RESUMEN
Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Top-down mass spectrometry (MS) has the potential to overcome challenges of other common approaches by minimizing sample preparation and preserving endogenous modifications. However, comprehensive mAb characterization requires generation of many, well-resolved fragments and remains challenging. While ETD retains modifications and cleaves disulfide bonds-making it attractive for mAb characterization-it can be less effective for precursors having high m/z values. Activated ion electron transfer dissociation (AI-ETD) uses concurrent infrared photoactivation to promote product ion generation and has proven effective in increasing sequence coverage of intact proteins. Here, we present the first application of AI-ETD to mAb sequencing. For the standard NIST mAb, we observe a high degree of complementarity between fragments generated using standard ETD with a short reaction time and AI-ETD with a long reaction time. Most importantly, AI-ETD reveals disulfide-bound regions that have been intractable, thus far, for sequencing with top-down MS. We conclude AI-ETD has the potential to rapidly and comprehensively analyze intact mAbs.
Asunto(s)
Anticuerpos Monoclonales/química , Técnicas Electroquímicas/métodos , Secuencia de Aminoácidos , Transporte de Electrón , Iones/químicaRESUMEN
Photoactivation and photodissociation have long proven to be useful tools in tandem mass spectrometry, but implementation often involves cumbersome and potentially dangerous configurations. Here, we redress this problem by using a fiber-optic cable to couple an infrared (IR) laser to a mass spectrometer for robust, efficient, and safe photoactivation experiments. Transmitting 10.6 µm IR photons through a hollow-core fiber, we show that such fiber-assisted activated ion-electron transfer dissociation (AI-ETD) and IR multiphoton dissociation (IRMPD) experiments can be carried out as effectively as traditional mirror-based implementations. We report on the transmission efficiency of the hollow-core fiber for conducting photoactivation experiments and perform various intact protein and peptide analyses to illustrate the benefits of fiber-assisted AI-ETD, namely, a simplified system for irradiating the two-dimensional linear ion trap volume concurrent with ETD reactions to limit uninformative nondissociative events and thereby amplify sequence coverage. We also describe a calibration scheme for the routine analysis of IR laser alignment and power through the fiber and into the dual cell quadrupolar linear ion trap. In all, these advances allow for a more robust, straightforward, and safe instrumentation platform, permitting implementation of AI-ETD and IRMPD on commercial mass spectrometers and broadening the accessibility of these techniques.
Asunto(s)
Mioglobina/análisis , Fibras Ópticas , Péptidos/análisis , Ubiquitina/análisis , Animales , Calibración , Bovinos , Caballos , Rayos Láser , Espectrometría de Masas , Procesos FotoquímicosRESUMEN
Freshwater- and seawater-acclimated Fundulus heteroclitus were exposed to acute hypoxia (10% air saturation, 3 h), followed by normoxic recovery (3 h). In both salinities, ventilation increased and heart rate fell in the classic manner, while MO2 initially declined by â¼50%, with partial restoration by 3 h of hypoxia, and no O2 debt repayment during recovery. Gill paracellular permeability (measured with [14C] PEG-4000) was 1.4-fold higher in seawater, and declined by 50% during hypoxia with post-exposure overshoot to 188%. A similar pattern with smaller changes occurred in freshwater. Drinking rate (also measured with [14C] PEG-4000) was 8-fold higher in seawater fish, but declined by â¼90% during hypoxia in both groups, with post-exposure overshoots to â¼270%. Gill diffusive water flux (measured with 3H2O) was 1.9-fold higher in freshwater fish, and exhibited a â¼35% decrease during hypoxia, which persisted throughout recovery, but was unchanged during hypoxia in seawater fish. Nevertheless, freshwater killifish gained mass while seawater fish lost mass during hypoxia, and these changes were not corrected during normoxic recovery. We conclude that this hypoxia-tolerant teleost beneficially reduces gill water permeability in a salinity-dependent fashion during hypoxia, despite attempting to simultaneously improve MO2 , but nevertheless incurs a net water balance penalty in both freshwater and seawater.
