel, the coincidental presence of potential danger signals was shown for HSP-60 and HSP-70. The presence of both antigen and danger signal allowed a successful induction of an immune response in a murine model. CONCLUSION: The verified tumor heterogeneity indicates the need for a multi-epitope approach for the successful immunotherapy in renal cell carcinoma.
RESUMO
BACKGROUND: Today, dementias are diagnosed late in the course of disease. Future treatments have to start earlier in the disease process to avoid disability requiring new diagnostic tools. The objective of this study is to develop a new method for the differential diagnosis and identification of new biomarkers of Alzheimer's disease (AD) using capillary-electrophoresis coupled to mass-spectrometry (CE-MS) and to assess the potential of early diagnosis of AD. METHODS AND FINDINGS: Cerebrospinal fluid (CSF) of 159 out-patients of a memory-clinic at a University Hospital suffering from neurodegenerative disorders and 17 cognitively-healthy controls was used to create differential peptide pattern for dementias and prospective blinded-comparison of sensitivity and specificity for AD diagnosis against the Criterion standard in a naturalistic prospective sample of patients. Sensitivity and specificity of the new method compared to standard diagnostic procedures and identification of new putative biomarkers for AD was the main outcome measure. CE-MS was used to reliably detect 1104 low-molecular-weight peptides in CSF. Training-sets of patients with clinically secured sporadic Alzheimer's disease, frontotemporal dementia, and cognitively healthy controls allowed establishing discriminative biomarker pattern for diagnosis of AD. This pattern was already detectable in patients with mild cognitive impairment (MCI). The AD-pattern was tested in a prospective sample of patients (nâ=â100) and AD was diagnosed with a sensitivity of 87% and a specificity of 83%. Using CSF measurements of beta-amyloid1-42, total-tau, and phospho(181)-tau, AD-diagnosis had a sensitivity of 88% and a specificity of 67% in the same sample. Sequence analysis of the discriminating biomarkers identified fragments of synaptic proteins like proSAAS, apolipoprotein J, neurosecretory protein VGF, phospholemman, and chromogranin A. CONCLUSIONS: The method may allow early differential diagnosis of various dementias using specific peptide fingerprints and identification of incipient AD in patients suffering from MCI. Identified biomarkers facilitate face validity for the use in AD diagnosis.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Proteínas do Tecido Nervoso/análise , Mapeamento de Peptídeos , Sinapses/química , Adulto , Idoso , Inteligência Artificial , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Diagnóstico Diferencial , Eletroforese Capilar , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica/métodos , Sensibilidade e Especificidade , Adulto JovemRESUMO
About 30% of renal cell carcinomas (RCC) will develop recurrence after surgery. Despite evidence for a significantly improved survival by autologous tumour cell vaccination therapy, the procedure has not become standard. Between August 1993 and December 1996, 1,267 RCC patients undergoing radical nephrectomy in 84 German hospitals were subsequently treated by autologous tumour cell vaccination therapy. The study group comprised 692 patients with complete follow-up (stages pT2-3, pNx-2, M0 based on the TNM classification, 4th edition). Subsequent propensity-score matching according to 7 defined criteria with 861 control patients undergoing nephrectomy alone without adjuvant treatment at the Carl-Thiem-Hospital Cottbus, resulted in 495 matched pairs. Overall and stage-specific survival rates were analysed after a median follow-up of 131 months. The 5- and 10-year overall survival (OS) rates were 80.6 and 68.9% in the vaccine group and 79.2 and 62.1% in the control group (p = 0.066). Patients with pT3 stage RCC revealed 5- and 10-year OS rates of 71.3 and 53.6% in the study group and 65.4 and 36.2% in the control group (p = 0.022). In multivariable analysis, patients in the vaccine group showed a significantly improved survival both in the whole study group (HR = 1.28, p = 0.030) and in the subgroup presenting with pT3 stage tumours (HR = 1.67, p = 0.011). Adjuvant treatment with autologous vaccination therapy resulted in a significantly improved overall survival in pT3 stage RCC patients, suggesting benefit especially in this subgroup. However, controlled clinical trials integrating the recent TNM classification and further risk constellations are required to define additional patient groups that may derive benefit from this treatment.
