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1.
J Mass Spectrom ; 55(8): e4535, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32725946
3.
J Mass Spectrom ; 55(8): e4524, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32558000

ABSTRACT

The development of a reliable quantitative method for a specific application requires consideration of several important principles. Although mass spectrometry has become the "gold standard" for sensitive and precise multicomponent quantification, it does not necessarily follow that all quantitative data generated by mass spectrometry are either precise or accurate. Depending on the nature of the sample and the goal of the assay, the endeavor can be challenging. With attention to some critical concerns, valid results can be obtained; without attention to these concerns, results can be misleading and, in some instances, invalid. For almost 10 years, beginning in 1996, the authors of this article, together with their friend and colleague Al Yergey, taught an American Society for Mass Spectrometry (ASMS) Short Course on the principles of quantitative mass spectrometry. In this Special Feature, we revisit and update some of these principles. We pay special attention to the contributions of Al Yergey to the course that not only enriched our own understanding of quantitative analysis but also had a measurable impact on the entire field of mass spectrometry.

4.
Methods Mol Biol ; 2138: 49-81, 2020.
Article in English | MEDLINE | ID: mdl-32219740

ABSTRACT

The apolipoproteins are well known for their roles in both health and disease, as components of plasma lipoprotein particles, such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), chylomicrons, and metabolic, vascular- and inflammation-related disorders, such as cardiovascular disease, atherosclerosis, metabolic syndrome, and diabetes. Increasingly, their roles in neurovascular and neurodegenerative disorders are also being elucidated. They play major roles in lipid and cholesterol transport between blood and organs and are, therefore, critical to maintenance and homeostasis of the lipidome, with apolipoprotein-lipid interactions, including cholesterol, fatty acids, triglycerides, phospholipids, and isoprostanes. Further, they have important pleiotropic roles related to aging and longevity, which are largely managed through their many structural variants, including multiple isoforms, and a diversity of post-translational modifications. Consequently, tools for the characterization and accurate quantification of apolipoproteins, including their diverse array of variant forms, are required to understand their salutary and disease related roles. In this chapter we outline three distinct quantitative approaches suitable for targeting apolipoproteins: (1) multiplex immunoassays, (2) mass spectrometric immunoassay, and (3) multiple reaction monitoring, mass spectrometric quantification. We also discuss management of pre-analytical and experimental design variables.


Subject(s)
Apolipoproteins/blood , Plasma/metabolism , Animals , Evaluation Studies as Topic , Humans , Immunoassay/methods , Mass Spectrometry/methods
5.
J Mass Spectrom ; 54(3): 288-300, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30676684

ABSTRACT

Increasingly, the activities of the practicing scientist inextricably intersect with the business world. Science is central to the development and delivery of many goods or services, and scientific discovery can directly, or indirectly, lead to substantial revenue generation. Scientists, however, have little training in the complex issues of intellectual property (IP), and often little understanding of their rights and obligations. Here, we define IP and disuss what it means to be an inventor or creator of IP. We define and differentiate between the primary forms of IP-trade secrets, copyright, trademarks, and patents-and discuss their implementation and implications for the practicing scientist.

6.
J Mass Spectrom ; 53(6): 525-540, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29532552

ABSTRACT

The practice of laser desorption/ionization (LDI) mass spectrometry continues to evolve. In the most commonly adopted manifestation of LDI, matrix assisted LDI, attention continues to be directed towards novel sample application strategies and modifications to the sample plate. Specifically, researchers continue to explore adaptations to the conventional, stainless steel sample plate that is the centerpiece of conventional LDI. Numerous variants of LDI-MS have been reported based on modifications of the plate surface, but none of these is widely adopted, either by end-users or by instrument manufacturers. Further, at this time, advances in surface engineering have had only modest impact on day-to-day operation. In this article, we review and discuss some of the numerous, but scattered reports on novel LDI strategies with an emphasis on modified sample support substrates and plates. We discuss and highlight innovations that have the potential to markedly enhance the utility of LDI-MS.

8.
J Am Soc Mass Spectrom ; 27(3): 532-41, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26733405

ABSTRACT

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.