Asunto(s)
Anaerobiosis , Fundulidae/fisiología , Branquias/fisiología , Osmorregulación/fisiología , Aclimatación , Animales , Femenino , Agua Dulce , Masculino , Oxígeno/metabolismo , Salinidad , Agua de Mar , Agua/fisiologíaRESUMEN
The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (Opsanus beta). Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to the apical membrane, which is negated by PKA inhibition. Moreover, RGN decreased HCO3- secretion, likely by limiting apical HCO3-/Cl- exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO3- secretion.
Asunto(s)
Batrachoidiformes/fisiología , Bicarbonatos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas de Peces/genética , Péptidos Natriuréticos/genética , Animales , Batrachoidiformes/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Peces/metabolismo , Péptidos Natriuréticos/metabolismoRESUMEN
Marine bony fish poses the unique ability to hydrate from imbibed seawater. They accomplish this, in part, by the precipitation of inorganic carbonate mineral in their intestine, which lowers luminal osmotic pressure and allows for water uptake. It has recently been described that in the Gulf toadfish (Opsanus beta) this Ca(Mg)CO3 precipitation occurs under the regulation of an organic matrix. To date no investigations have aimed to determine if this phenomenon applies more generally to marine fish. Here, intestinally derived precipitates were collected from gray snapper (Lutjanus griseus), white grunt (Haemulon plumieri), European flounder (Platichthys flesus), as well as Gulf toadfish, and their matrices were extracted. The ability of these matrices to regulate CaCO3 production was determined using an in vitro calcification assay, which revealed that the matrix derived from each of the tested species increased precipitation at low concentrations, while inhibiting it at higher concentrations in full agreement with the earlier studies on toadfish. Matrix extracted from European flounder precipitates was then analyzed by mass spectrometry, leading to the identification of over 50 unique proteins. When the identities of these proteins were compared to previous investigation of toadfish precipitate matrix, nearly 35% were found to overlap between the flounder and toadfish analyses, suggesting conserved mechanisms of precipitation control. The effects of using different sodium hypochlorite (NaOCl) solutions during precipitate purification on the resulting organic matrix are also discussed.
Asunto(s)
Carbonato de Calcio/metabolismo , Precipitación Química , Peces/metabolismo , Mucosa Intestinal/metabolismo , Animales , Agua Corporal/metabolismo , Calcificación Fisiológica , Proteínas de Peces/metabolismo , Peces/clasificación , Peces/fisiología , Espectrometría de Masas , Presión Osmótica , Agua de Mar , Hipoclorito de Sodio/química , Especificidad de la EspecieRESUMEN
The regulatory mechanisms behind the production of CaCO3 in the marine teleost intestine are poorly studied despite being essential for osmoregulation and responsible for a conservatively estimated 3-15% of annual oceanic CaCO3 production. It has recently been reported that the intestinally derived precipitates produced by fish as a byproduct of their osmoregulatory strategy form in conjunction with a proteinaceous matrix containing nearly 150 unique proteins. The individual functions of these proteins have not been the subject of investigation until now. Here, organic matrix was extracted from precipitates produced by Gulf toadfish (Opsanus beta) and the matrix proteins were fractionated by their charge using strong anion exchange chromatography. The precipitation regulatory abilities of the individual fractions were then analyzed using a recently developed in vitro calcification assay, and the protein constituents of each fraction were determined by mass spectrometry. The different fractions were found to have differing effects on both the rate of carbonate mineral production, as well as the morphology of the crystals that form. Using data collected from the calcification assay as well as the mass spectrometry experiments, individual calcification promotional indices were calculated for each protein, giving the first insight into the functions each of these matrix proteins may play in regulating precipitation.