Assuntos
Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/terapia , Neoplasias Renais/mortalidade , Neoplasias Renais/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/uso terapêutico , Carcinoma de Células Renais/patologia , Ensaios Clínicos como Assunto , Terapia Combinada , Ensaios de Uso Compassivo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Nefrectomia , Adulto JovemRESUMO
An important issue in congenital unilateral ureteropelvic junction (UPJ) obstruction, a frequent pathology in newborns, is whether infants should undergo surgery. Non-invasive biomarkers to reduce or replace the current invasive clinical exploration are not available. The objective of this study was to identify urinary markers of UPJ obstruction. We compared a number of proteome technologies to study the urinary proteome in UPJ obstruction and selected online capillary electrophoresis coupled to mass-spectrometry for the selection of non-invasive prognostic biomarkers. We selected 53 urinary biomarkers that were able to distinguish between different levels of UPJ obstruction. In a prospective study using these 53 biomarkers, we predicted with 97% accuracy, and several months in advance, the clinical outcome of 36 UPJ-obstruction patients. Some of the discriminating biomarkers were identified. A newly identified marker, proSAAS (proprotein convertase subtilisin/kexin type 1 inhibitor), generated a new hypothesis in the physiopathology of UPJ obstruction. These results show that analysis of urinary polypeptides in newborns with UPJ obstruction can predict their clinical outcome.
Assuntos
Biomarcadores/urina , Proteômica/métodos , Obstrução Ureteral/diagnóstico , Obstrução Ureteral/urina , Humanos , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , Doenças do Recém-Nascido/urinaRESUMO
Lithium remains the treatment of choice for many patients suffering from bipolar disorder. However, long-term treatment with lithium carries the potential to cause renal and thyroid dysfunction. Lithium-induced nephropathies are characterised by deterioration of urinary concentrating ability as well as, less frequently, a progressive and potentially irreversible decrease in glomerular filtration rate (GFR). Pathological changes after treatment with lithium include both tubulointerstitial and glomerular changes. Besides monitoring of the kidney-function, no screening-instruments exist for early identification of patients at risk of lithium-induced nephropathy. CE-MS (capillary electrophoresis coupled to a mass spectrometer) is a new technique that has been applied to the differential diagnosis of nephropathies. We sought to determine if CE-MS can be used to identify lithium-induced renal changes. A urine-sample was obtained from 14 subjects (7 males, 7 females, mean age 51.1 years) under long-term treatment with lithium (mean duration 17.4 years, range 8-35 years) without known nephropathy (mean creatinine 0.96 mg/dl; range 0.7-1.6). Urine samples were stored at -20 degrees C until analysis. CE-MS was performed according to standard procedures and a screen for nephropathies was used. Among the 14 urine samples, two subjects tested positive for a nephropathy. One further subject had a borderline result. Since 3/14 subjects with no known nephropathy showed some degree of pathological findings, CE-MS from a urine-sample may be helpful for the early detection of renal damage under treatment with lithium. However, a specific screen for lithium-induced nephropathies still needs to be developed.