Subject(s)
Glycated Hemoglobin/analysis , Hemoglobin Subunits/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Chromatography, High Pressure Liquid/methods , Diabetes Mellitus/diagnosis , Humans , Linear Models
9.
Clin Chem ; 62(1): 134-43, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26585930

ABSTRACT

BACKGROUND: MALDI-TOF mass spectrometry (MS) is set to make inroads into clinical chemistry because it offers advantages over other analytical platforms. These advantages include low acquisition and operating costs, ease of use, ruggedness, and high throughput. When coupled with innovative front-end strategies and applied to important clinical problems, it can deliver rapid, sensitive, and cost-effective assays. CONTENT: This review describes the general principles of MALDI-TOF MS, highlights the unique features of the platform, and discusses some practical methods based upon it. There is substantial potential for MALDI-TOF MS to make further inroads into clinical chemistry because of the selectivity of mass detection and its ability to independently quantify proteoforms. SUMMARY: MALDI-TOF MS has already transformed the practice of clinical microbiology and this review illustrates how and why it is now set to play an increasingly important role in in vitro diagnostics in particular, and clinical chemistry in general.


Subject(s)
Chemistry, Clinical/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Chemistry, Clinical/economics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/economics
10.
Biomed Res Int ; 2015: 519830, 2015.
Article in English | MEDLINE | ID: mdl-25789320

ABSTRACT

Alpha-linolenic acid (ALA) is plant-based essential omega-3 polyunsaturated fatty acids that must be obtained through the diet. This could explain in part why the severe deficiency in omega-3 intake pointed by numerous epidemiologic studies may increase the brain's vulnerability representing an important risk factor in the development and/or deterioration of certain cardio- and neuropathologies. The roles of ALA in neurological disorders remain unclear, especially in stroke that is a leading cause of death. We and others have identified ALA as a potential nutraceutical to protect the brain from stroke, characterized by its pleiotropic effects in neuroprotection, vasodilation of brain arteries, and neuroplasticity. This review highlights how chronic administration of ALA protects against rodent models of hypoxic-ischemic injury and exerts an anti-depressant-like activity, effects that likely involve multiple mechanisms in brain, and may be applied in stroke prevention. One major effect may be through an increase in mature brain-derived neurotrophic factor (BDNF), a widely expressed protein in brain that plays critical roles in neuronal maintenance, and learning and memory. Understanding the precise roles of ALA in neurological disorders will provide the underpinnings for the development of new therapies for patients and families who could be devastated by these disorders.


Subject(s)
Fatty Acids, Omega-3/metabolism , Neuroprotective Agents/metabolism , Stroke/prevention & control , alpha-Linolenic Acid/metabolism , Animals , Brain/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Humans , Stroke/metabolism
11.
Mol Cell Endocrinol ; 401: 142-54, 2015 Feb 05.
Article in English | MEDLINE | ID: mdl-25498962

ABSTRACT

Overweight (OW) and obese individuals are considered to be graded parts of the scale having increasing weight as a common feature. They may not, however, be part of the same continuum and may differ metabolically. In this study we applied an untargeted proteomic approach to compare protein abundances in mature adipocytes derived from the subcutaneous adipose tissue of overweight and morbidly obese female subjects to those of lean age matched controls. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both lean (L; n = 7, 23.3 ± 0.4 kg/m(2); mean BMI ± SD), overweight (OW; n = 8, 27.9 ± 0.6 kg/m(2); mean BMI ± SD) and morbidly obese (MOB; n = 7, 44.8 ± 3.8 kg/m(2); mean BMI ± SD) individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). One hundred and ten differentially expressed protein spots (i.e., fitting the statistical criteria ANOVA test, p < 0.05; fold-change ≥1.5) were detected, and of these, 89 were identified by MALDI-TOF mass spectrometry. Of these, 66 protein spots were common to both groups whereas 23 were unique to the MOB group. Significant differences were evident in the abundances of key proteins involved in glucose and lipid metabolism, energy regulation, cytoskeletal structure and redox control signaling pathways. Differences in the abundance of some chaperones were also evident. The differentially abundant proteins were investigated using Ingenuity Pathway Analysis (IPA) to establish their associations with known biological functions. The network identified in the OW group with the highest score relates to-: cell-to-cell signaling and interaction; in contrast, in the MOB group the major interacting pathways are associated with lipid metabolism, small molecule biochemistry and cancer. The differences in abundance of the differentially regulated proteins were validated by immunoblotting. These findings provide insights into metabolic differences in OW and MOB individuals.