Asunto(s)
Batrachoidiformes/metabolismo , Calcificación Fisiológica , Carbonato de Calcio/metabolismo , Proteínas de Peces/metabolismo , Animales , Carbonato de Calcio/química , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Intestinos/química , Osmorregulación , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
Obesity is associated with disrupted reproductive cycles in mares, but the impact of obesity on follicles and oocytes has received minimal attention. We investigated the impact of obesity on 1) expression of selected genes in follicle cells for carbohydrate metabolism, inflammatory cytokines, lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function; 2) follicular fluid content of metabolic hormones and metabolites; and 3) lipid fingerprint of oocytes. Mares (9-13 yr) were classified as control (n = 8, normal weight, body condition score [BCS] 5.1, 10.4% body fat) or obese (n = 9, BCS 7.9, 16.2% body fat). Gene expression from granulosa cells (GC) and cumulus cells (CC) was evaluated by RT-PCR. Serum and follicular fluid were evaluated for insulin, leptin, adiponectin, and metabolite profiling. Oocyte lipid fingerprints were acquired using matrix-assisted laser desorption/ionization mass spectrometry. Several genes for lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function were different between groups in GC and CC. Obese had (P < 0.05) or tended to have (0.05 < P < 0.1) lower insulin sensitivity and higher insulin and leptin in serum and follicular fluid. Many metabolites differed between control and obese in serum and/or follicular fluid and correlated with BCS and/or insulin sensitivity. Oocytes from control had greater concentrations of lipids consistent with phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins, while lipids consistent with triglycerides tended to be higher in obese. These findings suggest that maternal obesity causes alterations in the follicle and oocyte; the extent to which these alterations impact the conceptus and offspring is still to be determined.
Asunto(s)
Metabolismo de los Lípidos , Obesidad/metabolismo , Obesidad/patología , Oocitos/metabolismo , Oocitos/patología , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , Animales , Composición Corporal , Peso Corporal , Células del Cúmulo/metabolismo , Estrés del Retículo Endoplásmico , Femenino , Fase Folicular , Células de la Granulosa/metabolismo , Caballos , Insulina/sangre , Resistencia a la Insulina , Leptina/sangre , MetabolómicaRESUMEN
Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.
Asunto(s)
Acidosis/metabolismo , Citosol/metabolismo , Túbulos Renales Proximales/metabolismo , Proteoma/metabolismo , Animales , Hipertrofia/metabolismo , Corteza Renal/metabolismo , Masculino , Redes y Vías Metabólicas , Nefrectomía , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Increases in ambient salinity demand parallel increases in intestinal base secretion for maintenance of osmoregulatory status, which is likely the cause of a transient acidosis following transfer of euryhaline fish from freshwater to seawater. It was predicted that transfer of the marine Gulf toadfish (Opsanus beta) from seawater (35 ppt) to hypersaline (60 ppt) seawater (HSW) would lead to a transient acidosis that would be compensated by increases in branchial acid excretion to offset the acid-base disturbance. Toadfish exposed to HSW showed a significant decrease in blood pH and [HCO3-] but no increase in pCO2, followed by a full recovery after 48-96 h. A similar metabolic acidosis and recovery was found when fish were exposed to 60-ppt HCO3--free seawater (HEPES-buffered), which may suggest that compensation for intestinal base loss during hypersaline treatment is from gill H+ excretion rather than gill HCO3- uptake. However, we cannot rule out that reduced branchial HCO3- excretion contributed to an increase in net acid excretion. Since colchicine prevents full compensation, translocation of H+ and/or HCO3- transporters between cytosolic compartments and plasma membrane fractions might be involved in compensating for the hypersalinity-induced acidosis. Translocation of transporters rather than de novo synthesis may represent a faster and less energetically demanding response to rapidly fluctuating and high salinities encountered by toadfish in their natural environment.
Asunto(s)
Acidosis , Batrachoidiformes , Acidosis/veterinaria , Animales , Transporte Biológico , Branquias , Salinidad , Agua de Mar , AguaRESUMEN
Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance, to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavorable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs-the gills and intestine. Using immunofluorescence, we localized aquaporin-3 (AQP3) protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial AQP3 messenger-RNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter AQP3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish AQP3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater and facilitates intestinal permeability in seawater and freshwater.
