RESUMEN
Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as ß- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in ß- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens ßB2-crystallin, the main component of the ß-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in ßB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of ßB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of ßB2-crystallin and thus likely to have an impact on crystallin subunit-subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.
Asunto(s)
Ácido Aspártico/química , Cristalino/química , Cadena B de beta-Cristalina/química , Anciano , Envejecimiento , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Humanos , Estereoisomerismo , Espectrometría de Masas en TándemRESUMEN
In prostate cancer diagnosis, PSA test has greatly contributed to the early detection of prostate cancer; however, expanding overdiagnosis and unnecessary biopsies have emerged as serious issues. To explore plasma biomarkers complementing the specificity of PSA test, we developed a unique proteomic technology QUEST-MS (Quick Enrichment of Small Targets for Mass Spectrometry). The QUEST-MS method based on 96-well formatted sequential reversed-phase chromatography allowing efficient enrichment of <20 kDa proteins quickly and reproducibly. Plasma from 24 healthy controls, 19 benign prostate hypertrophy patients, and 73 prostate cancer patients were purified with QUEST-MS and analyzed by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed prostate cancer specific detection pattern, which included a neurotransmitter polypeptide neuropeptide-Y (NPY). We further validated the screening results by targeted multiple reaction monitoring technology using independent sample set (n = 110). The ROC curve analysis revealed that logistic regression-based combination of NPY, and PSA showed 81.5% sensitivity and 82.2% specificity for prostate cancer diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput profiling of plasma polypeptides and had potential to effectively uncover very low abundant tumor-derived small molecules, such as neurotransmitters, peptide hormones, or cytokines.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neuropéptido Y/sangre , Neoplasias de la Próstata/sangre , Anciano , Secuencia de Aminoácidos , Biomarcadores de Tumor/química , Estudios de Casos y Controles , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Peso Molecular , Clasificación del Tumor , Neoplasias de la Próstata/patología , Proteoma/química , Proteoma/metabolismo , Espectrometría de Masas en TándemRESUMEN
Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.
Asunto(s)
COVID-19 , Fibrosis Pulmonar Idiopática , Enfermedad Pulmonar Obstructiva Crónica , Animales , Ratones , SARS-CoV-2 , Enfermedad Pulmonar Obstructiva Crónica/genética , Pulmón , Fibrosis Pulmonar Idiopática/genéticaRESUMEN
Numerous studies have suggested that the different histological subtypes of ovarian carcinoma (i.e. clear cell, endometrioid, mucinous, and serous) have distinct clinical histories and characteristics; however, most studies that have aimed to determine biomarker have not performed comprehensive analyses based on subtype specificity. In the present study, we performed two-dimensional gel electrophoresis-based differential proteomic analysis of the different histological subtypes of ovarian carcinoma using tissue specimens from 39 patients. Seventy-seven protein spots (55 unique proteins) were found to be up- or downregulated in a subtype-specific manner. The most significant difference was observed for: (i) annexin-A4 (ANXA4) and phosphoserine aminotransferase (PSAT1), which are expressed strongly in clear cell carcinoma; (ii) cellular retinoic acid-binding protein 2 (CRABP2), which is expressed specifically in serous carcinoma; and (iii) serpin B5 (SPB5), which is upregulated in mucinous carcinoma. Validation of these candidates by western blotting using a 34 additional test sample set resulted in an expression pattern that was consistent with the screening and revealed that differential expression was independent of cancer stage or tumor grade within each subtype. Thus, the present study reinforces the notion that ovarian cancer subtypes can be clearly delineated on a molecular basis into four histopathological groups, and we propose that ANXA4, PSAT1, CRABP2, and SPB5 are candidate subtype-specific biomarkers that can help define the basis of tumor histology at a molecular level.
Asunto(s)
Anexina A4/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Ováricas/diagnóstico , Receptores de Ácido Retinoico/metabolismo , Serpinas/metabolismo , Transaminasas/metabolismo , Adenocarcinoma Mucinoso , Electroforesis en Gel Bidimensional , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , PronósticoRESUMEN
Site-specific characterization of glycoform heterogeneity currently requires glycan structure assignment and glycopeptide quantification in two independent experiments. We present here a new method combining multiple reaction monitoring mass spectrometry with energy-resolved structural analysis, which we termed "energy-resolved oxonium ion monitoring". We demonstrated that monitoring the yields of oligosaccharide-derived fragment ions (oxonium ions) over a wide range of collision induced dissociation (CID) energy applied to a glycopeptide precursor exhibits a glycan structure-unique fragmentation pattern. In the analysis of purified immunoglobulin glycopeptides, the energy-resolved oxonium ion profile was shown to clearly distinguish between isomeric glycopeptides. Moreover, limit of detection (LOD) of glycopeptide detection was 30 attomole injection, and quantitative dynamic range spanned 4 orders magnitude. Therefore, both quantification of glycopeptides and assignment of their glycan structures were achieved by a simple analysis procedure. We assessed the utility of this method for characterizing site-specific N-glycan microheterogeneity on therapeutic antibodies, including validation of lot-to-lot glycoform variability. A significant change in the degree of terminal galactosylation was observed in different production lots of trastuzumab and bevacizumab. Cetuximab Fab glycosylation, previously known to cause anaphylaxis, was also analyzed, and several causative antigens including Lewis X motifs were quantitatively detected. The data suggests that energy-resolved oxonium ion monitoring could fulfill the regulatory requirement on the routine quality control analysis of forthcoming biosimilar therapeutics.