Subject(s)
Adipocytes/metabolism , Obesity, Morbid/metabolism , Proteome/isolation & purification , Proteomics/methods , Subcutaneous Fat, Abdominal/cytology , Body Mass Index , Female , Gene Expression Regulation , Humans , Lipid Metabolism , Obesity, Morbid/pathology , Protein Interaction Maps , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Subcutaneous Fat, Abdominal/metabolism , Subcutaneous Fat, Abdominal/pathology
12.
Exp Biol Med (Maywood) ; 239(5): 568-80, 2014 May.
Article in English | MEDLINE | ID: mdl-24668551

ABSTRACT

Bariatric surgery is the most successful therapeutic approach to weight loss, but how it leads to weight loss, and how it resolves obesity-related complications, including type-2 diabetes, are poorly understood. This study, comprising two groups of individuals, one on a low-calorie diet (n = 5) and one undergoing bariatric surgery (n = 7), used both targeted and untargeted proteomic approaches to determine changes in protein levels pre- and post-intervention (i.e. 3-6 months later). Changes were observed in both circulating and excreted proteins following weight loss. Targeted multiplexed biochip arrays measured 12 plasma peptides/proteins involved in metabolism and inflammation: C-peptide, ferritin, interleukin-6, interleukin-1 alpha, resistin, insulin, tumor necrosis factor alpha, leptin, plasminogen-activator inhibitor-1, adiponectin, cystatin C, and C-reactive protein. Following a low-calorie diet, plasma insulin and C-reactive protein levels were significantly reduced (P = 0.045 and P = 0.030, respectively); adiponectin increased and leptin decreased following surgery (P = 0.014 and P = 0.005, respectively). Untargeted proteomic analysis employing 2D difference in-gel electrophoresis (DIGE) showed 28 protein spots with ≥1.5-fold changes in expression following weight loss by a low-calorie diet; comparison of pre- and post-intervention urine samples from the bariatric surgery group showed changes in excretion of 110 protein spots. The combination of targeted protein analysis by multiplexed arrays and an exploratory (i.e. an unbiased or discovery) proteomic assessment of hundreds of proteins offers valuable insights into the mechanistic differences between alternative weight-loss strategies. This is a powerful hypothesis-generating approach to study complex, multifactorial syndromes such as obesity. The findings that arise from these studies can then be validated in targeted, hypothesis-directed investigations.


Subject(s)
Bariatric Surgery , Blood Proteins/analysis , Caloric Restriction , Obesity/therapy , Proteome/analysis , Urine/chemistry , Adult , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Male , Obesity/surgery , Protein Array Analysis , Weight Loss
13.
Mass Spectrom Rev ; 33(4): 237-76, 2014.
Article in English | MEDLINE | ID: mdl-24167057

ABSTRACT

Reactive-nitrogen species (RNS) such as peroxynitrite (ONOO(-)), that is, the reaction product of nitric oxide ((•)NO) and superoxide (O2(-•)), nitryl chloride (NO2Cl) and (•)NO2 react with the activated aromatic ring of tyrosine to form 3-nitrotyrosine. This modification, which has been known for more than a century, occurs to both the free form of the amino acid (i.e., soluble/free tyrosine) and to tyrosine residues covalently bound within the backbone of peptides and proteins. Nitration of tyrosine is thought to be of biological significance and has been linked to health and disease, but determining its role has proved challenging. Several key questions have been the focus of much of the research activity: (a) to what extent is free/soluble tyrosine nitrated in biological tissues and fluids, and (b) are there specific site(s) of nitration within peptides/proteins and to what extent (i.e., stoichiometry) does this modification occur? These issues have been addressed in a wide range of sample types (e.g., blood, urine, CSF, exhaled breath condensate and various tissues) and a diverse array of physiological/pathophysiological scenarios. The accurate determination of nitrated tyrosine is, however, a stumbling block. Despite extensive study, the extent to which nitration occurs in vivo, the specificity of the nitration reaction, and its importance in health and disease, remain unclear. In this review, we highlight the analytical challenges and discuss the approaches adopted to address them. Mass spectrometry, in combination with either gas chromatography (GC-MS, GC-MS/MS) or liquid chromatography (LC-MS/MS), has played the central role in the analysis of 3-nitrotyrosine and tyrosine-nitrated biological macromolecules. We discuss its unique attributes and highlight the role of stable-isotope labeled 3-nitrotyrosine analogs in both accurate quantification, and in helping to define the biological relevance of tyrosine nitration. We show that the application of sophisticated mass spectrometric techniques is advantageous if not essential, but that this alone is by no means a guarantee of accurate findings. We discuss the important analytical challenges in quantifying 3-nitrotyrosine, possible workarounds, and we attempt to make sense of the disparate findings that have been reported so far.


