Identification of Active-Site in a Neurotoxic Snake Venom by Affinity Labelling and State-of-the-Art Tandem Mass Spectrometry Technology

1989 ◽  
pp. 149-160
Author(s):  
Thaiya Krishnamurthy ◽  
Marguerite E. Brooks ◽  
Donald F. Hunt ◽  
Jeffrey Shabanowitz ◽  
Shuian Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Snake Venom ◽  
Active Site ◽  
State Of The Art ◽  
Tandem Mass ◽  
Mass Spectrometry Technology ◽  
Affinity Labelling

scholarly journals A High-Calcium and Phosphate Rescue Diet and VDR-Expressing Transgenes Normalize Serum Vitamin D Metabolite Profiles and Renal Cyp27b1 and Cyp24a1 Expression in VDR Null Mice

Endocrinology ◽  
2015 ◽  
Vol 156 (12) ◽  
pp. 4388-4397 ◽  
Author(s):  
Martin Kaufmann ◽  
Seong Min Lee ◽  
J. Wesley Pike ◽  
Glenville Jones
Keyword(s):  
Mass Spectrometry ◽  
Vitamin D ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Cytochrome P450 Enzyme ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
High Calcium ◽  
Mass Spectrometry Technology ◽  
Liquid Chromatography Tandem Mass

Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca++, PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis.

Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry

2011 ◽  
Vol 6 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Oliver Schilling ◽  
Pitter F Huesgen ◽  
Olivier Barré ◽  
Ulrich auf dem Keller ◽  
Christopher M Overall
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Active Site ◽  
Peptide Libraries ◽  
Tandem Mass

scholarly journals A 6 Second Analytical Method for Quantitation of Tacrolimus in Whole Blood by Use of Laser Diode Thermal Desorption Tandem Mass Spectrometry

2019 ◽  
Vol 3 (6) ◽  
pp. 965-973
Author(s):  
Stephen D Merrigan ◽  
Kamisha L Johnson-Davis
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Thermal Desorption ◽  
Laser Diode ◽  
Analytical Method ◽  
Whole Blood ◽  
Turnaround Time ◽  
Tandem Mass ◽  
Mass Spectrometry Technology ◽  
Analytical Time

Abstract Background Therapeutic drug monitoring of immunosuppressive drugs is imperative for organ transplant recipients. High-performance LC-MS/MS is considered gold standard; however, immunoassays provide rapid turnaround time. New technology was developed to reduce mass spectrometry analytical run-time. The laser diode thermal desorption source coupled with tandem mass spectrometry (LDTD-MS/MS) eliminates chromatographic separation to increase analytical throughput. Methods A rapid, 6 second, LDTD-MS/MS analytical method was developed for the quantification tacrolimus in whole blood. Whole blood samples were lysed, followed by protein precipitation and solid-phase extraction. Extracted samples with desorption solution were spotted onto a LazWell plate then dried and loaded into the LDTD source for analysis with an AB SCIEX 5500 mass spectrometer in positive multiple reaction monitoring mode. The LDTD laser profile ramps from 0% to 65% of full power over 3 s and is held at 65% for 1 s before returning to initial conditions for 2 s. Results Data presented include tacrolimus by LDTD-MS/MS comparison to LC-MS/MS, sensitivity, imprecision, interference, linearity, and stability. Method comparison between LDTD-MS/MS and a validated in-house LC-MS/MS assay yielded the following: (LDTD-MS/MS) = 1.119 (LC-MS/MS) + 0.23 ng/mL, Sy/x = 1.26, r = 0.9871 (n = 122). The limit of quantification by LDTD-MS/MS for tacrolimus was <0.3 ng/mL and total imprecision was <10%. Conclusions Laser diode thermal desorption tandem mass spectrometry technology can provide rapid turnaround time to result for tacrolimus. The analytical time for LDTD-MS/MS was 6 s compared to 135 s by LC-MS/MS, a >95% decrease in analytical time.

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