Asunto(s)
Anticuerpos/química , Espectrometría de Masas/métodos , Oligosacáridos/química , Polisacáridos/análisis , Polisacáridos/química , Anticuerpos/uso terapéutico , Sitios de Unión , Glicopéptidos/química , Glicosilación , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/uso terapéutico , Termodinámica , Tripsina/metabolismoRESUMEN
BACKGROUND: Serum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. However, serum is featured by high level of protein glycosylations that often cause ionization suppression and confound accurate quantification analysis by mass spectrometry. Here we investigated the effect of N-glycan and sialic acid removal from serum proteins on the performance of label-free quantification results. RESULTS: Serum tryptic digests with or without deglycosylation treatment were analyzed by LC-MALDI MS and quantitatively compared on the Expressionist Refiner MS module. As a result, 345 out of 2,984 peaks (11.6%) showed the specific detection or the significantly improved intensities in deglycosylated serum samples (P < 0.01). We then applied this deglycosylation-based sample preparation to the identification of lung cancer biomarkers. In comparison between 10 healthy controls and 20 lung cancer patients, 40 peptides were identified to be differentially presented (P < 0.01). Their quantitative accuracies were further verified by multiple reaction monitoring. The result showed that deglycosylation was needed for the identification of some unique candidates, including previously unreported O-linked glycopeptide of complement component C9. CONCLUSIONS: We demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.
RESUMEN
One-step detection of biological molecules is one of the principal techniques for clinical diagnosis, and the potential of mass spectrometry for biomarker detection has been a promising new approach in the field of medical sciences. We demonstrate here a new and high-sensitivity method that we termed immunobeads-mass spectrometry (iMS), which combines conventional immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The key feature of iMS is the MS-compatible condition of immunoprecipitation using detergents with a monosaccaride-C8 alkyl chain or a disaccharide-C10 alkyl chain, and the minimized number of steps required for high-sensitivity detection of target peptides in serum or biological fluid. This was achieved by optimizing the wash buffer and subjecting the immunobeads directly to MALDI-TOF MS analysis. Using this method, we showed that 1 fmol of amyloid beta peptide spiked in serum was readily detectable, demonstrating the powerful tool of iMS as a biomarker detection method.
Asunto(s)
Antígenos/sangre , Inmunoprecipitación/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Péptidos beta-Amiloides/sangre , Biomarcadores/sangre , Detergentes/química , Humanos , Límite de Detección , MicroesferasRESUMEN
Bile acids have recently emerged as versatile signaling molecules, and their signaling pathway is a promising target for the treatment of metabolic diseases. Here, we developed a highly sensitive and high-throughput quantification method for six taurine- and glycine-conjugated bile acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after solid-phase extraction (SPE-MALDI-TOF MS). In a carbon tetrachloride (CCl4)-induced liver injury/regeneration model in mice, serum bile acid profiles were monitored, and the same samples were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and protein spots that significantly changed in quantity in a serial time points were identified by MALDI-TOF MS. Serum taurocholic acid (TCA) concentration was significantly elevated earlier than the increase of serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT) activity, a potentially sensitive marker for minimal hepatic damage. Furthermore, TCA peaked at 20 h after treatment when massive serum proteins appeared in circulation. It should be noted that direct MALDI-imaging mass spectrometry (IMS) has succeeded in showing a hepatic lobular distribution change of TCA, predominantly seen in zone 1 area whereas necrotic changes were dominant in zone 3 area. The in-depth analysis of bile acid profiles in circulation with hepatic lobular distribution is a strong basis to understand the serum proteome in CCl4-induced liver injury model.
Asunto(s)
Ácidos y Sales Biliares/sangre , Proteínas Sanguíneas/análisis , Intoxicación por Tetracloruro de Carbono/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Regeneración Hepática/fisiología , Proteómica/métodos , Alanina Transaminasa/metabolismo , Análisis de Varianza , Animales , Aspartato Aminotransferasas/metabolismo , Biomarcadores/sangre , Electroforesis en Gel Bidimensional , Glicina/metabolismo , Histocitoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Taurina/metabolismo , Ácido Taurocólico/sangreRESUMEN
The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Estudios de Casos y Controles , Cromatografía Liquida , Humanos , Curva ROC , Espectrometría de Masas en TándemRESUMEN
Significant advances have been made in the past decade in the field of mass spectrometry imaging (MS imaging). It is a relatively unestablished technique aimed at direct, high-sensitive and spatially exclusive detection of biological molecules from the surface of tissue sections, so that semi-quantitative distribution map of the analyte can be reconstituted from the mass spectra obtained. There is tremendous potential in its application especially in clinical field, such as biomarker discovery or pharmacokinetic study. However, vast majority of the work has been performed on frozen tissue sections, while it remains generally unpractical to produce frozen sections with clinically resected tumor samples. Here we report our novel sample preparation technique that enabled MS imaging from formalin-fixed paraffin embedded (FFPE) tissue section, including retrospective archive as old as 11 years. FFPE sections were first dewaxed with pre-warmed xylene, and exposed tissue surface was enzymatically digested in nanoliter scale droplets to retain analyte localization. As a result, we succeeded in obtaining MS images of peptide peaks derived from several proteins, identified by MS/MS analysis, using ovarian cancer FFPE sections. The qualities of mass spectra obtained by this method were not significantly different from those obtained from frozen sections. By this, we opened the door to retrospective study of past clinical cases in aim to discover molecular biomarker.