Functional Proteomics of Regulatory and Epigenetic Signaling in Normal and Malignant Hematopoiesis

Abstract Post-translational modifications of proteins are increasingly recognized as key regulators of functional complexes assembly and cellular enzymes activity, processes that control both normal and aberrant cell growth and development. To enable comprehensive, sensitive and quantitative analysis of these processes, we adapted recently developed high-efficiency nanoscale multidimensional liquid chromatography with high-resolution Orbitrap mass spectrometry. Termed accumulated ion monitoring (AIM), this mass spectrometry method achieved nearly 7 orders of magnitude of quantitative accuracy and absolute limit of detection of 600 molecules per scan, enabling the study of rare cell populations (Fig. 1). We leveraged AIM mass spectrometry to develop a panel of 1583 synthetic reference peptides, based on global and published proteomics maps of normal human and leukemia cells. This Quantitative Cell Proteomics Atlas (QCPA) profiles 384 key effectors of cell surface signaling, proliferation, quiescence, stress response, and epigenetic control of gene expression (Fig. 2). QCPA enables unprecedented accuracy and sensitivity for the functional analysis of rare cells, with focus on protein regulation via phosphorylation, acetylation and methylation, as well as on concentration (http://alexkentsis.net/qcpa). By using functional proteomics profiling of primary human CD34+ and acute myeloid leukemia (AML) cells, we identify new pathways controlling erythrocyte differentiation and AML therapy resistance, as facilitated by a newly developed program ProteoModlR (http://github.com/kentsisresearchgroup/ProteoModlR). AIM mass spectrometry and QCPA functional proteomics are rapidly adaptable and generalizable tools for the investigation of regulatory and epigenetic signaling in normal and diseased cells. Figure 1. Limits of detection and quantitation for a serially diluted synthetic peptide from human Myocyte-specific Enhancer Factor 2C. Figure 1. Limits of detection and quantitation for a serially diluted synthetic peptide from human Myocyte-specific Enhancer Factor 2C. Figure 2. Functional classification of proteins included in the QCPA. Figure 2. Functional classification of proteins included in the QCPA. Disclosures Bauer: Editas Medicine: Consultancy; Biogen: Research Funding.

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Functional Proteomic Analysis of Human Nucleolus

Molecular Biology of the Cell ◽

10.1091/mbc.e02-05-0271 ◽

2002 ◽

Vol 13 (11) ◽

pp. 4100-4109 ◽

Cited By ~ 316

Author(s):

Alexander Scherl ◽

Yohann Couté ◽

Catherine Déon ◽

Aleth Callé ◽

Karine Kindbeiter ◽

...

Keyword(s):

Proteomic Analysis ◽

Ribosome Biogenesis ◽

Molecular Mechanisms ◽

Convincing Evidence ◽

Biological Processes ◽

Functional Classification ◽

Control Of Gene Expression ◽

Nucleolar Proteins ◽

Nuclear Domain

The notion of a “plurifunctional” nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of ∼350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.

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Functional classification of proteins using mass spectrometry data and exploration of their frequency of identification in proteomic analysis

10.12681/eadd/26473 ◽

2009 ◽

Author(s):

Panagiotis Bougioukos

Keyword(s):

Mass Spectrometry ◽

Proteomic Analysis ◽

Mass Spectrometry Data ◽

Functional Classification

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LC-MS-MS vs ELISA: Validation of a Comprehensive Urine Toxicology Screen by LC-MS-MS and a Comparison of 100 Forensic Specimens

Journal of Analytical Toxicology ◽

10.1093/jat/bkz066 ◽

2019 ◽

Vol 43 (9) ◽

pp. 734-745 ◽

Cited By ~ 4

Author(s):

Kristin W Kahl ◽

Joshua Z Seither ◽

Lisa J Reidy

Keyword(s):

