Publications (8)29.27 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Use of labeled 15N proteins and peptides as internal standards in isotope-dilution mass spectrometry for the quantification of proteins has been increasing and is now accepted as a gold standard for this analysis. As a necessary reagent in this process, stable heavy isotope-labeled internal standards must be rigorously characterized in a number of ways including identity, concentration, purity, and structure. Additionally, the degree of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are 15N labeled, the percentage of incorporation is a valid measurement used to assess the fitness-to-purpose of the material. This measurement should be objective, repeatable, and based on empirical analysis. One means of assigning this value is to compare a mass spectrum of the isotopic profile of a peptide against a series of theoretical profiles containing different enrichment rates. This comparison can be made using the Pearson product-moment correlation coefficient (r) to find the best match between the empirical and theoretical profiles. Theoretical profiles can be generated using probability multinomial analysis but are computationally intensive and require the use of computers for practical use. The method described in this chapter describes the development and use of a computer program to calculate the percentage of 15N enrichment of a labeled internal standard. Additionally, methods will be described for the empirical determination of an isotopic profile using a variety of mass spectrometry techniques.
- [Show abstract] [Hide abstract] ABSTRACT: Perchlorate, an inorganic anion, has recently been recognized as an environmental contaminant by the US Environmental Protection Agency. Urine is the preferred matrix for assessment of human exposure to perchlorate. Although the measurement technique for perchlorate in urine was developed in 2005, the calibration and quality assurance aspects of the metrology infrastructure for perchlorate are still lacking in that there is no certified reference material (CRM) traceable to the International System of Units. To meet the quality assurance needs in biomonitoring measurements of perchlorate and the related anions that affect thyroid health, the National Institute of Standards and Technology (NIST), in collaboration with the Centers for Disease Control and Prevention (CDC), developed Standard Reference Material (SRM) 3668 Mercury, Perchlorate, and Iodide in Frozen Human Urine. SRM 3668 consists of perchlorate, nitrate, thiocyanate, iodine, and mercury in urine at two levels that represent the 50th and 95th percentiles, respectively, of the concentrations (with some adjustments) in the US population. It is the first CRM being certified for perchlorate. Measurements leading to the certification of perchlorate were made collaboratively at NIST and CDC using three methods based on liquid or ion chromatography tandem mass spectrometry. Potential sources of bias were analyzed, and results were compared for the three methods. Perchlorate in SRM 3668 Level I urine was certified to be 2.70 ± 0.21 μg L−1, and for SRM 3668 Level II urine, the certified value is 13.47 ± 0.96 μg L−1. Figure Producing a urine SRM for certification of perchlorate-an emerging environmental contaminant and a potential health hazard
- [Show abstract] [Hide abstract] ABSTRACT: Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID-MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of (15)N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC-MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of (15)N-rCRP as an internal standard in ID-MS. Standardization of CRP assays should help validate the relationship between CRP and human health.
- [Show abstract] [Hide abstract] ABSTRACT: Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.
- [Show abstract] [Hide abstract] ABSTRACT: NIST has performed preliminary research on applying a calibration methodology based on the method of standard additions to the quantification of peptides via reverse-phase liquid chromatography coupled to inductively coupled plasma mass spectrometry (RPLC-ICP-MS). A microwave-assisted lanthanide labeling procedure was developed and applied to derivatize peptides using the macrocyclic bifunctional chemical chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which significantly improved the lanthanide labeling yield and reduced reaction times compared to benchtop labeling procedures. Biomolecular MS technologies of matrix-assisted laser desorption ionization (MALDI)-MS and electrospray ionization (ESI)-MS/MS were used in concert with ICP-MS to confirm the results of microwave labeling, sample cleanup and standard additions experiments for several test peptides. The calibration scheme is outlined in detail and contextualized against complementary high accuracy calibration strategies currently employed for ICP-MS detection of biomolecules. Standard additions experiments using native, non-isotopic peptide calibrants confirm the simplicity of the scheme and the potential of applying a blending (recombined sample and spike) procedure, facilitating calibration via co-elution of lanthanide labeled peptides. Ways to improve and fully leverage the analytical methodology are highlighted.
- [Show abstract] [Hide abstract] ABSTRACT: Accurate assessment of selenium status and selenium nutritional requirement in humans through the detection, identification and quantification of human plasma selenoprotein P (SelP) and glutathione peroxidase 3 (GPx3) has been an on-going effort and long term goal. Although several HPLC-ICP-MS analyses of human plasma/serum have reported SelP and GPx3 measurements, none of them have yet to demonstrate unambiguous mass spectrometry-based identification of these proteins. This study explored the potential of mass spectrometry techniques for the detection and identification of selenoproteins in a human plasma candidate Standard Reference Material (SRM) 1950 Metabolites in Human Plasma, with a total selenium concentration of 105.5 ± 2.3 ng g−1. Since the classical proteomic shotgun approach of depleted human plasma is not specific and sensitive enough to identify low abundant selenoproteins, a laser ablation (LA) inductively coupled plasma mass spectroscopy (ICP-MS) method was developed. The challenge was to develop a highly sensitive method to target selenoproteins at physiological concentrations by using a conventional laser device and a sensitive LA-ICP-MS method easily transposable to other equipped laboratories. Through a combination of better sample preparation, by concentrating selenoproteins onto membranes, and increased sensitivity of Se detection, by humidifying ICP with an organic solution, a LA-ICP-MS method 80 times more sensitive compared to classical LA ICP-MS methods was developed. This method was successfully applied to the detection of SelP and GPx3 selenoproteins at physiological concentrations in samples of human plasma. Once detected, these low abundant selenoproteins were unambiguously identified by tandem mass spectrometry. This study highlights the importance of an approach combining ICP-MS and tandem mass spectrometry for unequivocal selenoprotein detection and identification at physiological concentrations. This procedure can be easily performed in other laboratories to study selenoproteins or other heteroatom-tagged proteins in humans or other biological samples using widely available instrumentation.
- [Show abstract] [Hide abstract] ABSTRACT: This paper describes the development of a reference measurement procedure to quantify human C-reactive protein (CRP) in serum using affinity techniques prior to tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the certification of reference materials in clinically relevant ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and LC-MS/MS analysis of CRP tryptic peptides. The effectiveness of intact protein affinity purification was evaluated through the measurement of CRP in several serum-based CRP control materials, yielding levels that were comparable to their expected mean concentration values. Quantitative results were confirmed with an external calibration approach. This study demonstrates the feasibility of affinity purification with LC-MS/MS for the reference measurement procedure development of low abundance serum protein analytes.
National Institute of Standards and Technology
GAI, Maryland, United States
- Analytical Chemistry Division