Asunto(s)
Acuaporina 3/metabolismo , Fundulidae/fisiología , Oxígeno/metabolismo , Agua/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Complementario , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Agua Dulce , Regulación de la Expresión Génica/fisiología , Hipoxia , Permeabilidad , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Salinidad , Agua de MarRESUMEN
Members of the marine bivalve family Limidae are known for their bright appearance. In this study, their colourful tissues were examined as a defence mechanism towards predators. We showed that when attacked by the peacock mantis shrimp (Odontodactylus scyllarus), the 'disco' clam, Ctenoides ales, opened wide to expose brightly coloured tissues to the predator. The predator also significantly preferred to consume the internal, non-colourful clam tissues than the external, colourful tissues. Mass spectrometry-based metabolomic analysis confirmed that colourful tissues had significantly different chemical compositions than the non-colourful ones. The internal, non-colourful tissues had metabolite profiles more similar to an outgroup bivalve than to the species' own colourful external tissues. A number of the compounds that differentiated the colourful tissues from the non-colourful tissues appeared to be peptide-like, which potentially serve as the underlying defensive compounds. This is the first study demonstrating that colourful bivalve tissues are used for chemical defence.
RESUMEN
Marine bony fish live in a hyperosmotic environment and maintain osmotic homeostasis by drinking seawater, and absorbing salt and water across their gastrointestinal tract. Although the ion and water transport mechanisms in the intestine have been the subject of much study, numerous questions remain unanswered. To address some of these questions, a shotgun proteomics methodology employing isobaric tandem mass tags (TMT) was used to interrogate the anterior intestine, posterior intestine, and intestinal fluid proteomes of Gulf toadfish (Opsanus beta) acclimated to normal (35â¯ppt) or hypersaline (60â¯ppt) seawater. Relative protein abundance between tissues was also investigated using label free quantitation. Protein products from nearly 3000 unique toadfish loci were identified and quantified between the tissues, and pathway analysis was performed to gain insight into biological significance. Numerous proteins potentially involved in ion transport, digestion, nutrient absorption, and intestinal CaCO3 precipitation were found to respond to changing salinity, providing additional insight into the molecular mechanisms behind these processes. Intestinal protein heterogeneity was also observed with proteins involved in ion transport responding to hypersalinity exposure primarily in the anterior intestine, and proteins involved in digestion and nutrient absorption showing higher abundance in the anterior intestine, regardless of salinity.
Asunto(s)
Batrachoidiformes/fisiología , Carbonato de Calcio/metabolismo , Proteínas de Peces/metabolismo , Mucosa Intestinal/metabolismo , Osmorregulación , Aclimatación , Animales , Calcificación Fisiológica , Transporte Iónico , Proteoma/metabolismo , Salinidad , Equilibrio HidroelectrolíticoRESUMEN
Due to ineffective wastewater treatment technologies, pharmaceuticals such as the selective serotonin reuptake inhibitors (SSRIs)-a common class of antidepressants which inhibit the serotonin transporter (SERT)-can be found in surface waters and marine receiving waters near wastewater effluents. Understanding how exposure to these chemicals might impact non-target organisms, especially combined with other environmental stressors like hypoxia, is essential in order to thoroughly evaluate environmental risk. It was hypothesized that both acute and chronic exposure to the SSRI fluoxetine (FLX) would interfere with the metabolic hypoxia response of the Gulf toadfish, Opsanus beta. Here we demonstrate that acute intraperitoneal treatment with 50⯵gâ¯g-1 FLX significantly reduces the regulation index, or degree of metabolic regulation, in toadfish. Acute FLX exposure significantly reduced SERT mRNA expression in the first and third gill arches, but mRNA expression was not affected in heart tissues or in the second gill arch. In contrast, the regulation index was unaffected by 14-17â¯day waterborne FLX exposure to environmentally relevant (0.01⯵gâ¯L-1) and approximately 1000-fold higher (8.5⯵gâ¯L-1) concentrations. However, the higher concentration was sufficient to induce a systemic elevation in plasma serotonin concentrations. Chronic FLX exposure did not alter SERT mRNA expression in heart or gill tissues. The results of this study implicate the involvement of 5-HT pathways in hypoxia tolerance but demonstrate that current environmental levels of FLX are insufficient to impair the metabolic hypoxia response in marine fish.