Assuntos
Eletroforese Capilar/métodos , Lítio , Espectrometria de Massas/métodos , Nefrite/induzido quimicamente , Adolescente , Adulto , Criança , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Masculino , Nefrite/classificação , Nefrite/urina , Fatores de TempoRESUMO
We evaluated the utility of SDS-PAGE/Western blot and CE coupled with MS (CE-MS) for detection of urinary polypeptide biomarkers of renal disease in patients with IgA-associated glomerulonephritides. In a reference cohort of 402 patients with various renal disorders and 207 healthy controls, we defined CE-MS patterns of renal damage and IgA nephropathy (IgAN). In a blinded analysis of a separate cohort of patients with IgAN (n = 10), Henoch-Schoenlein purpura (HSP) with nephritis (n = 10), and IgA-associated glomerulonephritis due to hepatitis C virus (HCV)-induced cirrhosis (n = 9), and healthy controls (n = 12), we compared SDS-PAGE/Western blot and CE-MS against clinical urinalysis for detection of urinary proteins/polypeptides. Urinalysis indicated proteinuria for 50, 90, and 33% of patients, respectively, and for none of the healthy controls. SDS-PAGE/Western blot showed urinary polypeptides abnormality for 90, 80, and 67% of patients, respectively, and for none of the healthy controls. CE-MS indicated a Renal Damage Pattern in 80, 80, and 100 of patients, respectively, and in 17% of healthy controls, with the more specific IgAN Pattern in 90, 90, and 1%, respectively, and in none of the healthy controls. Based on differences in CE-MS patterns, the disease mechanisms may differ among various IgA-associated glomerulonephritides. These exploratory findings should be evaluated in a prospective study with contemporaneous renal biopsy and urinary testing. If validated, it may be feasible to adapt the CE-MS methodology to develop novel tests to detect renal injury at earlier stages, assess clinical manifestations, and monitor responses to therapy in patients with IgA-associated renal diseases.
Assuntos
Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Glomerulonefrite por IGA/urina , Imunoglobulina A/urina , Peptídeos/urina , Proteinúria/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/urina , Biópsia/métodos , Criança , Pré-Escolar , Creatinina/sangue , Fibrose/imunologia , Fibrose/urina , Glomerulonefrite por IGA/imunologia , Hepatite C/imunologia , Hepatite C/urina , Humanos , Vasculite por IgA/imunologia , Vasculite por IgA/urina , Imunoglobulina A/isolamento & purificação , Espectrometria de Massas , Pessoa de Meia-Idade , Peptídeos/isolamento & purificação , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
IgA nephropathy is the most common primary glomerulonephritis and is a frequent cause for chronic kidney disease in children and young adults. Glomerular deposition of IgA also characterizes other renal disorders, including Henoch-Schoenlein purpura nephritis and immune-complex glomerulonephritis afflicting patients with liver disease due to chronic infection with the hepatitis C virus. Several treatment options are often considered, with the goal to prevent end-stage renal failure. Unfortunately, the diagnosis currently requires an invasive procedure, a renal biopsy. Because of the inherent risks, repetitive renal biopsy is frequently foregone as a means to monitor the clinical course or response to treatment. Recent advances in the analysis of the urinary proteome suggest that the excreted polypeptides include disease-specific patterns. We review recent studies of the various techniques for the identification and validation of such urinary biomarkers of IgA-associated glomerulonephritides. Currently, capillary electrophoresis coupled with mass spectrometry (MS) offers the greatest promise. To date, it seems more likely that disease-specific urinary polypeptide biomarkers are comprised of a panel of several distinct and well-defined peptides rather than a single molecule. Even most patients in clinical remission with normal clinical testing (dipstick urinalysis and quantitative proteinuria) were correctly classified by the pattern of polypeptides identified by capillary electrophoresis coupled with MS. With confirmation and refinement, such urinary testing may provide a tool for the diagnosis and monitoring of patients with IgA-associated renal diseases that is more sensitive than current standard clinical testing and far less risky than renal biopsy.
Assuntos
Glomerulonefrite por IGA/urina , Vasculite por IgA , Proteômica/métodos , Biomarcadores/urina , Regulação para Baixo , Eletroforese Capilar/métodos , Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite por IGA/fisiopatologia , Humanos , Vasculite por IgA/complicações , Vasculite por IgA/fisiopatologia , Espectrometria de Massas/métodos , Regulação para CimaRESUMO
CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.
