μL Of Human Serum Research Articles

Proteomic profiling of breast cancer serum via two-dimensional liquid-chromatography—tandem mass spectrometry

542 Background: Discovering biomarkers for early cancer detection is a major goal of translational research. We have applied modern proteomic technology, two-dimensional liquid-chromatography-tandem mass spectrometry (2-D LC-MS/MS), to large-scale breast cancer serum profiling. Methods: 189 human sera from breast disease or cancer patients (14 atypical hyperplasia, 63 benign, 29 DCIS, and 83 invasive CA) were analyzed via 2-D LC-MS/MS method. Patient ages ranged from 19 to 97 years, with a mean of 55. Ethnicities included African American, Asian, Hispanic, Native American, and Caucasian. This methodology utilizes only 10 μL of human serum per specimen. Every sample was run three times to ensure reproducibility and to maximize identification. The protein identification was conducted using the Bioworks Browser 3.1 and the IPI_human protein database (version 6.23.04) and filtered via HUPO criteria to ensure confidence. Results: 230 high-confidence proteins were identified from analyses of 189 sera. 26 common serum proteins were detected in all samples and 17 were found in two- or three- diagnostic stages. 83 proteins such as insulin receptor substrate 4, serine/threonine-protein kinase WNK4, DNA mismatch repair protein MSH6, and signal recognition particle 72 kDa protein were detected only in invasive sera. 30 proteins including breast carcinoma amplified sequence 3, uncharacterized bone marrow protein BM032, fibroblast growth factor receptor 2, and tumor necrosis factor receptor superfamily member 4 were detected only in DCIS sera. The 15 proteins detected only in atypical hyperplasia sera included cholecystokinin type A receptor and cell division cycle 2-related protein kinase 7. 59 proteins including serine/threonine protein phosphatase 6, probable transcription factor CST, insulin-like growth factor IA, and connective tissue growth factor were found in benign samples. Conclusions: Identification of signaling pathway/transcriptions proteins and cell cycle related proteins in breast cancer sera, and identification of breast cancer amplified sequence 3 proteins in the DCIS sera, suggest that high-throughput proteomic technologies like 2-D LC - MS/MS may facilitate the discovery of diagnostically useful breast cancer biomarkers. No significant financial relationships to disclose.

Determination of Digitoxin in Human Serum Using Stable Isotope Diluted Liquid Chromatography/Electrospray Ionization‐Tandem Mass Spectrometry

A method for the determination of digitoxin in human serum using a liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS) technique is reported. Digitoxin and the internal standard, [21,21,22‐2H3]digitoxin, were extracted from 200 µL of human serum using a solid phase extraction cartridge and analyzed by LC/ESI‐MS/MS in the selected reaction monitoring mode. The intra‐ and inter‐assay reproducibility and accuracy were satisfactory within the quantification range of 5–100 ng/mL, which was sensitive enough to measure the digitoxin in real samples. The concentrations of digitoxin in serum samples obtained from digitalized patients (n=19) were in the range of 5.3–24.1 ng/mL, and these correlated well with those obtained by radioimmunoassay using a very specific antiserum.

Phosphorimetric microassay for succinyltrialanyl- p-nitroanilide-hydrolyzing enzyme activity in human serum

A phosphorimetric assay of succinyltrialanyl- p-nitroanilide-hydrolyzing enzyme activity in human serum is described. p-Nitroaniline formed enzymatically is extracted with ether and determined phosphorimetrically in an ether/ethanol glass. The method is precise and very sensitive, requiring as little as 5 μl of human serum. The limit of detection is 10 pmol.

Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the assay of angiotensin-converting enzyme in human serum and for the separation of angiotensins and their analogues after pre-column fluorescence derivatization with benzoin. Angiotensin II, formed enzymatically from angiotensin I, is converted into a fluorescent derivative which is then separated isocratically from the substrate and biological substances in the enzyme reaction mixture on a reversed-phase column (TSK gel ODS-120T). The lower limit of detection for angiotensin II is 0.66 pmol per enzyme assay tube. The method is simple and sensitive, and requires as little as 5 μl of human serum. Angiotensin analogues can also be separated and quantified by the chromatographic technique, and thus this method permits the use of the analogues of angiotensin I as substrates.

