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1.
Retention Time Prediction and Protein Identification.
Henneman, A, Palmblad, M
Methods in molecular biology (Clifton, N.J.). 2020;:115-132
Abstract
In bottom-up proteomics, proteins are typically identified by enzymatic digestion into peptides, tandem mass spectrometry and comparison of the tandem mass spectra with those predicted from a sequence database for peptides within measurement uncertainty from the experimentally obtained mass. Although now decreasingly common, isolated proteins or simple protein mixtures can also be identified by measuring only the masses of the peptides resulting from the enzymatic digest, without any further fragmentation. Separation methods such as liquid chromatography and electrophoresis are often used to fractionate complex protein or peptide mixtures prior to analysis by mass spectrometry. Although the primary reason for this is to avoid ion suppression and improve data quality, these separations are based on physical and chemical properties of the peptides or proteins and therefore also provide information about them. Depending on the separation method, this could be protein molecular weight (SDS-PAGE), isoelectric point (IEF), charge at a known pH (ion exchange chromatography), or hydrophobicity (reversed phase chromatography). These separations produce approximate measurements on properties that to some extent can be predicted from amino acid sequences. In the case of molecular weight of proteins without posttranslational modifications this is straightforward: simply add the molecular weights of the amino acid residues in the protein. For IEF, charge and hydrophobicity, the order of the amino acids, and folding state of the peptide or protein also matter, but it is nevertheless possible to predict the behavior of peptides and proteins in these separation methods to a degree which renders such predictions useful. This chapter reviews the topic of using data from separation methods for identification and validation in proteomics, with special emphasis on predicting retention times of tryptic peptides in reversed-phase chromatography under acidic conditions, as this is one of the most commonly used separation methods in bottom-up proteomics.
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2.
Vitamin D Assays.
Bikle, DD
Frontiers of hormone research. 2018;:14-30
Abstract
The number of requests for vitamin D metabolite measurements has increased dramatically over the past decade leading commercial laboratories to develop rapid high throughput assays. The measurement of 25-hydroxyvitamin D (25[OH]D) and to a lesser extent 1,25-dihydroxyvitamin D (1,25[OH]2D) dominates these requests, but requests for multiple metabolite measurements in the same sample are also increasing. The most commonly used methods include immunoassays and liquid chromatography/mass spectrometry (LC-MS). Each method has its advantages and disadvantages, but with improvements in technology, especially in LC-MS, this method is gaining ascendance due to its greater precision and flexibility. The use of standards from the National Institutes of Standards and Technology has substantially reduced the variability from laboratory to laboratory, thereby improving the reliability of these measurements. Although the current demand is for measurement of total vitamin D metabolite levels, these metabolites circulate in blood tightly bound to vitamin D binding protein (DBP) and albumin with less than 1% free. The free concentration may be a more accurate indicator of vitamin D status especially in individuals with DBP levels that deviate from the normal population. Thus, methods to measure the free concentration at least of 25(OH)D are becoming available and may supplement if not replace measurements of total levels.
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3.
Targeting human urinary metabolome by LC-MS/MS: a review.
Rodríguez-Morató, J, Pozo, ÓJ, Marcos, J
Bioanalysis. 2018;(7):489-516
Abstract
Urine is a biological matrix that contains hundreds of metabolic end products which constitute the urinary metabolome. The development and advances on LC-MS/MS have revolutionized the analytical study of biomolecules by enabling their accurate identification and quantification in an unprecedented manner. Nowadays, LC-MS/MS is helping to unveil the complexity of urine metabolome, and the results obtained have multiple biomedical applications. This review focuses on the targeted LC-MS/MS analysis of the urine metabolome. In the first part, we describe general considerations (from sample collection to quantitation) required for a proper targeted metabolic analysis. In the second part, we address the urinary analysis and recent applications of four relevant families: amino acids, catecholamines, lipids and steroids.
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4.
The evolution of methods for urinary steroid metabolomics in clinical investigations particularly in childhood.