Assuntos
Biomarcadores/análise , Eletroforese Capilar/métodos , Análise de Sequência de Proteína/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Sequência de Aminoácidos , Biomarcadores/sangue , Biomarcadores/urina , Líquidos Corporais/química , Humanos , Dados de Sequência Molecular , Sistemas On-Line , Peptídeos/análise , Proteômica/métodosRESUMO
We analyzed urinary polypeptides from individuals with neonatal ureteropelvic junction (UPJ) obstruction to predict which individuals with this condition will evolve toward obstruction that needs surgical correction. We identified polypeptides that enabled diagnosis of the severity of obstruction and validated these biomarkers in urine collected in a prospective blinded study. Using these noninvasive biomarkers, we were able to predict, several months in advance and with 94% precision, the clinical evolution of neonates with UPJ obstruction.
Assuntos
Biomarcadores/urina , Pelve Renal/patologia , Proteoma/análise , Obstrução Ureteral/congênito , Obstrução Ureteral/diagnóstico , Estudos de Casos e Controles , Método Duplo-Cego , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Espectrometria de Massas , Prognóstico , Estudos Prospectivos , Padrões de Referência , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Manejo de Espécimes , Fatores de Tempo , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Obstrução Ureteral/fisiopatologiaRESUMO
BACKGROUND: Non-invasive methods for diagnosis of urothelial carcinoma have reduced specificity in patients with non-malignant genitourinary disease or other disorders. We aimed to use mass spectrometry and bioinformatics to define and validate a cancer-specific proteomic pattern. METHODS: We used capillary-electrophoresis-coupled mass spectrometry to obtain polypeptide patterns from urine samples of 46 patients with urothelial carcinoma and 33 healthy volunteers. From signatures of polypeptide mass, we established a model for predicting the presence of cancer. The model was refined further by use of 366 urine samples obtained from other healthy volunteers and patients with malignant and non-malignant genitourinary disease. We estimated the proportion of correct classifications from the refined model by applying it to a masked group containing 31 patients with urothelial carcinoma, 11 healthy individuals, and 138 patients with non-malignant genitourinary disease. We also sequenced several diagnostic polypeptides for urothelial carcinoma. FINDINGS: We identified a diagnostic urothelial-carcinoma pattern of 22 polypeptide masses. On masked assessment, prediction models based on these polypeptides correctly classified all samples of urothelial carcinoma (sensitivity 100% [95% CI 87-100) and all healthy samples (specificity 100% [84-100]). Correct identification of patients with urothelial carcinoma from those with other malignant and non-malignant genitourinary disease ranged from 86% to 100%. A prominent polypeptide from the diagnostic pattern for urothelial carcinoma was identified as fibrinopeptide A-a known biomarker of ovarian cancer and gastric cancer. INTERPRETATION: Validation of a highly specific biomarker pattern for urothelial carcinoma in a large group of patients with various urological disorders could be used in the diagnosis of other diseases that are identified in urine samples or in other body fluids.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma/diagnóstico , Carcinoma/patologia , Proteômica , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/patologia , Carcinoma/genética , Estudos de Casos e Controles , Biologia Computacional , Diagnóstico Diferencial , Eletroforese Capilar , Feminino , Humanos , Masculino , Espectrometria de Massas , Sensibilidade e Especificidade , Neoplasias Urológicas/genética , UrotélioRESUMO
Here we describe a mass spectrometry (MS) approach for biomarker discovery and structural characterization, based on both top-down and bottom-up analyses. Capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS serves to separate and mass-measure the thousands of polypeptides contained in human urine. Statistical analysis of the differences between healthy control samples and patients with focal-segmental glomerulosclerosis, membranous glomerulonephritis, minimal change disease, IgA nephropathy, and diabetic nephropathy validates multiple biomarkers for the control and each of the diseases. To identify those biomarkers, we employ preparative CE, enabling direct infusion ESI MS analysis, followed by sample manipulation and reanalysis where necessary. We show how tandem Fourier transform ion cyclotron resonance (FT-ICR) MS identifies these sometimes large (>8 kDa) biomarkers. Critically, we maintain connectivity between the CE TOF MS data and the ICR data used for biomarker identification.
