RESUMO
Analysis of serum protein glycovariants has the potential to identify new biomarkers of human disease. However, the inability to rapidly quantify glycans in a site-specific fashion remains the major barrier to applying such biomarkers clinically. Advancements in sample preparation and glycopeptide quantification are thus needed to better bridge glycoscience with biomarker discovery research. We present here the successful utilization of several sample preparation techniques, including multienzyme digestion and glycopeptide enrichment, to increase the repertoire of glycopeptides that can be generated from serum glycoproteins. These techniques combined with glycopeptide retention time prediction and UHPLC-QqQ conditions optimization were then used to develop a dynamic multiple-reaction monitoring (dMRM)-based strategy to simultaneously monitor over 100 glycosylation sites across 50 serum glycoproteins. In total, the abundances of over 600 glycopeptides were simultaneously monitored, some of which were identified by utilizing theoretically predicted ion products and presumed m/ z values. The dMRM method was found to have good sensitivity. In the targeted dMRM mode, the limit of quantitation (LOQ) of nine standard glycoproteins reached femtomole levels with dynamic ranges spanning 3-4 orders of magnitude. The dMRM-based strategy also showed high reproducibility with regards to both instrument and sample preparation performance. The high coverage of the serum glycoproteins that can be quantitated to the glycopeptide level makes this method especially suitable for the biomarker discovery from large sample sets. We predict that, in the near future, biomarkers, such as these, will be deployed clinically, especially in the fields of cancer and autoimmunity.
Assuntos
Doenças Autoimunes/sangue , Glicoproteínas/sangue , Neoplasias/sangue , Proteômica , Doenças Autoimunes/diagnóstico , Biomarcadores/sangue , Glicosilação , Humanos , Neoplasias/diagnósticoRESUMO
A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS-compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF-MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real-time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N-linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF-MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.
Assuntos
Anticorpos Monoclonais/análise , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Espectrometria de Massas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Anticorpos Monoclonais/química , Medicamentos Biossimilares/análise , Medicamentos Biossimilares/química , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Proteínas Recombinantes/análise , Proteínas Recombinantes/químicaRESUMO
Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, tyrosylprotein sulfotransferase-1 and -2. These enzymes catalyze the transfer of sulfate from the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residues to form tyrosine O-sulfate ester and PAP. More than 60 proteins have been identified to be tyrosine sulfated including several G protein-coupled receptors, such as CC-chemokine receptor 8 (CCR8) that is implicated in allergic inflammation, asthma, and atherogenesis. However, the kinetic properties of purified tyrosylprotein sulfotransferase (TPST)-1 and -2 have not been previously reported. Moreover, currently there is no available quantitative TPST assay that can directly monitor individual sulfation of a series of tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both isozymes using CCR8 peptides as substrates that have three tyrosine residues in series. Our method can differentiate between mono- and disulfated products, and our results show that the K(m,app) for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs and in determining its catalytic mechanism.
Assuntos
Proteínas de Membrana/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sulfotransferases/química , Catálise , Cromatografia Líquida/métodos , Humanos , Cinética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoadenosina Fosfossulfato/química , Fosfoadenosina Fosfossulfato/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
We measured lead and mercury in samples collected from 31 homes in communities near the former Clark Air Base, Philippines during May and October 2002. Sample media included water used for drinking and cooking, house dust and entryway soil. Composite samples of 15 food items purchased at local markets were also collected. Samples were analyzed for total lead (Pb) and total mercury (Hg) to evaluate the relative importance of each media to residential exposure concentrations in the community adjacent to Clark (Community A) versus a control community 5 km away (Community B). In general, we measured low (e.g. background) to undetectable levels of the target analytes in all media sampled with two important exceptions. First, the Hg concentrations we measured in canned mackerel composites, which were within the range reported for mackerel from other locations worldwide, may pose a risk to pregnant women who are frequent consumers (e.g. one or more cans per day). Second, we measured Pb above the USEPA residential screening concentration (400 mug/g) in dust and soil from two homes, illustrating the need for periodic residential lead monitoring in these and other communities in the Philippines. We found no significant difference between Communities A and B with respect to Pb and Hg concentrations in water or food, although we were not able to detect very low levels of Pb in most of the foods we sampled because of trace Pb contamination added during sample homogenization. Although the Pb levels we measured in dust and soil from Community A homes were higher on average than Community B homes, the levels in both communities were low (e.g. background) thus we did not investigate the difference further. To our knowledge, these are the first reported measurements of Pb in house dust in the Philippines. The concentrations of Pb we measured in house dust were significantly higher than those in entryway soil from both communities, adding empirical support to the assertion that yard soil should not be considered a proxy for house dust in exposure studies in the Philippines or elsewhere.
Assuntos
Poluentes Ambientais/análise , Contaminação de Alimentos , Chumbo/análise , Mercúrio/análise , Adolescente , Adulto , Poeira/análise , Monitoramento Ambiental , Feminino , Habitação , Humanos , Pessoa de Meia-Idade , Filipinas , Saúde da População Rural , Solo/análise , Saúde da População Urbana , Água/análiseRESUMO
Human tyrosylprotein sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network, affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this study, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites, and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident sulfotransferase.