Asunto(s)
Batrachoidiformes/metabolismo , Fluoxetina/toxicidad , Hipoxia , Contaminantes Químicos del Agua/toxicidad , Animales , Fluoxetina/análisis , Branquias/efectos de los fármacos , Branquias/metabolismo , Corazón/efectos de los fármacos , Espectrometría de Masas , Miocardio/metabolismo , Serotonina/sangre , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/toxicidad , Contaminantes Químicos del Agua/análisisRESUMEN
This corrects the article DOI: 10.1038/srep34494.
RESUMEN
Marine teleost fish produce CaCO3 in their intestine as part of their osmoregulatory strategy. This precipitation is critical for rehydration and survival of the largest vertebrate group on earth, yet the molecular mechanisms that regulate this reaction are unknown. Here, we isolate and characterize an organic matrix associated with the intestinal precipitates produced by Gulf toadfish (Opsanus beta). Toadfish precipitates were purified using two different methods, and the associated organic matrix was extracted. Greater than 150 proteins were identified in the isolated matrix by mass spectrometry and subsequent database searching using an O. beta transcriptomic sequence library produced here. Many of the identified proteins were enriched in the matrix compared to the intestinal fluid, and three showed no substantial homology to any previously characterized protein in the NCBI database. To test the functionality of the isolated matrix, a micro-modified in vitro calcification assay was designed, which revealed that low concentrations of isolated matrix substantially promoted CaCO3 production, where high concentrations showed an inhibitory effect. High concentrations of matrix also decreased the incorporation of magnesium into the forming mineral, potentially providing an explanation for the variability in magnesium content observed in precipitates produced by different fish species.
Asunto(s)
Batrachoidiformes/metabolismo , Carbonato de Calcio/metabolismo , Proteínas de Peces/metabolismo , Animales , Batrachoidiformes/genética , Proteínas de Peces/genética , Absorción Intestinal , Mucosa Intestinal/metabolismo , Magnesio/metabolismo , Espectrometría de MasasRESUMEN
The superior sensitivity of current mass spectrometers makes them prone to contamination issues, which can have deleterious effects on sample analysis. Here, bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate (marketed under the name Tinuvin 770) is identified as a major contaminant in applications using liquid chromatography coupled with mass spectrometry (LC-MS). Tinuvin 770 is often added to laboratory and medical plastics as a UV stabilizer. One particular lot of microcentrifuge tubes was found to have an excess of this compound that would leach into samples and drastically interfere with LC-MS data acquisition. Further analysis found that Tinuvin 770 readily leached into polar and nonpolar solvents from the contaminated tube lot. Efforts to remove Tinuvin 770 from contaminated samples were unsuccessful. A prescreening method using MALDI-TOF MS is presented to prevent system contamination and sample loss.
Asunto(s)
Ácidos Decanoicos/aislamiento & purificación , Contaminación de Equipos , Piperidinas/aislamiento & purificación , Plásticos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Cromatografía Liquida , Ácidos Decanoicos/química , Piperidinas/químicaRESUMEN
A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC-MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone, dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of microfluidics allowed for reduction of the chromatographic flow rate to 3µl/min with overall method run times comparable to standard flow LC-MS/MS methods reported in the literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a simple sample preparation protocol was employed requiring injection of only 0.5µl of sample, corresponding to a 100-400 fold increase in on-column sensitivity as compared to published standard flow assays. The measured LOQ for both testosterone and progesterone was 0.4ng/mL, representing an improvement over reported literature values obtained by standard flow methods employing comparable sample preparation and large injection volumes. The LOQs for cortisol (1.9ng/mL), cortisone (0.3ng/mL), and dihydrotestosterone (1.4ng/mL) were all within a biologically relevant range. A comparison of clinical serum samples was performed for the analysis of testosterone using this microfluidic LC-MS/MS assay and the Beckman Access II automated antibody-based measurement system. The immunoassay results were systematically higher due to matrix interference which was easily resolved with the increased chromatographic resolution obtained in the microflow LC-MS/MS assay.