Mass Spectrometry ◽

Limit Of Detection ◽

Screening Method ◽

Driving Under The Influence ◽

Cost Comparison ◽

Enzyme Linked Immunosorbent Assay ◽

Screening Methods ◽

Limits Of Detection ◽

The Cost ◽

Urine Specimens

Abstract Toxicology laboratories commonly employ immunoassay methodologies to perform an initial drug screen on urine specimens to direct confirmatory testing. Due to limitations of immunoassay testing and the need to screen for a broader range of drugs with lower limits of detection at a lower cost, mass spectrometry screening techniques have gained favor in the toxicology field. A liquid chromatography–tandem mass spectrometry (LC-MS-MS) urine screening panel was developed and validated for 52 drugs and metabolites. A simple dilute-and-shoot with enzymatic hydrolysis technique was utilized to prepare the urine specimens for analysis. Limit of detection, interference, ionization suppression/enhancement, carryover and stability of processed specimens were assessed during validation. To evaluate the toxicological results obtained from utilizing the LC-MS-MS in comparison with the laboratory’s current enzyme-linked immunosorbent assay (ELISA) panel, 100 authentic urine specimens from suspected driving under the influence and drug-facilitated crime cases were analyzed using both methodologies and the results were compared. In addition, the cost of each methodology was evaluated and compared. The validated LC-MS-MS method had limits of detection that were equal to or lower than the concentrations validated for ELISA cutoffs, had fewer exogenous interferences, and the cost of screening per specimen was reduced by ~70% when compared to ELISA. Comparing the toxicology results of forensic urine specimens demonstrated that by only using ELISA, the laboratory was unable to detect benzoylecgonine in 26%, lorazepam in 33% and oxymorphone in 60% of the positive specimens. Additional analytes detected using the LC-MS-MS method were zolpidem and/or metabolite, gabapentin, tramadol and metabolite, methadone and metabolite, meprobamate and phentermine. The results of the validation, the toxicological result comparison and the cost comparison showed that the LC-MS-MS screening method is a simple, sensitive and cost-effective alternative to ELISA screening methods for urine specimens.

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Measurement of Fibrosis Marker Xylosyltransferase I Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2006.071167 ◽

2006 ◽

Vol 52 (12) ◽

pp. 2243-2249 ◽

Cited By ~ 26

Author(s):

Joachim Kuhn ◽

Christian Prante ◽

Sylvia Schön ◽

Christian Götting ◽

Knut Kleesiek

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Human Serum ◽

Electrospray Ionization ◽

Synthetic Peptide ◽

Limit Of Detection ◽

Accurate Determination ◽

Connective Tissue Diseases ◽

Tandem Mass ◽

Ionization Tandem Mass Spectrometry

Abstract Background: Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients. Methods: We developed an HPLC electrospray ionization tandem mass spectrometry method to assay XT-I activity in serum by use of a synthetic peptide (Bio–BIK-F) as the XT-I substrate. On the basis of XT-I-mediated transfer of D-xylose from UDP-D-xylose to the synthetic peptide to form Bio-BIK-F-Xyl, we determined XT-I activity in human serum samples. Results: Multiple calibration curves for the analysis of Bio-BIK-F-Xyl exhibited consistent linearity and reproducibility in the range of 0.20–20 mg/L, corresponding to XT-I activity of 1.14–114 mU/L under assay conditions. The mean (SD, range) XT-I activity values in 30 blood donor sera were 18.4 (3.0, 8.7–24.8) mU/L. The limit of detection and lower limit of quantification were 8.5 μg/L (0.05 mU/L) and 163 μg/L Bio-BIK-F-Xyl (0.93 mU/L XT-I activity), respectively. Interassay imprecision (CV) was 5.4%–26.1% in the range of 0.64 to 129 mU/L, and mean recovery was 107% (range, 96%–129%). Method comparison with the radiochemical assay showed a moderate correlation (r = 0.79). The Passing–Bablok regression line was: radiochemical assay = 0.045 LC-MS/MS + 0.061 mU/L, Sy|x = 0.186. Conclusions: This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.