Subject(s)
Mass Spectrometry/methods , Tyrosine/analogs & derivatives , Amino Acids/chemistry , Animals , Artifacts , Body Fluids/chemistry , Carbon Dioxide/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Esters/chemistry , Gas Chromatography-Mass Spectrometry/methods , Glutathione/physiology , Humans , Hydrogen-Ion Concentration , Mass Spectrometry/trends , Models, Chemical , Molecular Structure , Nitrosation , Peroxynitrous Acid/pharmacology , Phenols/chemistry , Proteins/chemistry , Proteins/drug effects , Reactive Nitrogen Species/analysis , Sulfhydryl Compounds/chemistry , Tyrosine/analysis , Tyrosine/isolation & purification , Validation Studies as Topic
14.
J Mass Spectrom ; 48(7): 779-94, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23832934

ABSTRACT

The milk of the one-humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two-dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography-tandem mass spectrometry (LC-MS/MS) following one-dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy-chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel-eluted liquid fraction entrapment electrophoresis and (2) small-scale electrophoretic separation. Extracts were analyzed by LC-MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals.


Subject(s)
Camelus , Milk Proteins/analysis , Peptide Fragments/analysis , Peptide Mapping/methods , Spectrometry, Mass, Electrospray Ionization/methods , Amino Acid Sequence , Animals , Electrophoresis, Gel, Two-Dimensional , Female , Milk Proteins/chemistry , Milk Proteins/classification , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/classification
15.
J Proteomics ; 75(17): 5479-92, 2012 Sep 18.
Article in English | MEDLINE | ID: mdl-22771520

ABSTRACT

INTRODUCTION: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints. METHODS: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography. RESULTS: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p<0.005). CONCLUSION: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.


Subject(s)
Arthritis, Juvenile/diagnosis , Vitamin D-Binding Protein/physiology , Adolescent , Amino Acid Sequence , Arthritis, Juvenile/metabolism , Biomarkers/analysis , Biomarkers/metabolism , Child , Child, Preschool , Cohort Studies , Disease Progression , Female , Humans , Infant , Male , Models, Biological , Molecular Sequence Data , Prognosis , Protein Isoforms/analysis , Protein Isoforms/metabolism , Protein Isoforms/physiology , Proteome/analysis , Proteome/metabolism , Synovial Fluid/chemistry , Synovial Fluid/metabolism , Vitamin D-Binding Protein/analysis , Vitamin D-Binding Protein/metabolism
16.
J Mass Spectrom ; 47(6): 795-809, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22707172

ABSTRACT

The mass spectrometry community has expanded as instruments became more powerful, user-friendly, affordable and readily available. This opens up opportunities for novice users to perform high impact research, using highly advanced instrumentation. This introductory tutorial is targeted at the novice user working in a research setting. It aims to offer the benefit of other people's experiences and to help newcomers avoid known pitfalls and problematic issues. It discusses some of the essential features of sound analytical chemistry and highlights the need to use validated analytical methods that provide high quality results along with a measure of their uncertainty. Examples are used to illustrate potential pitfalls and their consequences.


Subject(s)
Mass Spectrometry/methods , Mass Spectrometry/standards , Proteomics/methods , Proteomics/standards , Reproducibility of Results , Research , Sensitivity and Specificity
17.
PLoS One ; 7(4): e34591, 2012.
Article in English | MEDLINE | ID: mdl-22514641

ABSTRACT

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(ß) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.


Subject(s)
Corynebacterium diphtheriae/metabolism , Enterobactin/analogs & derivatives , Siderophores/chemistry , Siderophores/isolation & purification , Biological Transport/drug effects , Citrates/chemistry , Corynebacterium diphtheriae/drug effects , Enterobactin/chemistry , Enterobactin/isolation & purification , Enterobactin/pharmacology , Ferric Compounds/chemistry , Iron/metabolism , Magnetic Resonance Spectroscopy , Ornithine/analogs & derivatives , Ornithine/chemistry , Siderophores/pharmacology , Spectrometry, Mass, Electrospray Ionization
18.
Rheumatology (Oxford) ; 51(3): 423-33, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22179724

ABSTRACT

This review examines the biomarker development process by using rheumatic disorders as the disease model for discussion. We evaluate the current role of biomarkers in the practice of rheumatology and discuss their likely role in the future. We define the essential components of the biomarker development pipeline and discuss the issue of fitness for purpose, i.e. what the biomarker(s) might offer in a clinical setting. As a component of this review we also highlight several emerging technologies that are beginning to provide practical solutions to support biomarker validation. In the process, we highlight some scenarios where additional biomarkers would add considerable value to clinical practice, and we review appropriate methods for each. We also emphasize some important but infrequently discussed considerations, including the need for protein variant verification. Ultimately, the adroit application of the methods of proteomics will transform the practice rheumatology and allow personalized clinical practice to become a reality.


Subject(s)
Biomarkers/analysis , Rheumatology , Humans , Proteomics/methods , Proteomics/trends , Rheumatology/trends
19.
Int J Proteomics ; 2011: 894618, 2011.
Article in English | MEDLINE | ID: mdl-22110951
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