A specific iron stain for iron-binding proteins in polyacrylamide gels: Application to transferrin and lactoferrin

A new method for specifically staining the iron atoms present in transferrin and lactoferrin after polyacrylamide gel electrophoresis and isoelectric focusing is described. The stain, 3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid))-1,2,4-triazine, disodium salt, or Ferene S, will detect transferrin in 5 μl of human serum, lactoferrin in 10 μl of human whey, and 10 μg of purified primate ( Macaca fascicularis) transferrin. This method of staining is very rapid as the serum transferrin bands can be seen within 5 to 10 min of staining.

An ultramicro assay for γ-glutamyl transpeptidase in biological materials by phosphorimetry

Summary A highly sensitive phosphorimetric method for the assay of γ-glutamyl transpeptidase (EC 2.3.2.2) in biological samples is described. p -Nitroaniline, formed enzymatically from γ-L-glutamyl- p -nitroanilide with an incubation time of 15 min, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanol. The method is precise and very sensitive, requiring as little as 0.1 μl of human serum or 0.01–0.1 μg of protein of rat tissue. The limit of detection for the p -nitroaniline formed is 5 pmol.

Phosphorimetric assay for β-glucuronidase in biological materials

A highly sensitive phosphorimetric method for the assay of β-glucuronidase in biological samples is described. p-Nitrophenol, formed enzymatically from p-nitrophenyl β-D-glucuronide, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanolic potassium hydroxide. The method is rapid, precise, and very sensitive, requiring as little as 0.5–5 μl of human serum or urine, or 0.3–3.0 μg of protein of rat tissue. The limit of detection for the p-nitrophenol formed is 20 pmol.

A new fluorescence assay for dipeptidyleptidase IV using tripeptide l-prolyl- l-prolyl- l-alanine as substrate

We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, l-prolyl- l-prolyl- l-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolosis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 μl of human serum and in crevicular fluid obtained from human gingival sulcus. The K m value for the tripeptide was 1.7 · 10 −5 M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards.

Dehydroisoandrosterone sulfate in peripheral blood of premenopausal, pregnant and postmenopausal women and men

A radioimmunoassay specific for the direct measurement of dehydroisoandrosterone sulfate in 0.5 or 1 μl of human serum was developed using an antibody directed against dehydroisoandrosterone hemisuccinate coupled to thyroglobulin. Dehydroisoandrosterone sulfate levels were measured in pregnant women throughout gestation starting with the 6th week of pregnancy, and in young ovulatory women, postmenopausal women and in men. During pregnancy the concentrations of dehydroisoandrosterone sulfate in maternal serum decreased steadily with advancing gestation [1825 ± 68 ng/ml (mean and S.E.) at 6–8 weeks gestation vs 706 ± 141 ng/ml at 36–41 weeks gestation], and the most striking decrease was observed between 14 and 16 weeks of gestation. The mean concentrations of dehydroiso-androsterone sulfate were higher in men than in young nonpregnant women (2679 ±196 ng/ml vs 2020 ± 108 ng/ml respectively). In postmenopausal women, mean serum dehydroisoandrosterone sulfate concentrations (764 ± 28 ng/ml) were considerably lower than those in young premenopausal women P < 0.001).

Fluorescence assay of X-prolyl dipeptidyl-aminopeptidase activity with a new fluorogenic substrate

We have synthesized a new fluorogenic substrate, 7-(Gly-Pro)-4-methylcoumarinamide, for X-prolyl dipeptidyl-aminopeptidase. Release of 7-amino-4-methylcoumarin by the enzymatic hydrolysis was determined fluorometrically. This new assay was highly sensitive; the enzyme activity in as little as 0.01 μl of human serum was measured, and it was discovered in human cerebrospinal fluid. The specific activity in human parotid saliva was about 17-fold higher than that in serum.

Photometric Assay of Dopamine-β-Hydroxylase Activity in Human Blood

Abstract We describe a simple, rapid, and sensitive procedure for measuring dopamine-β-hydroxylase activity in human blood. The assay is based on the enzymatic conversion of tyramine to octopamine [1-(p-hydroxyphenyl)-2-aminoethanol], which is then oxidized to p-hydroxybenzaldehyde and determined photometrically. Activities are assayed on 2-20 µl of human serum or plasma obtained from fingertip blood samples. The method should prove useful in clinical studies, including the field of pediatrics.