Assuntos
Nefropatias/urina , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Biomarcadores/urina , Eletroforese Capilar , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Peso Molecular , Sistemas On-Line , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
This study investigates proteomic analysis of urinary samples as a non-invasive method to detect acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to analyze urinary samples in 19 patients with different grades of subclinical or clinical acute rejection (BANFF Ia to IIb), 10 patients with urinary tract infection and 29 patients without evidence of rejection or infection. A distinct urinary polypeptide pattern identified 16 out of 17 cases of acute tubolointerstitial rejection, but was absent in two cases of vascular rejection. Urinary tract infection resulted in a different polypeptide pattern that allowed to differentiate between infection and acute rejection in all cases. Potentially confounding variables such as acute tubular lesions, tubular atrophy, tubulointerstitial fibrosis, calcineurin inhibitor toxicity, proteinuria, hematuria, allograft function and different immunosuppressive regimens did not interfere with test results. Blinded analysis of samples with and without rejection showed correct diagnosis by CE-MS in the majority of cases. Detection of acute rejection by CE-MS offers a promising non-invasive tool for the surveillance of renal allograft recipients. Further investigation is needed to establish polypeptide patterns in vascular rejection and to explore whether changes in the urinary proteome occur before the onset of histologically discernible rejection.
Assuntos
Química Clínica/métodos , Rejeição de Enxerto/diagnóstico , Transplante de Rim/métodos , Túbulos Renais/patologia , Proteinúria/diagnóstico , Proteinúria/patologia , Adulto , Idoso , Biópsia , Eletroforese Capilar , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Peptídeos/urina , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Fatores de Tempo , Infecções Urinárias/diagnóstico , Urina/química , Sistema Urogenital/patologiaRESUMO
The introduction of fast, sensitive, and robust techniques for proteomic analysis into clinical practice represents a major step toward a new diagnostic approach of body fluids. In addition, proteomics emerges as a key technology for the discovery of disease biomarkers in various body fluids. However, even in relatively protein-deprived body fluids such as urine, the complexity and wide dynamic range of protein expression pose a considerable challenge to both separation and identification technologies. In the present review we discuss from a clinical point-of-view recent advances of the use of proteomics in clinical diagnosis as well as therapy evaluation. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS) and discuss CE-MS from an application point of view evaluating its merits and vices with regard to biomarker discovery. This review further presents examples of clinical applications of CE-MS for detection and identification of biomarkers in urine.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Proteoma/análise , Urinálise/métodos , Biomarcadores/urina , Humanos , Peptídeos/análise , Análise de Sequência de Proteína , SoftwareRESUMO
We describe the use of capillary electrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47 patients who underwent prostate biopsy. Of this group, 21 patients had benign pathology and 26 with CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47 urine samples were compared to that of 41 young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474 samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Peptídeos/urina , Neoplasias da Próstata/diagnóstico , Idoso , Sequência de Aminoácidos , Biomarcadores/urina , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Projetos Piloto , Neoplasias da Próstata/patologia , Análise de Sequência de ProteínaRESUMO
BACKGROUND: IgA nephropathy (IgAN) is the most common chronic glomerular disease in adults. End-stage renal disease (ESRD) develops in about 30% of the patients. Early intervention and consequent therapy may prevent or at least delay the development of ESRD in these patients. Up to now, the diagnosis could only be achieved with a renal biopsy. METHODS: The urine of 45 patients with IgAN was collected and screened for protein/polypeptide patterns with a novel high throughput method, capillary electrophoreses on-line coupled to a mass spectrometer (CE-MS). CE-MS allows the fast and accurate evaluation of up to 2000 polypeptides in one urine sample. The results in IgAN were compared to findings in 13 patients with membranous nephropathy (MN) and 57 healthy individuals. RESULTS: In the patients with IgAN, even when urinary protein excretion was within the normal range of regular tests, the polypeptide pattern in urine differed significantly from that of healthy controls and patients with MN, indicating a specific "IgAN" pattern of polypeptide excretion. Classification regarding discrimination of IgAN from healthy controls and from MN had a sensitivity of 100% and 77%, respectively. Specificity was 90% and 100%, respectively. Compared to patterns established earlier in patients with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), or diabetic nephropathy (DN), sensitivity and specificity were 100%. Treatment of the patients was associated with changes of the pattern, possibly indicating a therapeutic effect. CONCLUSION: Proteomic analysis with CE-MS coupling permits fast and accurate identification and differentiation of polypeptide patterns in the urine of patients with IgAN, allowing differentiation from healthy controls and, probably, other renal diseases.