Honour, JW, Conway, E, Hodkinson, R, Lam, F
The Journal of steroid biochemistry and molecular biology. 2018;:28-51
Abstract
The metabolites of cortisol, and the intermediates in the pathways from cholesterol to cortisol and the adrenal sex steroids can be analysed in a single separation of steroids by gas chromatography (GC) coupled to MS to give a urinary steroid profile (USP). Steroids individually and in profile are now commonly measured in plasma by liquid chromatography (LC) coupled with MS/MS. The steroid conjugates in urine can be determined after hydrolysis and derivative formation and for the first time without hydrolysis using GC-MS, GC-MS/MS and liquid chromatography with mass spectrometry (LC-MS/MS). The evolution of the technology, practicalities and clinical applications are examined in this review. The patterns and quantities of steroids changes through childhood. Information can be obtained on production rates, from which children with steroid excess and deficiency states can be recognised when presenting with obesity, adrenarche, adrenal suppression, hypertension, adrenal tumours, intersex condition and early puberty, as examples. Genetic defects in steroid production and action can be detected by abnormalities from the GC-MS of steroids in urine. New mechanisms of steroid synthesis and metabolism have been recognised through steroid profiling. GC with tandem mass spectrometry (GC-MS/MS) has been used for the tentative identification of unknown steroids in urine from newborn infants with congenital adrenal hyperplasia. Suggestions are made as to areas for future research and for future applications of steroid profiling. As routine hospital laboratories become more familiar with the problems of chromatographic and MS analysis they can consider steroid profiling in their test repertoire although with LC-MS/MS of urinary steroids this is unlikely to become a routine test because of the availability, cost and purity of the internal standards and the complexity of data interpretation. Steroid profiling with quantitative analysis by mass spectrometry (MS) after chromatography now provides the most versatile of tests of adrenal function in childhood.
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5.
25-Hydroxyvitamin D Testing: Immunoassays Versus Tandem Mass Spectrometry.
Garg, U
Clinics in laboratory medicine. 2018;(3):439-453
Abstract
Vitamin D has been associated with many health conditions. Because of widespread deficiency in the general population, laboratory testing of vitamin D has increased exponentially in recent years. Currently, 25-hydroxyvitamin D (25[OH]D) is considered the best marker of vitamin D status. Automated immunoassays and tandem mass spectrometry are the most widely used assays for the measurement of 25(OH)D. Because a medical decision of vitamin D deficiency and treatment are made based on specific levels, it is important that different 25(OH)D assays are harmonized. Despite standardization efforts, significant differences remain among various methods and laboratories for the measurement of 25(OH)D.
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6.
Generic method approaches for monoclonal antibody therapeutics analysis using both ligand binding and LC-MS/MS techniques.
Lee, JW
Bioanalysis. 2016;(1):19-27
Abstract
Monoclonal antibody (mAb) and mAb-derived biotherapeutics are being developed to interact with specific target molecule(s) to intervene disease formation or progression. LC-MS/MS methods have emerged to compensate for the limitations of conventional ligand-binding assays. Application of a generic LC-MS/MS method to multiple mAb candidates can save method development time as most mAb biotherapeutics are IgG 1, 2 and 4 isotypes. Three common components are essential to a generic LC-MS/MS method: a common workflow, a common surrogate peptide and the corresponding stable isotope-labeled internal standard. The generic LC-MS/MS method is translatable from a single- into a multiple-analyte method, and from a generic into a specific method. Strategies and caveats on method applications are discussed in this chapter.
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7.
Application of mass spectrometry in newborn screening: about both small molecular diseases and lysosomal storage diseases.
Hwu, WL, Chien, YH, Lee, NC, Wang, SF, Chiang, SC, Hsu, LW
Topics in current chemistry. 2014;:177-96
Abstract
Many genetic diseases, especially the inborn errors of metabolism, have very low incidences, so developing a newborn screening test for each disease is not practical. This obstacle was overcome by employing the tandem mass spectrometry (MS/MS) technology. In the analysis, the samples can be injected directly into the flowing system without passing through a column, and both acylcarnitine and amino acid profiles can be obtained at the same time. MS/MS newborn screening has been shown to improve the outcome of patients affected by a number of inborn errors of metabolism. Recently, MS/MS analytical methods were developed for second-tier tests of newborn screening; new substrates have also been developed to measure the activity of lysosomal enzymes so lysosomal storage diseases can be diagnosed by MS/MS method now.
