Relative Quantification of Biomarkers Using Mixed-Isotope Labeling Coupled With Ms

Chapman, Heidi M; Schutt, Katherine L.; Dieter, Emily; Lamos, Shane M.

doi:10.4155/bio.12.208

Cited by 13 publications

(19 citation statements)

References 117 publications

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“…Chemical labeling through ICPL has also been made available by multiplex proteomic analysis. This multiplexed approach enables the comparison of multiple samples, three in this case, within one analytical run, with accuracy and precision that were comparable to that of the binary analyses [ 83 ]. This technique was used by Lecube et al to carry out the proteomic analysis of cerebrospinal fluid from obese and non-obese women with idiopathic intracranial hypertension to assess the hypothalamic control on body weight regulation.…”

Section: Separation Techniquesmentioning

confidence: 99%

Obesity Proteomics: An Update on the Strategies and Tools Employed in the Study of Human Obesity

Masood

Alfadda

2018

High-Throughput

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Proteomics has become one of the most important disciplines for characterizing cellular protein composition, building functional linkages between protein molecules, and providing insight into the mechanisms of biological processes in a high-throughput manner. Mass spectrometry-based proteomic advances have made it possible to study human diseases, including obesity, through the identification and biochemical characterization of alterations in proteins that are associated with it and its comorbidities. A sizeable number of proteomic studies have used the combination of large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography in combination with mass spectrometry, for high-throughput protein identification. These studies have applied proteomics to comprehensive biochemical profiling and comparison studies while using different tissues and biological fluids from patients to demonstrate the physiological or pathological adaptations within their proteomes. Further investigations into these proteome-wide alterations will enable us to not only understand the disease pathophysiology, but also to determine signature proteins that can serve as biomarkers for obesity and related diseases. This review examines the different proteomic techniques used to study human obesity and discusses its successful applications along with its technical limitations.

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Section: Separation Techniquesmentioning

confidence: 99%

Obesity Proteomics: An Update on the Strategies and Tools Employed in the Study of Human Obesity

Masood

Alfadda

2018

High-Throughput

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“…Relative quantification refers to determination of expression of the target gene compared to that of a reference gene; since the level of expression of the reference gene is constant, it can be used for comparison of expression differences from different sources. Interfering factors like impurities or leftover reagents can be eliminated by mathematical calculation; therefore, the method is simple, fast and reliable (Chapman et al, 2012). Therefore, to ensure accuracy of our results, we used a relative quantification approach with GAPDH as the internal reference gene to determine the amount of chTERT mRNA in the sample.…”

Section: Influence Of the Target Gene Local Structurementioning

confidence: 99%

shRNA inhibits the expression of chicken telomerase reverse transcriptase in MDCC-MSB1 cells

et al. 2016

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ABSTRACT. Here, we investigated the effects of blocking chicken telomerase reverse transcriptase (chTERT) in MDCC-MSB1 cells, using small-hairpin RNAs (shRNAs) to interfere with gene expression. shRNAs specific to chTERT mRNA were designed, cloned into DNA plasmid vectors, and transfected into MDCC-MSB1 cells. The transfected chTERT RNAs were expressed by the RNA polymerase machinery of the MDCC-MSB1 cells. mRNA expression in transfected MDCC-MSB1 cells was detected using real-time PCR. After transfection, telomerase activity was monitored via a modified telomeric repeat amplification protocol assay, and cell cycle analysis was performed using a flow cytometer. At 72 h after transfection, chTERT expression was considerably reduced in cells transfected with shRNA; the highest inhibition rate was 89%. Compared with the control group, telomerase activity was significantly reduced and the cells failed to progress to S phase. shRNA effectively reduced telomerase activity and prohibited the transition of MDCC-MSB1 cells from G2/M to S phase.

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“…Because the removal of the proton occurs at the α ‐methylene, we devised a method to distinguish between protons on the α ‐methylenes of N ‐benzyl and alkyl moieties. Isotopic labeling is a versatile tool that provides deep insight into the kinetics, regioselectivity and mechanisms of reactions . The loss of a deuterium atom or a proton will lead to a difference in one mass number that can be identified by MALDI‐TOF MS.…”

Section: Introductionmentioning

confidence: 99%

Dehydrogenation and dehalogenation of amines in MALDI‐TOF MS investigated by isotopic labeling

Kang

Zhou

et al. 2013

J. Mass Spectrom.

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Secondary and tertiary amines have been reported to form [M-H](+) that correspond to dehydrogenation in matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this investigation, we studied the dehydrogenation of amines in MALDI-TOF MS by isotopic labeling. Aliphatic amines were labeled with deuterium on the methylene of an N-benzyl group, which resulted in the formation of [M-D](+) and [M-H](+) ions by dedeuteration and dehydrogenation, respectively. This method revealed the proton that was removed. The spectra of most tertiary amines with an N-benzyl group showed high-intensity [M-D](+) and [M-H](+) ion peaks, whereas those of secondary amines showed low-intensity ion peaks. Ratios between the peak intensities of [M-D](+) and [M-H](+) greater than 1 suggested chemoselective dehydrogenation at the N-benzyl groups. The presence of an electron donor group on the N-benzyl groups enhanced the selectivity. The dehalogenation of amines with an N-(4-halobenzyl) group was also observed alongside dehydrogenation. The amino ions from dehalogenation can undergo second dehydrogenation. These results provide the first direct evidence about the position at which dehydrogenation of an amine occurs and the first example of dehalogenation of haloaromatic compounds in MALDI-TOF MS. These results should be helpful in the structural identification and elucidation of synthetic and natural molecules.

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Relative Quantification of Biomarkers Using Mixed-Isotope Labeling Coupled With Ms

Cited by 13 publications

References 117 publications

Obesity Proteomics: An Update on the Strategies and Tools Employed in the Study of Human Obesity

Obesity Proteomics: An Update on the Strategies and Tools Employed in the Study of Human Obesity

shRNA inhibits the expression of chicken telomerase reverse transcriptase in MDCC-MSB1 cells

Dehydrogenation and dehalogenation of amines in MALDI‐TOF MS investigated by isotopic labeling

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