Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry

Guan, Fuyu; Uboh, Cornelius E.; Soma, Lawrence R.; Luo, Yang; Rudy, Jeffrey A.; Tobin, Thomas

doi:10.1016/j.jchromb.2005.09.045

Cited by 89 publications

(85 citation statements)

References 35 publications

(49 reference statements)

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“…Although full-scan MS/MS spectra are not acquired in this mode of operation [termed selected reaction monitoring (SRM) and, its extension, multiple reaction monitoring (MRM)], the method provides high selectivity by monitoring chromatographic coelution of multiple transitions for a given peptide. It is important to note that SRM and MRM have already been successfully applied to a variety of biological applications, including quantification of DNA adducts purified from tissue (8) and detection of doping substances in human urine and plasma (9)(10)(11)(12). To date, the use of SRM and MRM in proteomics is less widespread, although MRM has been used to quantify protein expression (13), to find protein biomarkers for disease severity in rheumatoid arthritis (14), and for detection and quantitative analysis of protein phosphorylation (15)(16)(17).…”

mentioning

confidence: 99%

Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks

Wolf-Yadlin

Hautaniemi

Lauffenburger

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

473

434

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Although recent developments in MS have enabled the identification and quantification of hundreds of phosphorylation sites from a given biological sample, phosphoproteome analysis by MS has been plagued by inconsistent reproducibility arising from automated selection of precursor ions for fragmentation, identification, and quantification. To address this challenge, we have developed a new MS-based strategy, based on multiple reaction monitoring of stable isotope-labeled peptides, that enables highly reproducible quantification of hundreds of nodes (phosphorylation sites) within a signaling network and across multiple conditions simultaneously. We have applied this strategy to quantify temporal phosphorylation profiles of 222 tyrosine phosphorylated peptides across seven time points following EGF treatment, including 31 tyrosine phosphorylation sites not previously known to be regulated by EGF stimulation. With this approach, 88% of the signaling nodes were reproducibly quantified in four analyses, as compared with only 34% by typical information-dependent analysis. As a result of the improved reproducibility, full temporal phosphorylation profiles were generated for an additional 104 signaling nodes with the multiple reaction monitoring strategy, an 88% increase in our coverage of the signaling network. This method is broadly applicable to multiple signaling networks and to a variety of samples, including quantitative analysis of signaling networks in clinical samples. Using this approach, it should now be possible to routinely monitor the phosphorylation status of hundreds of nodes across multiple biological conditions. epidermal growth factor receptor ͉ mass spectrometry ͉ signal transduction ͉ tyrosine phosphorylation L igand binding to cell surface receptors activates multiple protein tyrosine phosphorylation-mediated signaling cascades that regulate many cell biological processes, including proliferation, differentiation, migration, and cell death (1-3). To mechanistically define the relationship between signaling networks and downstream biological responses, it is necessary to quantify the dynamics of protein phosphorylation sites across multiple cell states and to correlate this information to quantitative phenotypic measurements. Until recently, antibody-based assays (e.g., FACS, Western blots, tissue microarrays) have been favored for most signaling network studies. These assays provide excellent quantitative information on the phosphorylation status of selected nodes within the network, but require a priori knowledge of the proteins and phosphorylation sites to be studied and are limited by the need to have high-quality, non-cross-reactive antibodies recognizing specific sites within the network. By comparison, analysis of protein phosphorylation by MS provides the capability of identifying novel phosphorylation sites on novel proteins within the network, requires minimal a priori knowledge, and is therefore compatible with both well and poorly characterized signaling networks. Recent developments in M...

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mentioning

confidence: 99%

Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks

Wolf-Yadlin

Hautaniemi

Lauffenburger

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

473

434

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“…Such fragments are often detected in ESI-MS analyses of testosterone and related compounds having the 4-ene-3-one structure [48 -50]. The ion at m/z 109 amu is formed by so-called B ring fission producing a residue of C 7 H 9 O [50], while the ion at m/z 97 amu represents a residue of C 6 H 9 O [50]. Owing to nearly identical ESI-MS and ESI-MS/MS behaviors of testosterone and epitestosterone, an efficient separation method was required to obtain reliable identification.…”

Section: Discussionmentioning

confidence: 99%

Comparison of partial filling MEKC analyses of steroids with use of ESI‐MS and UV spectrophotometry

Amundsen

Kokkonen

Sirén³

2008

J of Separation Science

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Until now, partial filling micellar EKC-ESI-MS (PF-MEKC-ESI-MS) has seldom been applied for human biomolecules. In this study, determination of three electrically neutral endogenous steroid hormones, namely androstenedione, testosterone, and epitestosterone, by PF-MEKC-ESI-MS was investigated. Since ESI-MS and ESI-MS/MS behaviors of testosterone and epitestosterone proved to be nearly identical, efficient separation of the two compounds was required to obtain reliable identification. The chemical conditions as well as the instrumental parameters affecting the PF-MEKC-ESI-MS analysis were researched. In optimal conditions, ESI-MS showed excellent selectivity, and all the steroids could be identified using SIM. LODs were 0.75-5 microg/mL. The results obtained by PF-MEKC-ESI-MS were compared with those obtained by corresponding PF-MEKC-UV. PF-MEKC-UV provided better resolution, repeatability, and more than ten-fold higher sensitivity, in terms of LODs, than PF-MEKC-ESI-MS. The reasons for this were carefully examined. In comparison with PF-MEKC-UV, PF-MEKC-ESI-MS suffered from greater band broadening owing to the sheath-liquid interface. Resolution was also decreased owing to the elevated capillary temperature. Finally, we discovered that in the analysis of electrically neutral compounds, in-capillary sample concentration by micellar sweeping could be more efficiently utilized in PF-MEKC-UV than in PF-MEKC-ESI-MS.

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“…The extraction method was described by Guan et al (2005) for equine plasma, with slight modifications. To 1 g faeces the internal standard NETA (10 ng/g), 3 ml of acetate buffer (pH 4.8) and 1 ml of 1N NaOH were added.…”

Section: Methodsmentioning

confidence: 99%

Simultaneous measurement of boldenone (α and β), ADD, testosterone, epitestosterone and AED in bovine faeces

et al. 2008

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Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry

Cited by 89 publications

References 35 publications

Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks

Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks

Comparison of partial filling MEKC analyses of steroids with use of ESI‐MS and UV spectrophotometry

Simultaneous measurement of boldenone (α and β), ADD, testosterone, epitestosterone and AED in bovine faeces

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