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8.
Open source libraries and frameworks for mass spectrometry based proteomics: a developer's perspective.
Perez-Riverol, Y, Wang, R, Hermjakob, H, Müller, M, Vesada, V, Vizcaíno, JA
Biochimica et biophysica acta. 2014;(1 Pt A):63-76
Abstract
Data processing, management and visualization are central and critical components of a state of the art high-throughput mass spectrometry (MS)-based proteomics experiment, and are often some of the most time-consuming steps, especially for labs without much bioinformatics support. The growing interest in the field of proteomics has triggered an increase in the development of new software libraries, including freely available and open-source software. From database search analysis to post-processing of the identification results, even though the objectives of these libraries and packages can vary significantly, they usually share a number of features. Common use cases include the handling of protein and peptide sequences, the parsing of results from various proteomics search engines output files, and the visualization of MS-related information (including mass spectra and chromatograms). In this review, we provide an overview of the existing software libraries, open-source frameworks and also, we give information on some of the freely available applications which make use of them. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.
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9.
Can we accurately measure the concentration of clinically relevant vitamin D metabolites in the circulation? The problems and their consequences.
Bartoszewicz, Z, Kondracka, A, Jaźwiec, R, Popow, M, Dadlez, M, Bednarczuk, T
Endokrynologia Polska. 2013;(3):238-45
Abstract
Increased interest in vitamin D measurements in clinical studies has contributed to the development in recent years of several new immunochemical assays (manual and for automatic analyzers). New methods, including HPLC (high performance liquid chromatography), and LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) have also been introduced into routine diagnostic laboratories. Because of the variety of assays and methods used, the question arises which one is the most accurate for the measurement of vitamin D metabolites concentration. In this review, we summarise the advantages and disadvantages of these methods, describe the complexity of vitamin D metabolites pattern in the circulation, and discuss the problem of accurate measuring its concentration.
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10.
Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review.
Gosetti, F, Mazzucco, E, Gennaro, MC, Marengo, E
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 2013;:22-36
Abstract
The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called "classical doping agents" (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the list of the prohibited substances of the World Anti-Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly reminded that assumption of doping agents not only leads to athletes the possible failing of doping tests but causes important health risk and WADA prohibited list establishes criteria to highlight the alteration of the natural steroid profile caused by exogenous administration. Doping control analyses are generally performed in urine, a matrix that provides a prolonged detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To identify the chemical structures of anabolic steroids the use of mass spectrometry detection is very advantageous. Gas chromatography-mass spectrometry (GC-MS) techniques allowed for the development of comprehensive screening methods. GC-MS methods are sensitive and robust but present the disadvantages of time-consuming sample pretreatment, that is often based on hydrolysis and derivatisation reactions. Liquid chromatography-mass spectrometry (LC-MS) methods have been successfully used to identify and determinate steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has become the technique of choice for steroid analysis. Due to its generally higher speed, sensitivity, reproducibility and specificity with respect to HPLC, it can be used to simultaneously separate and determinate multi component steroid mixtures. The technique is of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency enhancement due to the small particle (sub-2μm) column packing, which provides high peak capacity within analysis times even 5-10 fold shorter than conventional HPLC methods. Modern multiplex instruments can analyze thousands of samples per month so that, notwithstanding the generally high instrumental costs, the cost of the individual assay is affordable. In addition, the improved specificity and resolution offered by time-of-flight or quadrupole time-of-flight mass spectrometry allow their application in doping control analysis or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of the present review is to consider, compare and discuss the applications of the UHPLC/MS methods present in literature for the identification and determination of forbidden steroids and their metabolites in human biological matrices.