Assuntos
Glomerulonefrite por IGA/diagnóstico , Proteinúria/urina , Adulto , Idoso , Eletroforese Capilar , Feminino , Glomerulonefrite por IGA/urina , Glomerulonefrite Membranosa/diagnóstico , Glomerulonefrite Membranosa/urina , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Sensibilidade e EspecificidadeRESUMO
We report on our results using capillary electrophoresis coupled to mass spectrometry (CE-MS) to examine human bodyfluids. To demonstrate the versatility of this approach, data on two different bodyfluids, urine and cerebrospinal fluid, are shown. CE-MS analysis of human urine enables the identification of a series of polypeptides which serve as biomarkers for a variety of different renal diseases. The polypeptides are utilized to generate disease-specific polypeptide patterns. Diagnosis of these diseases is possible based on these polypeptide patters. Further, due to the high mass accuracy, polypeptides of interest can subsequently be identified using tandem MS (MS/MS) analysis. The patterns, which are based on distinct polypeptides, allow differentiation of even similar diseases like focal-segmental glomerulosclerosis (FSGS) and minimal change disease (MCD). We present preliminary data suggesting that the indicative polypeptides also enable to evaluate therapy success. Initial data obtained on human cerebrospinal fluid strongly suggest that CE-MS analysis of low-molecular-weight proteins and peptides reveals several potential biomarkers for schizophrenia as well as Alzheimer's disease. In conclusion, the data presented here indicate that CE-MS analysis, applied towards different human bodyfluids, holds the promise to allow diagnosis, staging, and evaluation of therapy success of a large number of diseases, due to its ability to display ca. 1000 individual native polypeptides within ca. 60 min.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Biomarcadores/urina , Eletroforese Capilar/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Humanos , Esquizofrenia/diagnóstico , Esquizofrenia/terapiaRESUMO
We describe the application of capillary electrophoresis (CE) coupled on-line to an electrospray ionization-time of flight-mass spectrometer (ESI-TOF-MS) to the analysis of human urine and serum for the identification of biomarkers for clinical diagnostics. CE-MS led to display > 1000 polypeptides present in complex biological samples within 45-60 min in a single analysis run. To extract the information of the CE-MS spectra in a timely fashion, a software was designed to automatically deconvolute and normalize the spectra. Both urine and serum contain several hundred polypeptides in samples from healthy individuals. Hence, it is possible to establish typical "normal urine" or "normal serum" polypeptide patterns. Samples from patients with different diseases display polypeptide patterns that differ significantly from those obtained from healthy individuals. Examining series of patients with the same disease allowed the establishment of polypeptide patterns typical for specific diseases. This permits the search for marker peptides specific for diseases. The data indicate that a single polypeptide present in all patients with the same disease, but absent in all healthy control individuals does not exist. The combination of several polypeptides found in either urine or serum or both are forming a specific pattern, which is indicative not only for the particular disease, but also for the stage of disease. CE-MS detects many polypeptides in single samples and the application of the software to the search of identical polypeptides excreted in urine allows the unbiased diagnosis based on a pattern and does not rely on single disease markers.