Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry—mass spectrometry

Lovelace, Jerry L.; Kusmierz, Jozef J.; Desiderio, Dominic M.

doi:10.1016/0378-4347(91)80609-g

Cited by 38 publications

(14 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although RIAs can be developed with the requisite detection limits (100 amol), maintaining this sensitivity for routine measurements can be difficult. Immunoassays also have questionable specificity because of cross-reactivity between the target peptides and related peptides such as precursors and metabolites 7, 8. Furthermore, RIAs are usually limited to measurement of a single peptide per sample.…”

Section: Introductionmentioning

confidence: 99%

Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS

Zubieta

Kennedy

2009

Anal. Chem.

View full text Add to dashboard Cite

A method using capillary LC-MS 3 to determine endogenous opioid peptides in microdialysis samples collected in vivo was developed, validated, and applied to measurements in the rat striatum. Peptides in dialysate rapidly degraded when stored at room temperature or −80 °C. Adding acetic acid to a final concentration of 5% stabilized the peptides for 5 days allowing storage of fractions and off-line measurements which proved more convenient and reliable than previously used on-line methods. Study of the effect of dialysis flow rate from 0.2 to 2 μL/min and column inner diameter (i.d.) from 25 to 75 μm on the relative signal obtained for peptides revealed that lowest flow rates and smallest column i.d. gave the highest relative signal. The method was tested for 10 different neuropeptides and LODs were from 0.5 pM to 60 pM (4 μL samples) for most. β-endorphin had an LOD of 5 nM when detected directly, but it could be quantitatively determined by detecting a characteristic peptide produced by tryptic digestion with an LOD of 3 pM. This approach may prove useful for other large neuropeptides as well. The method was used to determine met-enkephalin, leu-enkephalin, dynorphin A 1-8 , and β-endorphin in vivo. Endomorphin 1 and 2 were below the detection limit of the method in vivo. Quantitative determination of leu-enkephalin using external calibration was verified by standard addition experiments. The improvements over previous approaches using capillary LC-MS n make in vivo neuropeptide monitoring more practical and feasible for a variety of neuropeptides.

show abstract

Section: Introductionmentioning

confidence: 99%

Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS

Zubieta

Kennedy

2009

Anal. Chem.

View full text Add to dashboard Cite

show abstract

“…In order to clarify the molecular events that occur in pituitary macroadenoma formation, much of our research effort [1][2][3][4][5][6][7] has focused on the analysis of neuropeptidergic systems in the anterior pituitary, where, under regulation and input from hypothalamic-releasing and -inhibiting hormones, a variety of pituitary hormones are released into the blood stream to act at distal sites to regulate many important physiological functions. However, pathophysiological changes in the pituitary cause pituitary tumors that seriously impact the pituitary's functions [8].…”

Section: Introductionmentioning

confidence: 99%

Differences in the spatial and quantitative reproducibility between two second‐dimensional gel electrophoresis systems

Zhan¹,

Desiderio

2003

Electrophoresis

Self Cite

View full text Add to dashboard Cite

Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume.

show abstract

“…This sensitive technique is mainly performed on crude extracts and therefore allows many samples to be processed within a day. Nevertheless, the selectivity of any RIA could be hampered by interference with divalent cations, as well as by cross-reactions with macromolecules and structural analogs of the peptide of interest [15-161. Indeed, RIA depends on the interaction of the peptide with an antibody and therefore monitors a secondary structural parameter and not the amino acid sequence of the peptide [17]. Consequently, more specific analytical methods, in which primary structural parameters (amino acid composition and sequence) are monitored, are needed to determine neuropeptides in biological samples.…”

Section: Introductionmentioning

confidence: 99%

Detection of neurotensin in tissues by capillary zone electrophoresis

Denoroy

1994

Electrophoresis

View full text Add to dashboard Cite

An electrophoretic method for detecting neurotensin in tissues was developed. Homogenates of rat duodenum and adrenal glands were first extracted by solid-phase extraction and purified by reversed-phase high-performance liquid chromatography. The neurotensin-rich fraction was further analyzed by capillary zone electrophoresis (CZE) using a commercial instrument with UV detection. A minor peak was detected as neurotensin and its identity was further confirmed by performing several CZE analyses at different pH values. Such an approach could be used to analyze with good molecular specificity the neuropeptides in some human tissues.

show abstract

Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry—mass spectrometry

Cited by 38 publications

References 22 publications

Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS

Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS

Differences in the spatial and quantitative reproducibility between two second‐dimensional gel electrophoresis systems

Detection of neurotensin in tissues by capillary zone electrophoresis

Contact Info

Product

Resources

About