Search

Identifying and annotating the molecular composition of individual foods will improve scientific understanding of how foods impact human health and how much variation exists in the molecular composition of foods of the same species. The complexity of this task includes distinct varieties and variations in natural occurring pigments of foods. Lipidomics, a sub-field of metabolomics, has emerged as an effective tool to help decipher the molecular composition of foods. For this proof-of-principle research, we determined the lipidomic profiles of green, yellow and red bell peppers (Capsicum annuum) using liquid chromatography mass spectrometry and a novel tool for automated annotation of compounds following database searches. Among 23 samples analyzed from 6 peppers (2 green, 1 yellow, and 3 red), over 8000 lipid compounds were detected with 315 compounds (106 annotated) found in all three colors. Assessments of relationships between these compounds and pepper color, using linear mixed effects regression and false discovery rate (p-value = 7.4 × 10−5; FDR adjusted p-value = 0.0080). These results support lipidomics as a viable analytical technique to identify molecular compounds that can be used for unique characterization of foods.

OBJECTIVES: Astaxanthin, a predominately marine-source carotenoid, is the subject of a large number of studies for its antioxidant and anti-inflammatory properties. Astaxanthin is not generally a primary carotenoid in human plasma due to relatively low dietary intake. Salmon is the one of the few dietary sources of astaxanthin in typical American diets and the concentration may vary by the source of salmon foods. A study was performed to 1) Compare astaxanthin concentration in various sources of salmon; 2) Compare astaxanthin plasma concentrations before and after salmon consumption. METHODS: An assortment of salmon types and forms was purchased in the greater Denver, CO region: wild Pacific, farmed Atlantic, canned and pouch. Plasma samples were collected from five participants prior to and after a five week Mediterranean diet intervention study, which included two servings of salmon per week. Salmon samples were freeze-dried, then both salmon (in triplicate) and plasma samples were prepared by liquid-liquid extraction for untargeted liquid chromatography-mass spectrometry analysis. An accurate mass and retention time database was used to identify and quantify astaxanthin. ANOVA with Tukey multiple testing corrections was used to assess the relationship between astaxanthin and the different salmon products, and paired t-tests for astaxanthin in plasma. RESULTS: Astaxanthin concentration was significantly higher in fresh salmon compared to pouch packaged (23.0-fold; P = 1.70e-04) and canned (34.9-fold; P = 1.23e-08). Interestingly, astaxanthin levels were similar between fresh wild Pacific and fresh farmed Atlantic salmon (0.91-fold, P = 0.82) and by mode of cooking (i.e., fresh, cooked, frozen; P = 0.81). Astaxanthin concentration in plasma was significantly increased after farmed Atlantic salmon consumption (1.98-fold, P = 6.16e-09). CONCLUSIONS: Our data suggest that astaxanthin concentration varies among different processed salmon products compared to wild and farmed salmon. After salmon consumption, plasma astaxanthin concentration increased and may have potential as a biomarker of salmon consumption. FUNDING SOURCES: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

OBJECTIVES: The carotenoid β-cryptoxanthin is a natural pigment that is both an antioxidant and a precursor to retinol. Research supports that β-cryptoxanthin has greater bioavailability than β-carotene in humans. Red bell peppers have more than double the amount of β-cryptoxanthin than any of the top seven consumed vegetables, as ranked by the USDA. To determine if the amounts of β-cryptoxanthin in bell peppers are dependent upon the organic status, color, cooking, season or location that the fruit was grown within North America, β-cryptoxanthin was measured and compared in green, red and yellow bell peppers. METHODS: An assortment of bell peppers were purchased in the greater Denver, CO region. Green, red and yellow peppers; organic and non-organic; and peppers grown in Canada, the US and Mexico during two different seasons were selected for analysis. The effects of lightly sautéing compared to fresh peppers and season of growth were compared. Samples (100 mg/1 mL) were freeze-dried, then prepared by liquid-liquid extraction for untargeted liquid chromatography mass spectrometry (LC-MS)-based metabolomics analysis. An accurate mass and retention time (AMRT) database was used to identify and quantify β-cryptoxanthin. Linear regression was used to assess the relationship between β-cryptoxanthin and pepper qualities. RESULTS: β-cryptoxanthin concentration was significantly higher in red bell peppers compared to green (11.8-fold) and yellow peppers (7.1-fold) (P = 1.624e-11). β-cryptoxanthin concentration does not appear to be influenced by organic status, season or geographic location. Likewise, the cooked peppers were similar in β-cryptoxanthin content compared to their fresh counterparts. CONCLUSIONS: Our results suggest that the consumption of bell peppers as a source of β-cryptoxanthin is consistent across organic status, fresh, cooked, season and the location in which they were grown. While β-cryptoxanthin concentration in significantly higher in red bell peppers, more research is necessary in order to determine whether these differences result in any altered health outcomes. FUNDING SOURCES: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science , this issue p. 711

Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications: (1) immediate short-term training workshops and (2) long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute (NHLBI)-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health (NJH) and the University of Colorado School of Medicine (UCD). The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consistently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications.

Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

Background Recombinant human activated protein C (rhAPC) is associated with improved survival in high-risk patients with severe sepsis; however, the effects of both lipopolysaccharide (LPS) and rhAPC on the bronchoalveolar lavage fluid (BALF) proteome are unknown. Methods Using differential in gel electrophoresis (DIGE) we identified changes in the BALF proteome from 10 healthy volunteers given intrapulmonary LPS in one lobe and saline in another lobe. Subjects were randomized to pretreatment with saline or rhAPC. Results An average of 255 protein spots were detected in each proteome. We found 31 spots corresponding to 8 proteins that displayed abundance increased or decreased at least 2-fold after LPS. Proteins that decreased after LPS included surfactant protein A, immunoglobulin J chain, fibrinogen-γ, α1-antitrypsin, immunoglobulin, and α2-HS-glycoprotein. Haptoglobin increased after LPS-treatment. Treatment with rhAPC was associated with a larger relative decrease in immunoglobulin J chain, fibrinogen-γ, α1-antitrypsin, and α2-HS-glycoprotein. Conclusion Intrapulmonary LPS was associated with specific protein changes suggesting that the lung response to LPS is more than just a loss of integrity in the alveolar epithelial barrier; however, pretreatment with rhAPC resulted in minor changes in relative BALF protein abundance consistent with its lack of affect in ALI and milder forms of sepsis.