Top-down mass spectrometry on tissue extracts and biofluids with isoelectric focusing and superficially porous silica liquid chromatography.

Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ~7.0 femtomole detection limits and linear spectral response for proteins fractionated over ~4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5-6× increase in the number of unique monoisotopic masses observed <30 kDa and an ~4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate pI compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different pI. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues.

Thyroxine and triiodothyronine content in commercially available thyroid health supplements.

BACKGROUND: As defined by the Dietary Supplement Health and Education Act 1997, such substances as herbs and dietary supplements fall under general Food and Drug Administration supervision but have not been closely regulated to date. We examined the thyroid hormone content in readily available dietary health supplements marketed for "thyroid support." METHODS: Ten commercially available thyroid dietary supplements were purchased. Thyroid supplements were dissolved in 10 mL of acetonitrile and water with 0.1% trifloroacetic acid and analyzed using high-performance liquid chromatography for the presence of both thyroxine (T4) and triiodothyronine (T3) using levothyroxine and liothyronine as a positive controls and standards. RESULTS: The amount of T4 and T3 was measured separately for each supplement sample. Nine out of 10 supplements revealed a detectable amount of T3 (1.3-25.4 µg/tablet) and 5 of 10 contained T4 (5.77-22.9 µg/tablet). Taken at the recommended dose, 5 supplements delivered T3 quantities of greater than 10 µg/day, and 4 delivered T4 quantities ranging from 8.57 to 91.6 µg/day. CONCLUSIONS: The majority of dietary thyroid supplements studied contained clinically relevant amounts of T4 and T3, some of which exceeded common treatment doses for hypothyroidism. These amounts of thyroid hormone, found in easily accessible dietary supplements, potentially expose patients to the risk of alterations in thyroid levels even to the point of developing iatrogenic thyrotoxicosis. The current study results emphasize the importance of patient and provider education regarding the use of dietary supplements and highlight the need for greater regulation of these products, which hold potential danger to public health.

Changes in nitrate and nitrite concentrations over 24h for sweet basil and scallions.

Nitrate and nitrite concentrations were determined for sweet basil and scallions over 24h to determine if time of sampling or harvest impacts concentrations in raw vegetables. Also, nitrate and nitrite concentrations were determined separately for various edible parts of these plants. Basil had significant changes in nitrate and nitrite concentrations over a 24h period. Nitrate was correlated to changes in light intensity with a 3h lag time. The highest nitrate concentrations in basil (2777 ppm) occurred around 3h after the light intensity peaked and had low values (165-574 ppm) during the dark period. The scallion nitrate and nitrite concentrations were always low but nitrate showed a peak a few hours before sunrise. Nitrate and nitrite concentrations in some raw vegetables may be reduced by harvesting at the best time of day for each type of plant. Nitrate concentrations were different in the edible plant parts tested.