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Objective and parametric methods used in functional classification disabled swimmers

Physiotherapy ◽

10.2478/physio-2013-0022 ◽

2013 ◽

Vol 21 (3) ◽

Cited By ~ 1

Author(s):

Natalia Uścinowicz ◽

Wojciech Seidel ◽

Paweł Zostawa ◽

Sebastian Klich

Keyword(s):

Medical Diagnosis ◽

Olympic Games ◽

Objective Evaluation ◽

Subjective Assessment ◽

Fair Play ◽

Functional Classification ◽

Kinematic Parameters ◽

Athletes With Disabilities ◽

Rule Out

AbstractThe recent Olympic Games in London incited much interest in the competition of disabled athletes. Various people connected with swimming, including coaches and athletes, have speculated about the fairness of competitions of disabled athletes. A constant problem are the subjective methods of classification in disabled sport. Originally, athletes with disabilities were classified according to medical diagnosis. Due to the injustice which still affects the competitors, functional classification was created shortly after. In the present review, the authors show the anomalies in the structure of the classification. The presented discovery led to the suggestion to introduce objective methods, thanks to which it would be no longer necessary to rely on the subjective assessment of the classifier. According to the authors, while using objective methods does not completely rule out the possibility of fraud by disabled athletes in the classification process, it would certainly reduce their incidence. Some of the objective methods useful for the classification of disabled athletes are: posturography, evaluation of the muscle parameters, electrogoniometric assessment, surface electromyography, and analysis of kinematic parameters. These methods have provide objective evaluation in the diagnostic sense but only if they are used in tandem. The authors demonstrate the undeniable benefits of using objective methods. Unfortunately, there are not only advantages of such solution, there also several drawbacks to be found. The conclusion of the article is the statement by the authors that it is right to use objective methods which allow to further the most important rule in sport: fair-play.

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A Novel Thiosemicarbazide-Based Fluorescent Chemosensor for Hypochlorite in Near-Perfect Aqueous Solution and Zebrafish

Chemosensors ◽

10.3390/chemosensors9040065 ◽

2021 ◽

Vol 9 (4) ◽

pp. 65

Author(s):

Minji Lee ◽

Donghwan Choe ◽

Soyoung Park ◽

Hyeongjin Kim ◽

Soomin Jeong ◽

...

Keyword(s):

Mass Spectrometry ◽

Reactive Oxygen Species ◽

Aqueous Solution ◽

Fluorescence Quenching ◽

Limit Of Detection ◽

Fluorescent Sensor ◽

Ionization Mass ◽

Oxygen Species ◽

Marked Selectivity ◽

Uv Visible

A novel thiosemicarbazide-based fluorescent sensor (AFC) was developed. It was successfully applied to detect hypochlorite (ClO−) with fluorescence quenching in bis-tris buffer. The limit of detection of AFC for ClO− was analyzed to be 58.7 μM. Importantly, AFC could be employed as an efficient and practical fluorescent sensor for ClO− in water sample and zebrafish. Moreover, AFC showed a marked selectivity to ClO− over varied competitive analytes with reactive oxygen species. The detection process of AFC to ClO− was illustrated by UV–visible and fluorescent spectroscopy and electrospray ionization–mass spectrometry (ESI–MS).

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Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0612 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1461-1468 ◽

Cited By ~ 2

Author(s):

Jean-Claude Alvarez ◽

Pierre Moine ◽

Isabelle Etting ◽

Djillali Annane ◽

Islam Amine Larabi

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Half Life ◽

Limit Of Detection ◽

Treated Patient ◽

Internal Standard ◽

Therapeutic Drug ◽

Active Metabolites ◽

Limit Of Quantification ◽

Positive Mode

AbstractObjectivesA method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524.MethodsA simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.ResultsCalibration curves were linear in the 1–5000 μg/L range for remdesivir and 5–2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6–110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.ConclusionsThis method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

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